Aberrant Wound-Healing Response in Mitomycin C-Treated Leaking Blebs: A Histopathologic Study

PubMed Central

Elner, Victor M.; Newman-Casey, Paula Anne; Patil, A. Jayaprakash; Flint, Andrew; Biswas, Jyotirmay; Moroi, Sayoko E.; Pushparaj, Vaijayanthi; Edward, Deepak P.

2014-01-01

Objective To characterize histopathologic features of leaking mitomycin-C-treated blebs and aberrant wound healing that may lead to persistent conjunctival thinning and leakage. Methods Forty mitomycin C-treated filtering blebs excised for persistent leaks from 40 patients were examined histopathologically using hematoxylin-eosin, periodic acid-Schiff, Masson trichrome, and Alcian blue histochemical stains. Results Ninety percent of the leaking blebs contained epithelial-stromal domes with areas of acellular stroma covered by attenuated epithelium. Seventy-five percent of the blebs demonstrated varying degrees of fibrovascular repair growing from the bleb margin, either beneath or interdigitating with the acellular zone. A novel observation in 65% of specimens was Alcian blue-positive myxoid stroma at the interface between the fibrovascular proliferation and the epithelial-stromal dome. The association between the presence of fibrovascular proliferation and Alcian-blue staining myxoid stroma was significant by Fisher exact test (P=0.002). Conclusions A desirable filtration bleb requires a sufficient reparative fibrovascular response to maintain an epithelial-stromal barrier in order to prevent leakage. Fibroblasts must lay down a continuous collagen-rich fibrous layer, rather than merely myxoid stroma, beneath the conjunctival epithelium to promote bleb stability. Surgical techniques and postsurgical care should aim to attain this desired outcome. PMID:19667341

Application of alternative fixatives to formalin in diagnostic pathology

PubMed Central

Gatta, L. Benerini; Cadei, M.; Balzarini, P.; Castriciano, S.; Paroni, R.; Verzeletti, A.; Cortellini, V.; De Ferrari, F.; Grigolato, P.

2012-01-01

Fixation is a critical step in the preparation of tissues for histopathology. The aim of this study was to investigate the effects of different fixatives vs formalin on proteins and DNA, and to evaluate alternative fixation for morphological diagnosis and nucleic acid preservation for molecular methods. Forty tissues were fixed for 24 h with six different fixatives: the gold standard fixative formalin, the historical fixatives Bouin and Hollande, and the alternative fixatives Greenfix, UPM and CyMol. Tissues were stained (Haematoxylin-Eosin, Periodic Acid Schiff, Trichromic, Alcian-blue, High Iron Diamine stainings), and their antigenicity was determined by immunohistochemistry (performed with PAN-CK, CD31, Ki-67, S100, CD68, AML antibodies). DNA extraction, KRAS sequencing, FISH for CEP-17, and flow cytometry analysis of nuclear DNA content were applied. For cell morphology the alternative fixatives (Greenfix, UPM, CyMol) were equivalent to formalin. As expected, Hollande proved to be the best fixative for morphology. The morphology obtained with Bouin was comparable to the one with formalin. Hollande was the best fixative for histochemistry. Bouin proved to be equivalent to formalin. The alternative fixatives were equivalent to formalin, although with greater variability in haematoxylin-eosin staining. It proved the possibility to obtain immunohistochemical staining largely equivalent to that following formalin-fixation with the following fixatives: Greenfix, Hollande, UPM and CyMol. The tissues fixed in Bouin did not provide results comparable to those obtained with formalin. The DNA extracted from samples fixed with alternative fixatives was found to be suitable for molecular analysis. PMID:22688293

Embedding, serial sectioning and staining of zebrafish embryos using JB-4 resin.

PubMed

Sullivan-Brown, Jessica; Bisher, Margaret E; Burdine, Rebecca D

2011-01-01

Histological techniques are critical for observing tissue and cellular morphology. In this paper, we outline our protocol for embedding, serial sectioning, staining and visualizing zebrafish embryos embedded in JB-4 plastic resin-a glycol methacrylate-based medium that results in excellent preservation of tissue morphology. In addition, we describe our procedures for staining plastic sections with toluidine blue or hematoxylin and eosin, and show how to couple these stains with whole-mount RNA in situ hybridization. We also describe how to maintain and visualize immunofluorescence and EGFP signals in JB-4 resin. The protocol we outline-from embryo preparation, embedding, sectioning and staining to visualization-can be accomplished in 3 d. Overall, we reinforce that plastic embedding can provide higher resolution of cellular details and is a valuable tool for cellular and morphological studies in zebrafish.

DRAQ5 and Eosin (‘D&E’) as an Analog to Hematoxylin and Eosin for Rapid Fluorescence Histology of Fresh Tissues

PubMed Central

Elfer, Katherine N.; Sholl, Andrew B.; Wang, Mei; Tulman, David B.; Mandava, Sree H.; Lee, Benjamin R.; Brown, J. Quincy

2016-01-01

Real-time on-site histopathology review of biopsy tissues at the point-of-procedure has great potential for significant clinical value and improved patient care. For instance, on-site review can aid in rapid screening of diagnostic biopsies to reduce false-negative results, or in quantitative assessment of biospecimen quality to increase the efficacy of downstream laboratory and histopathology analysis. However, the only currently available rapid pathology method, frozen section analysis (FSA), is too time- and labor-intensive for use in screening large quantities of biopsy tissues and is too destructive for maximum tissue conservation in multiple small needle core biopsies. In this work we demonstrate the spectrally-compatible combination of the nuclear stain DRAQ5 and the anionic counterstain eosin as a dual-component fluorescent staining analog to hematoxylin and eosin intended for use on fresh, unsectioned tissues. Combined with optical sectioning fluorescence microscopy and pseudo-coloring algorithms, DRAQ5 and eosin (“D&E”) enables very fast, non-destructive psuedohistological imaging of tissues at the point-of-acquisition with minimal tissue handling and processing. D&E was validated against H&E on a one-to-one basis on formalin-fixed paraffin-embedded and frozen section tissues of various human organs using standard epi-fluorescence microscopy, demonstrating high fidelity of the staining mechanism as an H&E analog. The method was then applied to fresh, whole 18G renal needle core biopsies and large needle core prostate biospecimen biopsies using fluorescence structured illumination optical sectioning microscopy. We demonstrate the ability to obtain high-resolution histology-like images of unsectioned, fresh tissues similar to subsequent H&E staining of the tissue. The application of D&E does not interfere with subsequent standard-of-care H&E staining and imaging, preserving the integrity of the tissue for thorough downstream analysis. These results indicate that this dual-stain pseudocoloring method could provide a real-time histology-like image at the time of acquisition and valuable objective tissue analysis for the clinician at the time of service. PMID:27788264

Comparison between histochemical and immunohistochemical methods for diagnosis of sporotrichosis.

PubMed Central

Marques, M E; Coelho, K I; Sotto, M N; Bacchi, C E

1992-01-01

AIMS: To compare the efficacy of histochemical and immunohistochemical methods in detecting forms of Sporothrix schenckii in tissue. METHODS: Thirty five cutaneous biopsy specimens from 27 patients with sporotrichosis were stained by histochemical haematoxylin and eosin, periodic acid Schiff, and Gomori's methenamine silver methods and an immunohistochemical (avidin-biotin complex immunoperoxidase) (ABC) technique associated with a newly produced rabbit polyclonal antibody anti-Sporothrix schenckii. RESULTS: A total of 29 (83%) cases were positive by the ABC method used in association with anti-Sporothrix schenckii rabbit polyclonal antibodies. Histochemical methods, using silver staining, periodic acid Schiff, and conventional haematoxylin and eosin detected 37%, 23%, and 23% of forms of S schenckii, respectively. The ABC technique was significantly more reliable than periodic acid Schiff and silver staining techniques. CONCLUSIONS: It is concluded that immunostaining is an easy and rapid method which can efficiently increase the accuracy of the diagnosis of sporotrichosis in human tissue. Images PMID:1479036

Multimodal confocal mosaicing microscopy: an emphasis on squamous cell carcinoma

NASA Astrophysics Data System (ADS)

Chen, Nathaniel W.; Sensibaugh, Jordan; Ardeshiri, Ardaland; Blanchard, Adam; Jacques, Steven; Gareau, Daniel

2010-02-01

Our previous study reported a sensitivity of 96.6% and a specificity of 89.2% in rapidly detecting Basal Cell Carcinomas (BCCs) when nuclei were stained with acridine orange. Squamous Cell Carcinomas (SCCs) and infiltrative BCCs remain difficult to detect. More complete screening can be achieved utilizing both acridine orange for nuclei staining and eosin for cytoplasmic contrast, using two lasers to excite the two stains independently. Nuclear fluorescence is achieved by staining with acridine orange (0.5mM, 60 s), and cytoplasmic fluorescence is achieved by staining with eosin working solution (30 s). This work shows good morphological contrast of SCC and infiltrative BCC with eosin, acridine orange, and reflectance, and presents a means for rapid SCC and infiltrative BCC detection in fresh skin excisions using multimodal confocal microscopy. In addition, digital staining is shown to effectively simulate hematoxylin and eosin (H&E) histology with confocal mosaics.

Blind colour separation of H&E stained histological images by linearly transforming the colour space.

PubMed

Celis, R; Romo, D; Romero, E

2015-12-01

Blind source separation methods aim to split information into the original sources. In histology, each dye component attempts to specifically characterize different microscopic structures. In the case of the hematoxylin-eosin stain, universally used for routine examination, quantitative analysis may often require the inspection of different morphological signatures related mainly to nuclei patterns, but also to stroma distribution. Stain separation is usually a preprocessing operation that is transversal to different applications. This paper presents a novel colour separation method that finds the hematoxylin and eosin clusters by projecting the whole (r,g,b) space to a folded surface connecting the distributions of a series of [(r-b),g] planes that divide the cloud of H&E tones. The proposed method produces density maps closer to those obtained with the colour mixing matrices set by an expert, when comparing with the density maps obtained using nonnegative matrix factorization (NMF), independent component analysis (ICA) and a state-of-the-art method. The method has outperformed three baseline methods, NMF, Macenko and ICA, in about 8%, 12% and 52% for the eosin component, whereas this was about 4%, 8% and 26% for the hematoxylin component. © 2015 The Authors Journal of Microscopy © 2015 Royal Microscopical Society.

New concepts of histological changes in experimental augmentation cystoplasty: insights into the development of neoplastic transformation at the enterovesical and gastrovesical anastomosis.

PubMed

Gitlin, J S; Wu, X R; Sun, T T; Ritchey, M L; Shapiro, E

1999-09-01

To our knowledge the pathogenesis of malignancy associated with ileal cystoplasty, ureterosigmoidostomy and ileal conduits is currently unknown. To gain further insights into the mechanism of neoplastic transformation we studied histological changes in a canine augmentation cystoplasty model. Enterocystoplasty and gastrocystoplasty were performed using a 5 to 7 cm. patch of ileum in 8 dogs and gastric antrum in 6. Specimens were harvested 4 months postoperatively. Representative 3 microm sections of the enterovesical and gastrovesical junctions were stained with hematoxylin and eosin. Uroplakin expression was assessed using an indirect peroxidase method subjected to double staining with alcian blue and periodic acid-Schiffreagent. The bladder portion of the augmentation cystoplasty had 3 to 4 stratified cell layers covered with a distinctive umbrella cell layer. Strong uroplakin staining was visible in all cell layers except the basal layer. At the enterovesical and gastrovesical junctions 6 to 10 layers of hyperplastic, urothelial appearing cells covered the glandular epithelium of the ileal and gastric segments. These cells expressed uroplakins. At this junction zone there was a marked decrease of underlying enteric glands, which had atrophied in proportion to the degree of urothelial hyperplasia. Double staining of uroplakin stained sections with alcian blue and periodic acid-Schiff reagent revealed mucosubstances in hyperplastic urothelial cells covering the enteral segments, indicating that the cells co-expressed uroplakins and mucins. Histological changes in this experimental canine model of augmentation cystoplasty indicated that the overgrowth of hyperplastic transitional epithelium develops at the enterovesical and gastrovesical junctions. These cells express not only uroplakins, but also mucosubstances. Our results suggest that the migrated hyperplastic urothelial cells have undergone changes characteristic of the enteric and gastric epithelium, which may have important implications in the pathogenesis of malignancy in bladder augmentations.

The Use of Ancillary Stains in the Diagnosis of Barrett Esophagus and Barrett Esophagus-associated Dysplasia: Recommendations From the Rodger C. Haggitt Gastrointestinal Pathology Society.

PubMed

Srivastava, Amitabh; Appelman, Henry; Goldsmith, Jeffrey D; Davison, Jon M; Hart, John; Krasinskas, Alyssa M

2017-05-01

Barrett esophagus (BE) is a known risk factor for the development of esophageal adenocarcinoma. Pathologists play a critical role in confirming the diagnosis of BE and BE-associated dysplasia. As these diagnoses are not always straightforward on routine hematoxylin and eosin-stained slides, numerous ancillary stains have been used in an attempt to help pathologists confirm the diagnosis. On the basis of an in-depth review of the literature, the Rodger C. Haggitt Gastrointestinal Pathology Society provides recommendations regarding the use of ancillary stains in the diagnosis of BE and BE-associated dysplasia. Because goblet cells are almost always identifiable on routine hematoxylin and eosin-stained sections, there is insufficient evidence to justify reflexive use of Alcian blue (at pH 2.5) and/or periodic-acid Schiff stains on all esophageal biopsies to diagnose BE. In addition, the use of mucin glycoprotein immunostains and markers of intestinal phenotype (CDX2, Das-1, villin, Hep Par 1, and SOX9) are not indicated to aid in the diagnosis of BE at this time. A diagnosis of dysplasia in BE remains a morphologic diagnosis, and hence, ancillary stains are not recommended for diagnosing dysplasia. Although p53 is a promising marker for identifying high-risk BE patients, it is not recommended for routine use at present; additional studies are needed to address questions regarding case selection, interpretation, integration with morphologic diagnosis, and impact on clinical outcome. We hope that this review and our recommendations will provide helpful information to pathologists, gastroenterologists, and others involved in the evaluation of patients with BE and BE-associated dysplasia.

Protocol for vital dye staining of corneal endothelial cells.

PubMed

Park, Sunju; Fong, Alan G; Cho, Hyung; Zhang, Cheng; Gritz, David C; Mian, Gibran; Herzlich, Alexandra A; Gore, Patrick; Morganti, Ashley; Chuck, Roy S

2012-12-01

To describe a step-by-step methodology to establish a reproducible staining protocol for the evaluation of human corneal endothelial cells. Four procedures were performed to determine the best protocol. (1) To determine the optimal trypan blue staining method, goat corneas were stained with 4 dilutions of trypan blue (0.4%, 0.2%, 0.1%, and 0.05%) and 1% alizarin red. (2) To determine the optimal alizarin red staining method, goat corneas were stained with 2 dilutions of alizarin red (1% and 0.5%) and 0.2% trypan blue. (3) To ensure that trypan blue truly stains damaged cells, goat corneas were exposed to either 3% hydrogen peroxide or to balanced salt solution, and then stained with 0.2% trypan blue and 0.5% alizarin red. (4) Finally, fresh human corneal buttons were examined; 1 group was stained with 0.2% trypan blue and another group with 0.4% trypan blue. For the 4 procedures performed, the results are as follows: (1) trypan blue staining was not observed in any of the normal corneal samples; (2) 0.5% alizarin red demonstrated sharper cell borders than 1% alizarin red; (3) positive trypan blue staining was observed in the hydrogen peroxide exposed tissue in damaged areas; (4) 0.4% trypan blue showed more distinct positive staining than 0.2% trypan blue. We were able to determine the optimal vital dye staining conditions for human corneal endothelial cells using 0.4% trypan blue and 0.5% alizarin red.

Ultrastructural Investigation of Pelvic Peritoneum in Patients With Chronic Pelvic Pain and Subtle Endometriosis in Association With Chromoendoscopy.

PubMed

Mehdizadehkashi, Abolfazl; Tahermanesh, Kobra; Fazel Anvari-Yazdi, Abbas; Chaichian, Shahla; Azarpira, Negar; Nobakht, Maliheh; Abed, Seyedeh Mehr; Hashemi, Neda

2017-01-01

To evaluate the pelvic peritoneum under chromoendoscopy by scanning electron microscopy (SEM) as well as light microscopy with hematoxylin and eosin staining and immunohistochemistry (IHC) assays in patients with chronic pelvic pain (CPP) associated with subtle endometriosis. Case series study (Canadian Task Force classification II). A referral academic community tertiary medical center. Three women aged 29 to 37 years were referred to the obstetrics and gynecology clinic of the tertiary university hospital with CPP. They were suspicious for endometriosis, were not responding to medical treatments, and had undergone previous pelvic laparoscopy to determine the stage of endometriosis and preparation of peritoneal samples under the guidance of staining with methylene blue in 0.25% dilution. Comparison of stained and unstained pelvic peritoneal samples after the instillation of 0.25% methylene blue into the pelvic cavity. In 3 patients, laparoscopic examination showed minimal endometriosis. A total of 18 samples (9 stained and 9 unstained) from the 3 patients were prepared for SEM. Ten of the samples (55.6%) showed microstructural peritoneal destruction (7 of 9 stained [77.7%] and 3 of 9 [33.4%] unstained). Eighteen samples (9 stained and 9 unstained) from the 3 patients were also prepared for IHC. Six of these samples (33.3%) were S-100-positive, including 4 of 9 (44.4%) stained samples and 2 of 9 (22.2%) unstained samples. In general, in the context of CPP and endometriosis, there is no established relationship between the severity of pain and stage of endometriosis. In the pathophysiology of CPP associated with endometriosis, ultrastructural changes can play a significant role. Under methylene blue staining, some destroyed areas were detected, but the stained areas do not necessarily correlate with increased microstructural peritoneal destruction. Copyright © 2016 AAGL. Published by Elsevier Inc. All rights reserved.

Morphology and histochemistry of the "C" cells of guinea pig thyroid gland after treatment with STH preparation: Part II--young animals.

PubMed

Sawicki, B

1977-01-01

Investigations were carried out on 32 male guinea pigs 2 to 3 months of age. The STH (produced by BIOMED, Warszawa, Poland) was administered intramuscularly every other day, in 7 injections of 20 Evans's units (E. U.) or 100 E. U./kg body weight each. Thyroid gland sections were stained with heamatoxylin and eosin and with the Azan method. The C cells were detected with the modified silver method of Grimelius and with the HCl-toluidine blue and HCl-lead haemotoxylin techniques. Moreover, reactions were performed for succinate and alpha-glycerophosphate dehydrogenases and also for non-specific esterases and non-specific acetylcholinesterase. STH evoked proliferation of the C cells, changed their morphology and activity pattern of the enzymes present therein, probably testifying to an enhanced secretory activity of these cells.

Sentinel lymph node detection in early stage cervical cancer: a prospective study comparing preoperative lymphoscintigraphy, intraoperative gamma probe, and blue dye.

PubMed

Kara, P Pelin; Ayhan, Ali; Caner, Biray; Gültekin, Murat; Ugur, Omer; Bozkurt, M Fani; Usubutun, Alp

2008-07-01

The objective of this prospective study was to determine the feasibility of sentinel lymph node (SLN) detection in patients with cervical cancer using lymphoscintigraphy (LS), gamma probe, and blue dye. A total of 32 patients with early stage cervical cancer (FIGO IA2-IIA) who were treated with total abdominal hysterectomy and bilateral pelvic and paraortic lymphadenectomy underwent SLN biopsy. LS was performed on all the patients following the injection of 74 MBq technetium-99m-nanocolloid pericervically. The first appearing persistent focal accumulation on either dynamic or static images of LS was considered to be an SLN. Blue dye was injected just prior to surgical incision in 16 patients (50%) at the same locations as the radioactive isotope injection. During the operation, blue-stained node(s) were excised as SLNs. For gamma probe, a lymph node was accepted as an SLN, if its ex vivo radioactive counts were at least 10-fold above background radioactivity. SLNs, which were negative by routine hematoxylin and eosin (H&E) examination, were histopathologically reevaluated for the presence of micrometastases by step sectioning and immunohistochemical staining with pancytokeratin. At least one SLN was identified for each patient by gamma probe. Intraoperative gamma probe was the most sensitive method with a technical success rate of SLN detection of 100% (32/32), followed by LS 87.5% (28/32) and blue dye 68.8% (11/16), respectively. The average number of SLNs per patient detected by gamma probe was 2.09 (range 1-5). The localizations of the SLNs were external iliac 47.8%, obturatory 32.8%, common iliac 9%, paraaortic 4.4%, and paracervical 6%. Micrometastases, not detected by routine H&E were found by immunohistochemistry in one patient. On the basis of the histopathological analysis, the negative predictive value for predicting metastases was 100%, and there were no false-negative results. Preoperative LS with radiocolloids, intraoperative lymphatic mapping with blue dye and gamma probe are all feasible methods comparable with each other for SLN detection in early stage cervical cancer patients, but gamma probe is the most useful method in terms of technical success.

Application of immunohistochemical staining to detect antigen destruction as a measure of tissue damage.

PubMed

Onul, Abdullah; Colvard, Michael D; Paradise, William A; Elseth, Kim M; Vesper, Benjamin J; Gouvas, Eftychia; Deliu, Zane; Garcia, Kelly D; Pestle, William J; Radosevich, James A

2012-09-01

Electrocautery and directed energy devices (DEDs) such as lasers, which are used in surgery, result in tissue damage that cannot be readily detected by traditional histological methods, such as hematoxylin and eosin staining. Alternative staining methods, including 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to stain live tissue, have been reported. Despite providing superior detection of damaged tissue relative to the hematoxylin and eosin (H&E) method, the MTT method possesses a number of drawbacks, most notably that it must be carried out on live tissue samples. Herein, we report the development of a novel staining method, "antigen destruction immunohistochemistry" (ADI), which can be carried out on paraffin-embedded tissue. The ADI method takes advantage of epitope loss to define the area of tissue damage and provides many of the benefits of live tissue MTT staining without the drawbacks inherent to that method. In addition, the authors provide data to support the use of antibodies directed at a number of gene products for use in animal tissue for which there are no species-specific antibodies commercially available, as well as an example of a species-specific direct antibody. Data are provided that support the use of this method in many tissue models, as well as evidence that ADI is comparable to the live tissue MTT method.

Application of Immunohistochemical Staining to Detect Antigen Destruction as a Measure of Tissue Damage

PubMed Central

Onul, Abdullah; Colvard, Michael D.; Paradise, William A.; Elseth, Kim M.; Vesper, Benjamin J.; Gouvas, Eftychia; Deliu, Zane; Garcia, Kelly D.; Pestle, William J.

2012-01-01

Electrocautery and directed energy devices (DEDs) such as lasers, which are used in surgery, result in tissue damage that cannot be readily detected by traditional histological methods, such as hematoxylin and eosin staining. Alternative staining methods, including 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to stain live tissue, have been reported. Despite providing superior detection of damaged tissue relative to the hematoxylin and eosin (H&E) method, the MTT method possesses a number of drawbacks, most notably that it must be carried out on live tissue samples. Herein, we report the development of a novel staining method, “antigen destruction immunohistochemistry” (ADI), which can be carried out on paraffin-embedded tissue. The ADI method takes advantage of epitope loss to define the area of tissue damage and provides many of the benefits of live tissue MTT staining without the drawbacks inherent to that method. In addition, the authors provide data to support the use of antibodies directed at a number of gene products for use in animal tissue for which there are no species-specific antibodies commercially available, as well as an example of a species-specific direct antibody. Data are provided that support the use of this method in many tissue models, as well as evidence that ADI is comparable to the live tissue MTT method. PMID:22723525

Aberrant wound-healing response in mitomycin C-treated leaking blebs: a histopathologic study.

PubMed

Elner, Victor M; Newman-Casey, Paula Anne; Patil, A Jayaprakash; Flint, Andrew; Biswas, Jyotirmay; Moroi, Sayoko E; Pushparaj, Vaijayanthi; Edward, Deepak P

2009-08-01

To characterize histopathologic features of leaking mitomycin C-treated blebs and aberrant wound healing that may lead to persistent conjunctival thinning and leakage. Forty mitomycin C-treated filtering blebs excised for persistent leaks from 40 patients were examined histopathologically using hematoxylin-eosin, periodic acid-Schiff, Masson trichrome, and Alcian blue histochemical stains. Ninety percent of the leaking blebs contained epithelial-stromal domes with areas of acellular stroma covered by attenuated epithelium. Seventy-five percent of the blebs demonstrated varying degrees of fibrovascular repair growing from the bleb margin, either beneath or interdigitating with the acellular zone. A novel observation in 65% of specimens was Alcian blue-positive myxoid stroma at the interface between the fibrovascular proliferation and the epithelial-stromal dome. The association between the presence of fibrovascular proliferation and Alcian blue-staining myxoid stroma was significant by Fisher exact test (P = .002). A desirable filtration bleb requires a sufficient reparative fibrovascular response to maintain an epithelial-stromal barrier to prevent leakage. Fibroblasts must lay down a continuous collagen-rich fibrous layer, rather than merely myxoid stroma, beneath the conjunctival epithelium to promote bleb stability. Surgical techniques and postsurgical care should aim to attain this desired outcome.

Application of Giemsa stain for easy detection of Trichinella spiralis muscle larvae

PubMed Central

Ramírez-Melgar, Carmen; Gómez-Priego, Alberto

2007-01-01

The application of Giemsa technique to stain compressed diaphragm samples obtained from rodents experimentally infected with Trichinella spiralis is described. Diaphragm samples from rats heavily infected with 20 muscle larvae per gram of body weight (20 ML/gbw) were cut into several pieces and stained with Giemsa; on the other hand, whole diaphragms from slightly infected mice (1 ML/gbw) were also stained with Giemsa. Besides, muscle samples were also stained with Giemsa. Observation at 10 × magnification revealed that both ML and nurse cells (NC) look as bluish structures clearly contrasting with the pinkish color of the non-infected muscle fibers. NC in the diaphragms of mice could be easily observed at naked eye as blue points contrasting with the pink surrounding areas formed by the non-infected muscle fibers. Among NC observed in the diaphragms of rats infected with 20 ML/gbw, 4.4% was multiple infection. These findings were confirmed in sectioned and hematoxylin-eosin stained specimens. This data could be usefulness for a rapid diagnosis of trichinellosis in post-mortem mammals without magnification procedures. PMID:17374981

[Forensic medical implications of histomorphological changes in the bone and cartilage tissues under effect of radiation].

PubMed

Osipenkova-Vichtomova, T K

2013-01-01

The objective of the present work was to study roentgenological, microscopic, and histomorphological changes in the bone and cartilage tissues under effect of different doses of gamma-ray radiation from Gammatron-2 (GUT Co 400) and betatron bremsstrahlung radiation (25 MeV). The total radiation dose varied from 9.6 Gy to 120 Gy per unit area during 5-8 weeks. The study included 210 patients at the age from 7 to 82 years (97 men and 113 women). Histomorphological studies were carried out using samples of bone and cartilage tissues taken from different body regions immediately after irradiation and throughout the follow-up period of up to 4 years 6 months. Control samples were the unexposed bone and cartilage tissues from the same subjects (n = 14). The tissues were stained either with eosin and hematoxylin or by Van Gieson's and Mallory's methods. Gomori's nonspecific staining was used to detect acid and alkaline phosphatase activities. Moreover, argyrophilic substance was identified in the cartilaginous tissue. Best's carmine was used for glycogen staining and Weigert's stain for elastic fibers. Metachromasia was revealed by toluidine blue staining and fat by the sudan III staining technique. In addition, the ultrastructure of cartilaginous tissue was investigated. Taken together, these methods made it possible to identify the signs of radiation-induced damage to the bone and cartilage tissues in conjunction with complications that are likely to develop at different periods after irradiation including such ones as spontaneous fractures, deforming arthrosis and radiation-induced tumours.

Better detection of Demodex mites by Löffler’s alkaline methylene blue staining in patients with blepharitis

PubMed Central

Kiuchi, Katsuji

2018-01-01

Purpose To determine whether the Löffler’s alkaline methylene blue staining method is better than no staining in detecting Demodex mites in the eyelashes of patients with blepharitis. Materials and methods Eyelashes were collected from 22 patients with blepharitis. The mean age of the patients was 82.5±6.2 years (± SD) with a range from 71 to 93 years. Eyelashes were epilated by forceps and placed individually on microscope slides. The number of Demodex mites was determined by conventional optical microscopy before and immediately after the addition of the methylene blue staining solution. Results The mean Demodex count before the addition of the methylene blue solution was 2.9±2.9, and it was 4.4±3.9 after the addition of the methylene blue solution (P<0.01, Wilcoxon test). Conclusion The methylene blue staining method is a simple and useful method in detecting the presence and quantifying the number of Demodex mites. We recommend the methylene blue staining method not only for the diagnosis of the presence of Demodex mites but also to evaluate the therapeutic effects of medications to eliminate the mite infestation. PMID:29713140

[Hirschsprung's disease: the immunohistochemistry as ancillary method for the diagnosis.

PubMed

Menchaca-Cervantes, Celestina; Rodríguez-Velasco, Alicia; Ramón-García, Guillermo; Rendón-Macías, Mario Enrique

2013-01-01

Background: the confirmatory diagnosis of Hirschsprung's disease is made by histopathological study. However, this procedure is limited with only hematoxylin and eosin staining, especially in biopsies of premature babies or when non-expert pathologists make the evaluation. The immunohistochemistry from ganglia cell calretinin has been used to reduce the risk of misdiagnosis. Our objective was to show the benefits of this antibody in diagnosis of Hirschsprung's disease in biopsy specimens. Methods: we evaluated patients with histopathological diagnosis of Hirschsprung's disease made by hematoxylin and eosin staining. We determined if there was enough paraffin block for immunohistochemistry with two markers: calretinin and neurofilaments. Three controls of autopsy of children under 3 years of age with other diagnosis were included. Results: of a total of 48 cases with histopathological diagnosis of Hirschsprung's disease only 13 had adequate tissue for immunohistochemistry. The immunohistochemistry confirmed the diagnosis in nine cases. In the other four cases there were initial misdiagnosis due to evidence of calretinin (ganglion cells) and, thus, Hirschsprung's disease was discarded. Conclusions: the use of immunohistochemistry allows confirming the diagnosis of Hirschsprung's disease and reduce the risk of a false-positive result with only hematoxylin and eosin staining.

Better detection of Demodex mites by Löffler's alkaline methylene blue staining in patients with blepharitis.

PubMed

Kiuchi, Katsuji

2018-01-01

To determine whether the Löffler's alkaline methylene blue staining method is better than no staining in detecting Demodex mites in the eyelashes of patients with blepharitis. Eyelashes were collected from 22 patients with blepharitis. The mean age of the patients was 82.5±6.2 years (± SD) with a range from 71 to 93 years. Eyelashes were epilated by forceps and placed individually on microscope slides. The number of Demodex mites was determined by conventional optical microscopy before and immediately after the addition of the methylene blue staining solution. The mean Demodex count before the addition of the methylene blue solution was 2.9±2.9, and it was 4.4±3.9 after the addition of the methylene blue solution ( P <0.01, Wilcoxon test). The methylene blue staining method is a simple and useful method in detecting the presence and quantifying the number of Demodex mites. We recommend the methylene blue staining method not only for the diagnosis of the presence of Demodex mites but also to evaluate the therapeutic effects of medications to eliminate the mite infestation.

Comparison of methylene blue/gentian violet stain to Gram's stain for the rapid diagnosis of gonococcal urethritis in men.

PubMed

Taylor, Stephanie N; DiCarlo, Richard P; Martin, David H

2011-11-01

We compared a simple, one-step staining procedure using a mixture of methylene blue and gentian violet to Gram stain for the detection of gonococcal urethritis. The sensitivity and specificity of both Gram stain and methylene blue/gentian violet stain were 97.3% and 99.6%, respectively. There was a 100% correlation between the 2 methods.

The Resin-Embedded Cornea Prepared Via Rapid Processing Protocol : A Good Histomorphometric Target for Clinical Investigation in Ophthalmology and Optometry

PubMed Central

Cheah, Pike See; Mohidin, Norhani; Mohd Ali, Bariah; Maung, Myint; Latif, Azian Abdul

2008-01-01

This study illustrates and quantifies the changes on corneal tissue between the paraffin-embedded and resin-embedded blocks and thus, selects a better target in investigational ophthalmology and optometry via light microscopy. Corneas of two cynomolgus monkeys (Macaca fascicularis) were used in this study. The formalin-fixed cornea was prepared in paraffin block via the conventional tissue processing protocol (4-day protocol) and stained with haematoxylin and eosin. The glutaraldehyde-fixed cornea was prepared in resin block via the rapid and modified tissue processing procedure (1.2-day protocol) and stained with toluidine blue. The paraffin-embedded sample exhibits various undesired tissue damage and artifact such as thinner epithelium (due to the substantial volumic extraction from the tissue), thicker stroma layer (due to the separation of lamellae and the presence of voids) and the distorted endothelium. In contrast, the resin-embedded corneal tissue has demonstrated satisfactory corneal ultrastructural preservation. The rapid and modified tissue processing method for preparing the resin-embedded is particularly beneficial to accelerate the microscopic evaluation in ophthalmology and optometry. PMID:22570589

Digital enhancement of haematoxylin- and eosin-stained histological images for red-green colour-blind observers.

PubMed

Landini, G; Perryer, G

2009-06-01

Individuals with red-green colour-blindness (CB) commonly experience great difficulty differentiating between certain histological stain pairs, notably haematoxylin-eosin (H&E). The prevalence of red-green CB is high (6-10% of males), including among medical and laboratory personnel, and raises two major concerns: first, accessibility and equity issues during the education and training of individuals with this disability, and second, the likelihood of errors in critical tasks such as interpreting histological images. Here we show two methods to enhance images of H&E-stained samples so the differently stained tissues can be well discriminated by red-green CBs while remaining usable by people with normal vision. Method 1 involves rotating and stretching the range of H&E hues in the image to span the perceptual range of the CB observers. Method 2 digitally unmixes the original dyes using colour deconvolution into two separate images and repositions the information into hues that are more distinctly perceived. The benefits of these methods were tested in 36 volunteers with normal vision and 11 with red-green CB using a variety of H&E stained tissue sections paired with their enhanced versions. CB subjects reported they could better perceive the different stains using the enhanced images for 85% of preparations (method 1: 90%, method 2: 73%), compared to the H&E-stained original images. Many subjects with normal vision also preferred the enhanced images to the original H&E. The results suggest that these colour manipulations confer considerable advantage for those with red-green colour vision deficiency while not disadvantaging people with normal colour vision.

A comparative study to evaluate liquid dish washing soap as an alternative to xylene and alcohol in deparaffinization and hematoxylin and eosin staining

PubMed Central

Pandey, Pinki; Dixit, Alok; Tanwar, Aparna; Sharma, Anuradha; Mittal, Sanjeev

2014-01-01

Introduction: Our study presents a new deparaffinizing and hematoxylin and eosin (H and E) staining method that involves the use of easily available, nontoxic and eco-friendly liquid diluted dish washing soap (DWS) by completely eliminating expensive and hazardous xylene and alcohol from deparaffinizing and rehydration prior to staining, staining and from dehydration prior to mounting. The aim was to evaluate and compare the quality of liquid DWS treated xylene and alcohol free (XAF) sections with that of the conventional H and E sections. Materials and Methods: A total of 100 paraffin embedded tissue blocks from different tissues were included. From each tissue block, one section was stained with conventional H and E (normal sections) and the other with XAF H and E (soapy sections) staining method. Slides were scored using five parameters: Nuclear, cytoplasmic, clarity, uniformity, and crispness of staining. Z-test was used for statistical analysis. Results: Soapy sections scored better for cytoplasmic (90%) and crisp staining (95%) with a statistically significant difference. Whereas for uniformity of staining, normal sections (88%) scored over soapy sections (72%) (Z = 2.82, P < 0.05). For nuclear (90%) and clarity of staining (90%) total scored favored soapy sections, but the difference was not statistically significant. About 84% normal sections stained adequately for diagnosis when compared with 86% in soapy sections (Z = 0.396, P > 0.05). Conclusion: Liquid DWS is a safe and efficient alternative to xylene and alcohol in deparaffinization and routine H and E staining procedure. We are documenting this project that can be used as a model for other histology laboratories. PMID:25328332

Metastatic iridociliary adenocarcinoma in a labrador retriever.

PubMed

Zarfoss, M K; Dubielzig, R R

2007-09-01

An enucleated left eye from a 15-year-old female spayed Labrador Retriever was received by the Comparative Ocular Pathology Laboratory of Wisconsin (COPLOW) for histopathologic evaluation. Routine histologic preparation included staining with hematoxylin and eosin, and with alcian blue periodic acid-Schiff (PAS). At necropsy 9 months later, all grossly abnormal tissues (ipsilateral orbit and lung) were submitted to the COPLOW for histopathologic evaluation. Histopathologic evaluation of the globe revealed extensive invasion of the uvea and sclera by a pleomorphic cell population that formed disorganized cords and exhibited PAS-positive basement membrane material. Necropsy revealed a morphologically similar tumor in the ipsilateral orbit and lung. On immunohistochemical examination, the intraocular tumor stained diffusely immunopositive for vimentin, S-100, and neuron-specific enolase and multifocally, sparsely immunopositive for cytokeratin AE1/AE3. The orbital and thoracic tumors stained positively for vimentin but negatively for cytokeratin AE1/AE3. There are few reports of canine metastatic iridociliary adenocarcinoma in the literature; this is the first with immunohistochemical analysis.

Low vacuum scanning electron microscopy for paraffin sections utilizing the differential stainability of cells and tissues with platinum blue.

PubMed

Inaga, Sumire; Hirashima, Sayuri; Tanaka, Keiichi; Katsumoto, Tetsuo; Kameie, Toshio; Nakane, Hironobu; Naguro, Tomonori

2009-07-01

The present study introduces a novel method for the direct observation of histological paraffin sections by low vacuum scanning electron microscopy (LVSEM) with platinum blue (Pt-blue) treatment. Pt-blue was applied not only as a backscattered electron (BSE) signal enhancer but also as a histologically specific stain. In this method, paraffin sections of the rat tongue prepared for conventional light microscopy (LM) were stained on glass slides with a Pt-blue staining solution (pH 9) and observed in a LVSEM using BSE detector. Under LVSEM, overviews of whole sections as well as three-dimensional detailed observations of individual cells and tissues could be easily made at magnifications from x40 to x10,000. Each kind of cell and tissue observed in the section could be clearly distinguished due to the different yields of BSE signals, which depended on the surface structures and different affinities to Pt-blue. Thus, we roughly classified cellular and tissue components into three groups according to the staining intensity of Pt-blue observed by LM and LVSEM: 1) a strongly stained (deep blue by LM and brightest by LVSEM) group which included epithelial tissue, endothelium and mast cells; 2) a moderately stained (light blue and bright) group which included muscular tissue and nervous tissue; 3) an unstained or weakly stained (colorless and dark) group which included elastic fibers and collagen fibers. We expect that this method will prove useful for the three-dimensional direct observation of histological paraffin sections of various tissues by LVSEM with higher resolutions than LM.

Laparoscopic sentinel node procedure using a combination of patent blue and radiocolloid in women with endometrial cancer.

PubMed

Barranger, Emmanuel; Cortez, Annie; Grahek, Dany; Callard, Patrice; Uzan, Serge; Darai, Emile

2004-03-01

We assessed the feasibility of a laparoscopic sentinel node (SN) procedure based on the combined use of radiocolloid and patent blue labeling in patients with endometrial cancer. Seventeen patients (median age, 69 years) with endometrial cancer of stage I (16 patients) or stage II (1 patient) underwent a laparoscopic SN procedure based on combined radiocolloid and patent blue injected pericervically. After the SN procedure, all patients underwent complete laparoscopic pelvic lymphadenectomy and either laparoscopically assisted vaginal hysterectomy (16 patients) or laparoscopic radical hysterectomy (1 patient). SNs (mean number per patient, 2.6; range, 1-4) were identified in 16 (94.1%) of the 17 patients. Macrometastases were detected in three SNs from two patients by hematoxylin and eosin staining. In three other patients, immunohistochemical analysis identified six micrometastatic SNs and one SN containing isolated tumor cells. No false-negative SN results were observed. An SN procedure based on a combination of radiocolloid and patent blue is feasible in patients with early endometrial cancer. Combined use of laparoscopy and this SN procedure permits minimally invasive management of endometrial cancer.

Enabling Histopathological Annotations on Immunofluorescent Images through Virtualization of Hematoxylin and Eosin

PubMed Central

Lahiani, Amal; Klaiman, Eldad; Grimm, Oliver

2018-01-01

Context: Medical diagnosis and clinical decisions rely heavily on the histopathological evaluation of tissue samples, especially in oncology. Historically, classical histopathology has been the gold standard for tissue evaluation and assessment by pathologists. The most widely and commonly used dyes in histopathology are hematoxylin and eosin (H&E) as most malignancies diagnosis is largely based on this protocol. H&E staining has been used for more than a century to identify tissue characteristics and structures morphologies that are needed for tumor diagnosis. In many cases, as tissue is scarce in clinical studies, fluorescence imaging is necessary to allow staining of the same specimen with multiple biomarkers simultaneously. Since fluorescence imaging is a relatively new technology in the pathology landscape, histopathologists are not used to or trained in annotating or interpreting these images. Aims, Settings and Design: To allow pathologists to annotate these images without the need for additional training, we designed an algorithm for the conversion of fluorescence images to brightfield H&E images. Subjects and Methods: In this algorithm, we use fluorescent nuclei staining to reproduce the hematoxylin information and natural tissue autofluorescence to reproduce the eosin information avoiding the necessity to specifically stain the proteins or intracellular structures with an additional fluorescence stain. Statistical Analysis Used: Our method is based on optimizing a transform function from fluorescence to H&E images using least mean square optimization. Results: It results in high quality virtual H&E digital images that can easily and efficiently be analyzed by pathologists. We validated our results with pathologists by making them annotate tumor in real and virtual H&E whole slide images and we obtained promising results. Conclusions: Hence, we provide a solution that enables pathologists to assess tissue and annotate specific structures based on multiplexed fluorescence images. PMID:29531846

A comparative study to evaluate liquid dish washing soap as an alternative to xylene and alcohol in deparaffinization and hematoxylin and eosin staining.

PubMed

Pandey, Pinki; Dixit, Alok; Tanwar, Aparna; Sharma, Anuradha; Mittal, Sanjeev

2014-07-01

Our study presents a new deparaffinizing and hematoxylin and eosin (H and E) staining method that involves the use of easily available, nontoxic and eco-friendly liquid diluted dish washing soap (DWS) by completely eliminating expensive and hazardous xylene and alcohol from deparaffinizing and rehydration prior to staining, staining and from dehydration prior to mounting. The aim was to evaluate and compare the quality of liquid DWS treated xylene and alcohol free (XAF) sections with that of the conventional H and E sections. A total of 100 paraffin embedded tissue blocks from different tissues were included. From each tissue block, one section was stained with conventional H and E (normal sections) and the other with XAF H and E (soapy sections) staining method. Slides were scored using five parameters: Nuclear, cytoplasmic, clarity, uniformity, and crispness of staining. Z-test was used for statistical analysis. Soapy sections scored better for cytoplasmic (90%) and crisp staining (95%) with a statistically significant difference. Whereas for uniformity of staining, normal sections (88%) scored over soapy sections (72%) (Z = 2.82, P < 0.05). For nuclear (90%) and clarity of staining (90%) total scored favored soapy sections, but the difference was not statistically significant. About 84% normal sections stained adequately for diagnosis when compared with 86% in soapy sections (Z = 0.396, P > 0.05). Liquid DWS is a safe and efficient alternative to xylene and alcohol in deparaffinization and routine H and E staining procedure. We are documenting this project that can be used as a model for other histology laboratories.

pH influences the biocompatibility of methylene blue solutions.

PubMed

Gusman, David Jonathan Rodrigues; Cintra, Luciano Tavares Angelo; Novaes, Vivian Cristina Noronha; Matheus, Henrique Rinaldi; de Araujo, Nathália Januario; de Almeida, Juliano Milanezi

2018-01-01

The aim of this study was to investigate the biocompatibility of methylene blue at different pH levels through the method of implantation in subcutaneous tissue. Eighty-four sterilized polyethylene tubes were allocated in the subcutaneous tissue of 28 rats, each one receiving four tubes, set into four groups: group tube (G-T)-empty tube, fibrin group (G-F)-tube filled with fibrin sponge, group methylene blue pH 7 (G-MB/pH 7)-tube filled with fibrin sponge soaked by methylene blue (100 μg/ml) at pH 7.0, and group methylene blue pH 1 (G-MB/pH 1)-tube filled with fibrin sponge and soaked by methylene blue (100 μg/ml) at pH 1.0. After 7, 15, and 30 days, seven animals from each group were euthanized, and the tubes involved by the surrounding tissue were removed and fixed with 4% buffered formaldehyde solution. The collected pieces were processed and histological sections (4 μm) were stained with hematoxylin and eosin and analyzed by light microscopy. Scores were assigned to analysis of histopathologic parameters. The results were statistically analyzed by the Kruskal-Wallis test (p ≤ 0.05). At 7 and 30 days, the G-MB/pH 1 group showed no significant difference in the G-T control group, while G-MB/pH 7 had a significant increase on tissue reaction, also when compared to G-T. At 15 days, there was no statistical difference between the groups. Within the limits of this study, it is concluded that methylene blue at pH 1.0 provides better biocompatibility than at pH 7.0.

Porcine Intestinal Mast Cells. Evaluation of Different Fixatives for Histochemical Staining Techniques Considering Tissue Shrinkage

PubMed Central

Rieger, J.; Twardziok, S.; Huenigen, H.; Hirschberg, R.M.; Plendl, J.

2013-01-01

Staining of mast cells (MCs), including porcine ones, is critically dependent upon the fixation and staining technique. In the pig, mucosal and submucosal MCs do not stain or stain only faintly after formalin fixation. Some fixation methods are particularly recommended for MC staining, for example the fixation with Carnoy or lead salts. Zinc salt fixation (ZSF) has been reported to work excellently for the preservation of fixation-sensitive antigens. The aim of this study was to establish a reliable histological method for counting of MCs in the porcine intestinum. For this purpose, different tissue fixation and staining methods that also allow potential subsequent immunohistochemical investigations were evaluated in the porcine mucosa, as well as submucosa of small and large intestine. Tissues were fixed in Carnoy, lead acetate, lead nitrate, Zamboni and ZSF and stained subsequently with either polychromatic methylene blue, alcian blue or toluidine blue. For the first time our study reveals that ZSF, a heavy metal fixative, preserves metachromatic staining of porcine MCs. Zamboni fixation was not suitable for histochemical visualization of MCs in the pig intestine. All other tested fixatives were suitable. Alcian blue and toluidine blue co-stained intestinal goblet cells which made a prima facie identification of MCs difficult. The polychromatic methylene blue proved to be the optimal staining. In order to compare MC counting results of the different fixation methods, tissue shrinkage was taken into account. As even the same fixation caused shrinkagedifferences between tissue from small and large intestine, different factors for each single fixation and intestinal localization had to be calculated. Tissue shrinkage varied between 19% and 57%, the highest tissue shrinkage was found after fixation with ZSF in the large intestine, the lowest one in the small intestine after lead acetate fixation. Our study emphasizes that MC counting results from data using different fixation techniques can only be compared if the respective studyimmanent shrinkage factor has been determined and quantification results are adjusted accordingly. PMID:24085270

Sentinel lymph nodes in endometrial cancer: is hysteroscopic injection valid?

PubMed

Clement, D; Bats, A S; Ghazzar-Pierquet, N; Le Frere Belda, M A; Larousserie, F; Nos, C; Lecuru, F

2008-01-01

We aimed to describe hysteroscopic peritumoral tracer injection for detecting sentinel lymph nodes (SLNs) in patients with endometrial cancer and to evaluate tolerance of the procedure, detection rate and location of SLNs. Five patients with early endometrial cancer underwent hysteroscopic radiotracer injection followed by lymphoscintigraphy, then by surgery with hysteroscopic peritumoral blue dye injection, and radioactivity measurement using an endoscopic handheld gamma probe. SLNs and other nodes were sent separately to the pathology laboratory. SLNs were evaluated by hematoxylin-eosin-saffron staining and, when negative, by immunohistochemistry. Tolerance of the injection by the patients was poor (mean visual analog scale score, 8/10). SLNs were detected in only two patients (external iliac and common iliac+paraaortic, respectively). Detection rates were 1/5 by radiotracer, 1/5 by dye, and 2/5 by the combined method. One SLN was involved in a patient whose other nodes were negative. In three patients no SLNs were found by radiotracer or blue dye. Of the 83 non sentinel nodes removed from these patients, none was involved. Hysteroscopic peritumoral injection may be more difficult than cervical injection and, in our experience, carries a lower SLN detection rate.

A simple and rapid microwave-assisted hematoxylin and eosin staining method using 1,1,1 trichloroethane as a dewaxing and a clearing agent.

PubMed

Temel, S G; Noyan, S; Cavusoglu, I; Kahveci, Z

2005-01-01

The use and practicability of microwave-assisted staining procedures in routine histopathology has been well established for more than 17 years. In the study reported here, we aimed to examine an alternative approach that would shorten the duration of dewaxing and clearing steps of hematoxylin and eosin (H & E) staining of paraffin sections by using a microwave oven. Although xylene is one of the most popular dewaxing and clearing agents, its flammability restricts its use in a microwave oven; thus we preferred 1,1,1 trichloroethane, which is not flammable, as the dewaxing and clearing agent in the present study. In Group I and Group II (control groups), intestine was processed with xylene and 1,1,1 trichloroethane, respectively. The sections were then stained with H & E according to the conventional staining protocol at room temperature and subdivided into two groups according to the duration of dewaxing and clearing in xylene. In Groups III and IV (experimental groups) similar tissues were processed with xylene and 1,1,1 trichloroethane, respectively; however, sections from these groups were divided into four subgroups to study the period required for dewaxing and clearing in 1,1,1 trichloroethane, then stained with H & E in the microwave oven at 360 W for 30 sec. Our conventional H & E staining procedure, which includes dewaxing, staining and clearing of sections, requires approximately 90 min, while our method using 1,1,1 trichloroethane and microwave heating required only 2 min. Our alternative method for H & E staining not only reduced the procedure time significantly, but also yielded staining quality equal or superior to those stained the conventional way. Our results suggest that 1,1,1 trichloroethane can be used effectively and safely as a dewaxing and clearing agent for H & E staining in a microwave oven.

Rapid alkaline methylene blue supravital staining for assessment of anterior segment infections.

PubMed

Kiuchi, Katsuji

2016-01-01

To present the Löffler's alkaline methylene blue technique of staining eye discharges in eyes with anterior segment infections. The Löffler's alkaline methylene blue staining method is a simple staining technique that can be used to differentiate bacterial, viral, and fungal infections. It is a cationic dye that stains cells blue because the positively charged dye is attracted to negatively charged particles such as polyphosphates, DNAs, and RNAs. Specimens collected from patients by swabbing are smeared onto microscope slides and the methylene blue solution is dropped on the slide. The slide is covered with a glass cover slip and examined under a microscope. The entire time from the collection to the viewing is about 30 seconds. Histopathological images of the conjunctival epithelial cells and neutrophils in eye discharges were dyed blue and the nuclei were stained more intensely blue. Bacterial infections consisted mainly of neutrophils, and viral infections consisted mainly of lymphocytes. Löffler's alkaline methylene blue staining can be done in about 30 seconds for diagnosis. Even though this is a one color stain, it is possible to infer the cause of the infection by detection of the absence of bacteria and/or fungi in context of the differential distribution of neutrophils and lymphocytes.

A procedure for Alcian blue staining of mucins on polyvinylidene difluoride membranes.

PubMed

Dong, Weijie; Matsuno, Yu-ki; Kameyama, Akihiko

2012-10-16

The isolation and characterization of mucins are critically important for obtaining insight into the molecular pathology of various diseases, including cancers and cystic fibrosis. Recently, we developed a novel membrane electrophoretic method, supported molecular matrix electrophoresis (SMME), which separates mucins on a polyvinylidene difluoride (PVDF) membrane impregnated with a hydrophilic polymer. Alcian blue staining is widely used to visualize mucopolysaccharides and acidic mucins on both blotted membranes and SMME membranes; however, this method cannot be used to stain mucins with a low acidic glycan content. Meanwhile, periodic acid-Schiff staining can selectively visualize glycoproteins, including mucins, but is incompatible with glycan analysis, which is indispensable for mucin characterizations. Here we describe a novel staining method, designated succinylation-Alcian blue staining, for visualizing mucins on a PVDF membrane. This method can visualize mucins regardless of the acidic residue content and shows a sensitivity 2-fold higher than that of Pro-Q Emerald 488, a fluorescent periodate Schiff-base stain. Furthermore, we demonstrate the compatibility of this novel staining procedure with glycan analysis using porcine gastric mucin as a model mucin.

Discriminative staining methods for the nervous system: luxol fast blue--periodic acid-Schiff--hematoxylin triple stain and subsidiary staining methods.

PubMed

Goto, N

1987-09-01

This paper describes a new series of staining methods which can discriminatively demonstrate every structure of the nervous system, including axons and capillaries, in animal and human materials. Methods described in this paper consist of one primary stain, luxol fast blue-periodic acid Schiff-hematoxylin (LPH) and six different subsidiary staining methods. The LPH triple stain can precisely differentiate the following structures: neurons (Nissl bodies, cytoplasm, nuclear membrane and nucleolus), various kinds of nuclei (glia, ependyma, endothelium, leucocyte, connective tissue, etc.), myelin sheaths, neuronal processes (axons and dendrites), reacted glial cell bodies (protoplasmic astrocytes, foamy cells, etc.), blood vessels (arteries, veins and capillaries), meninges, intervening connective tissue, erythrocytes, lipofuscin granules, amyloid bodies, and others. Subsidiary staining methods are also described briefly. Applications are discussed in the context of staining technology and neuromorphological research.

Multiplication free neural network for cancer stem cell detection in H-and-E stained liver images

NASA Astrophysics Data System (ADS)

Badawi, Diaa; Akhan, Ece; Mallah, Ma'en; Üner, Ayşegül; ćetin-Atalay, Rengül; ćetin, A. Enis

2017-05-01

Markers such as CD13 and CD133 have been used to identify Cancer Stem Cells (CSC) in various tissue images. It is highly likely that CSC nuclei appear as brown in CD13 stained liver tissue images. We observe that there is a high correlation between the ratio of brown to blue colored nuclei in CD13 images and the ratio between the dark blue to blue colored nuclei in H&E stained liver images. Therefore, we recommend that a pathologist observing many dark blue nuclei in an H&E stained tissue image may also order CD13 staining to estimate the CSC ratio. In this paper, we describe a computer vision method based on a neural network estimating the ratio of dark blue to blue colored nuclei in an H&E stained liver tissue image. The neural network structure is based on a multiplication free operator using only additions and sign operations. Experimental results are presented.

Comparing The Efficacy of Hematoxylin and Eosin, Periodic Acid Schiff and Fluorescent Periodic Acid Schiff-Acriflavine Techniques for Demonstration of Basement Membrane in Oral Lichen Planus: A Histochemical Study

PubMed Central

Pujar, Ashwini; Pereira, Treville; Tamgadge, Avinash; Bhalerao, Sudhir; Tamgadge, Sandhya

2015-01-01

Background: Basement membrane (BM) is a thick sheet of extracellular matrix molecules, upon which epithelial cells attach. Various immunohistochemical studies in the past have been carried out but these advanced staining techniques are expensive and not feasible in routine laboratories. Although hematoxylin and eosin (H-E) is very popular among pathologists for looking at biopsies, the method has some limitations. This is where special stains come handy. Aims and Objectives: The aim of the present study was to demonstrate and compare the efficacy of H-E, periodic acid Schiff (PAS) and fluorescent periodic acid–acriflavine staining techniques for the basement membrane and to establish a histochemical stain which could be cost effective, less time consuming, and unambiguous for observation of the basement membrane zone. Materials and Methods: A total number of 40 paraffin-embedded tissue sections of known basement membrane containing tissues including 10 – Normal oral mucosa (NOM) and 30 – oral lichen planus (OLP) were considered in the study. Four-micron-thick sections of each block were cut and stained with H-E stain, PAS and fluorescent periodic acid–acriflavine stain. Sections were evaluated by three oral pathologists independently for continuity, contrast and pattern. Results: Though all the three stains showed favorable features at different levels, acriflavine stain was better than the other stains in demonstrating BM continuity, contrast and also the pattern followed by PAS stain. Acriflavine stain was the better in demonstrating a fibrillar pattern of a BM. Acriflavine stains a BM distinctly and is less time consuming and easy to carry out using readily available dyes as compared to other stains. Conclusion: The continuity and contrast along with the homogenous pattern and the afibrillar pattern of the BM was better demonstrated by acriflavine followed by the PAS stain. PMID:26538690

Characterization of Uterine Granular Cell Tumors in B6C3F1 Mice: A Histomorphologic, Immunohistochemical, and Ultrastructural Study

PubMed Central

Veit, A. C.; Painter, J. T.; Miller, R. A.; Hardisty, J. F.; Dixon, D.

2009-01-01

The granular cell tumor is most often a benign neoplasm of uncertain origin. Four uterine granular cell tumors in control and treated female B6C3F1 mice were identified in chronic studies at the National Toxicology Program. Two tumors occurred in untreated control animals and 2 in treated animals receiving different compounds. Tissue sections were evaluated histologically and stained with hematoxylin and eosin, periodic acid–Schiff with diastase resistance, Masson’s trichrome, toluidine blue, phosphotungstic acid–hematoxylin, and stained immunohistochemically with a panel of antibodies to muscle (desmin, alpha smooth muscle actin), neural (S-100, neuron specific enolase), epithelial (wide-spectrum cytokeratin), and macrophage (F4/80) markers. The main histomorphologic feature of tumor cells was the presence of abundant cytoplasmic eosinophilic granules that stained positive for periodic acid–Schiff with diastase resistance. Tumors varied in appearance and were comprised of sheets and nests of round to polygonal cells with distinct borders. Nuclei were hyperchromatic, pleomorphic, and centrally to eccentrically located and often contained single nucleoli. Occasional multinucleated giant cells were observed. Tumors were pale pink and homogeneous with trichrome stain and negative with toluidine blue. Three tumors had positive to weakly positive immunoreactivity for desmin, and 1 was positive for alpha smooth muscle actin. Expression of S-100, wide-spectrum cytokeratin, and neuron-specific enolase was negative for all tumors. Ultrastructurally, prominent electron-dense cytoplasmic granules were abundant and contained secondary lysosomes with heterogeneous lysosomal contents. The characteristics of these uterine granular cell tumors were suggestive of a myogenic origin. PMID:18725470

Sentinel lymph node detection following the hysteroscopic peritumoural injection of 99mTc-labelled albumin nanocolloid in endometrial cancer.

PubMed

Maccauro, Marco; Lucignani, Giovanni; Aliberti, Gianluca; Villano, Carlo; Castellani, Maria Rita; Solima, Eugenio; Bombardieri, Emilio

2005-05-01

The purpose of this study was to assess the feasibility of sentinel lymph node (SLN) detection in endometrial cancer patients with a dual-tracer procedure after hysteroscopic peritumoural injection. Twenty-six women with previously untreated endometrial adenocarcinoma underwent the hysteroscopic injection of 111 MBq 99mTc-Nanocoll and blue dye administered subendometrially around the lesion. On the same day, all 26 patients underwent lymphoscintigraphy, followed 3-4 h later by hysterotomy with bilateral salpingo-oophorectomy and pelvic lymphadenectomy. Para-aortic lymphadenectomy was also performed in cases of either serous or papillary carcinoma (n=7/26). All SLNs were removed and examined with haematoxylin and eosin staining and immunohistochemical techniques. The procedure was well tolerated by patients, only two experiencing transient vagal symptoms. The sensitivity of this technique for correct identification of SLNs was 100%. Lymph node metastases were found in 4 out of the 26 patients (15%), bilaterally in the external iliac region (n=1), unilaterally in the external iliac region (n=1), unilaterally in the common iliac region (n=1) and unilaterally in the para-aortic region (n=1). In all four cases, nodal metastases were located within SLNs detected by lymphoscintigraphy. Only 10 of the 26 patients (38%) had significant blue dye staining. All blue-stained SLNs were radioactive. In patients with endometrial cancer, it is feasible to use lymphatic mapping and SLN biopsy to define the topographic distribution of the lymphatic network and also to accurately detect lumbo-aortic and pelvic metastases within SLNs. In the majority of patients with early stage endometrial cancer, this procedure may avoid unnecessary radical pelvic lymphadenectomy. It may also guide para-aortic lymph node dissection on the basis of the SLN status.

Single step modified ink staining for Tzanck test: quick detection of herpetic giant cells in Tzanck smear.

PubMed

Mizutani, Hitoshi; Akeda, Tomoko; Yamanaka, Kei-Ichi; Isoda, Kenichi; Gabazza, Esteban C

2012-02-01

Tzanck test has been recently re-evaluated as a method for the diagnosis of herpes virus infection. Giemsa staining for the Tzanck test is time-consuming and laborious. There is a need to develop simple and quick staining methods for bedside diagnosis of this disease. We report a single step and quick method for staining herpes giant cells in Tzanck smears using routinely available inks and physiological saline. A keratinocyte cell line (HaCaT) was cultured on a slide glass and stained with various commercially available blue, blue-black and black inks serially diluted with physiological saline. Clinical smear samples from herpes lesions were also stained with these solutions without specific pretreatment. The nuclei of HaCaT were clearly stained showing high contrast with the cytoplasm using 5% Parker-Quink blue-black ink saline solution. Concentration of ink solution higher or lower than 5% resulted in less contrast. Blue or black inks or other manufacturers' inks can also be used, but staining of the cultured keratinocytes was less clear. Smear of clinical samples from herpes lesions were also stained with 5% ink solution. The nuclei of the multinucleated giant cells were clearly stained, and the sample could be immediately used for microscopic examination. One step staining of Tzanck smear using this diluted ink solution is an inexpensive and a convenient bedside diagnostic tool for the dermatologist. © 2011 Japanese Dermatological Association.

Variable Saline Concentrations for Initial Resuscitation Following Polytrauma

DTIC Science & Technology

2017-02-22

sagittal suture between the coronal and lambdoid suture lines. Head injury was induced by dropping a cylindrical rod weighing 400 grams from 1.5 cm...injury. Brain samples were perfusion fixed and evaluated for extent of injury with hematoxylin and eosin staining . 3.5 Capillary Leak and Blood...Distribution is unlimited. Cleared, 88PA, Case # 2017-2329, 15 May 2017. 4.8 Histology Hematoxylin and eosin staining demonstrated no differences

Comparison of fixation and processing methods for hairless guinea pig skin following sulfur mustard exposure. (Reannouncement with new availability information)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bryant, M.A.; Braue Jr, E.H.

1992-12-31

Ten anesthetized hairless guinea pigs Crl:IAF(HA)BR were exposed to 10 pi of neat sulfur mustard (HD) in a vapor cup on their skin for 7 min. At 24 h postexposure, the guinea pigs were euthanatized and skin sections taken for histologic evaluation. The skin was fixed using either 10% neutral buffered formalin (NBF), McDowell Trump fixative (4CF-IG), Zenker`s formol-saline (Helly`s fluid), or Zenker`s fluid. Fixed skin sections were cut in half: one half was embedded in paraffin and the other half in plastic (glycol methacrylate). Paraffin-embedded tissue was stained with hematoxylin and eosin; plastic-embedded tissue was stained with Lee`s methylenemore » blue basic fuchsin. Skin was also frozen unfixed, sectioned by cryostat, and stained with pinacyanole. HD-exposed skin was evaluated histologically for the presence of epidermal and follicular necrosis, microblister formation, epidermitis, and intracellular edema to determine the optimal fixation and embedding method for lesion preservation. The percentage of histologic sections with lesions varied little between fixatives and was similar for both paraffin and plastic embedding material. Plastic-embedded sections were thinner, allowing better histologic evaluation, but were more difficult to stain. Plastic embedding material did not infiltrate tissue fixed in Zenker`s fluid or Zenker`s formol-saline. Frozen tissue sections were prepared in the least processing time and lesion preservation was comparable to fixed tissue. It was concluded that standard histologic processing using formalin fixation and paraffin embedding is adequate for routine histopathological evaluation of HD skin lesions in the hairless guinea pig.... Sulfur mustard, Vesicating agents, Pathology, Hairless guinea pig model, Fixation.« less

Fluorescent staining for leukocyte chemotaxis. Eosinophil-specific fluorescence with aniline blue.

PubMed

McCrone, E L; Lucey, D R; Weller, P F

1988-11-10

To overcome problems associated with the quantitation of human eosinophil chemotaxis in micropore filters, we have developed a fluorescent method of specifically staining eosinophils in chemotactic filters. A neutral solution of aniline blue yielded bright green fluorescent staining of the cytoplasmic granules of eosinophils. Other leukocytes and contaminating neutrophils potentially present with eosinophils did not fluoresce with aniline blue. The fluorescent staining eosinophils within filters provided bright, non-fading images that facilitated visual microscopic counting and were of sufficiently high contrast, unlike those with conventional eosinophil stains, to allow image analyzer based enumeration of eosinophil chemotactic responses at levels through the filters. Although not cell type-specific, congo red and ethidium bromide also provided high contrast, fluorescent images of all leukocyte types within chemotactic filters. Fluorescent staining with aniline blue constitutes a rapid, stable and eosinophil-specific stain that facilitates the visual or image analyzer-based quantitation of eosinophil chemotaxis.

MRI-guided targeted blood-brain barrier disruption with focused ultrasound: histological findings in rabbits.

PubMed

McDannold, Nathan; Vykhodtseva, Natalia; Raymond, Scott; Jolesz, Ferenc A; Hynynen, Kullervo

2005-11-01

Focused ultrasound offers a method to disrupt the blood-brain barrier (BBB) noninvasively and reversibly at targeted locations. The purpose of this study was to test the safety of this method by searching for ischemia and apoptosis in areas with BBB disruption induced by pulsed ultrasound in the presence of preformed gas bubbles and by looking for delayed effects up to one month after sonication. Pulsed ultrasound exposures (sonications) were performed in the brains of 24 rabbits under monitoring by magnetic resonance imaging (MRI) (ultrasound: frequency = 1.63 MHz, burst length = 100 ms, PRF = 1 Hz, duration = 20 s, pressure amplitude 0.7 to 1.0 MPa). Before sonication, an ultrasound contrast agent (Optison, GE Healthcare, Milwaukee, WI, USA) was injected IV. BBB disruption was confirmed with contrast-enhanced MR images. Whole brain histologic examination was performed using haematoxylin and eosin staining for general histology, vanadium acid fuchsin-toluidine blue staining for ischemic neurons and TUNEL staining for apoptosis. The main effects observed were tiny regions of extravasated red blood cells scattered around the sonicated locations, indicating affected capillaries. Despite these vasculature effects, only a few cells in some of the sonicated areas showed evidence for apoptosis or ischemia. No ischemic or apoptotic regions were detected that would indicate a compromised blood supply was induced by the sonications. No delayed effects were observed either by MRI or histology up to 4 wk after sonication. Ultrasound-induced BBB disruption is possible without inducing substantial vascular damage that would result in ischemic or apoptotic death to neurons. These findings indicate that this method is safe for targeted drug delivery, at least when compared with the currently available invasive methods.

Enabling Histopathological Annotations on Immunofluorescent Images through Virtualization of Hematoxylin and Eosin.

PubMed

Lahiani, Amal; Klaiman, Eldad; Grimm, Oliver

2018-01-01

Medical diagnosis and clinical decisions rely heavily on the histopathological evaluation of tissue samples, especially in oncology. Historically, classical histopathology has been the gold standard for tissue evaluation and assessment by pathologists. The most widely and commonly used dyes in histopathology are hematoxylin and eosin (H&E) as most malignancies diagnosis is largely based on this protocol. H&E staining has been used for more than a century to identify tissue characteristics and structures morphologies that are needed for tumor diagnosis. In many cases, as tissue is scarce in clinical studies, fluorescence imaging is necessary to allow staining of the same specimen with multiple biomarkers simultaneously. Since fluorescence imaging is a relatively new technology in the pathology landscape, histopathologists are not used to or trained in annotating or interpreting these images. To allow pathologists to annotate these images without the need for additional training, we designed an algorithm for the conversion of fluorescence images to brightfield H&E images. In this algorithm, we use fluorescent nuclei staining to reproduce the hematoxylin information and natural tissue autofluorescence to reproduce the eosin information avoiding the necessity to specifically stain the proteins or intracellular structures with an additional fluorescence stain. Our method is based on optimizing a transform function from fluorescence to H&E images using least mean square optimization. It results in high quality virtual H&E digital images that can easily and efficiently be analyzed by pathologists. We validated our results with pathologists by making them annotate tumor in real and virtual H&E whole slide images and we obtained promising results. Hence, we provide a solution that enables pathologists to assess tissue and annotate specific structures based on multiplexed fluorescence images.

Photoinactivation effect of eosin methylene blue and chlorophyllin sodium-copper against Staphylococcus aureus and Escherichia coli.

PubMed

Caires, Cynthia S A; Leal, Cassia R B; Ramos, Carlos A N; Bogo, Danielle; Lima, Alessandra R; Arruda, Eduardo J; Oliveira, Samuel L; Caires, Anderson R L; Nascimento, Valter A

2017-07-01

The use of eosin methylene blue according to Giemsa as photosensitizer is presented for the first time in this paper. The present study evaluated the potential application of chlorophyllin sodium copper salt (CuChlNa) and eosin methylene blue according to Giemsa (EMB) as antimicrobial photosensitizers (aPS) for photodynamic inactivation (PDI) of Staphylococcus aureus (gram-positive) and Escherichia coli (gram-negative) bacteria. The experiments were performed using S. aureus stain ATCC 25923 and E. coli ATCC 25922 in which five aPS concentrations (0.0, 1.0, 2.5, 5.0, 10.0, and 20.0 μM for S. aureus and 0.0, 5.0, 10.0, 20.0, 40.0, and 50.0 μM for E. coli) were prepared and added in 2 mL of a saline solution containing the bacterial inoculum. After aPS incubation, the samples were divided into two groups, one kept in the dark and another submitted to the illumination. Then, the bacterial inactivation was determined 18 h after the incubation at 37 °C by counting the colony-forming units (CFU). The results revealed that both EMB and CuChlNa can be used as aPS for the photoinactivation of S. aureus, while only EMB was able to photoinactivate E. coli. Nevertheless, a more complex experimental setup was needed for photoinactivation of E. coli. The data showed that EMB and CuChlNa presented similar photoinactivation effects on S. aureus, in which bacterial growth was completely inhibited at photosensitizer (PS) concentrations over 5 μM, when samples were previously incubated for 30 min and irradiated by a light dose of 30 J cm -2 as a result of an illumination of 1 h at 8.3 mW cm -2 by using a red light at 625 nm with a 1 cm beam diameter and output power of 6.5 mW. In the case of E. coli, bacterial growth was completely inhibited only when combining a PS incubation period of 120 min with concentrations over 20 μM.

Unsupervised segmentation of H and E breast images

NASA Astrophysics Data System (ADS)

Hope, Tyna A.; Yaffe, Martin J.

2017-03-01

Heterogeneity of ductal carcinoma in situ (DCIS) continues to be an important topic. Combining biomarker and hematoxylin and eosin (HE) morphology information may provide more insights than either alone. We are working towards a computer-based identification and description system for DCIS. As part of the system we are developing a region of interest finder for further processing, such as identifying DCIS and other HE based measures. The segmentation algorithm is designed to be tolerant of variability in staining and require no user interaction. To achieve stain variation tolerance we use unsupervised learning and iteratively interrogate the image for information. Using simple rules (e.g., "hematoxylin stains nuclei") and iteratively assessing the resultant objects (small hematoxylin stained objects are lymphocytes), the system builds up a knowledge base so that it is not dependent upon manual annotations. The system starts with image resolution-based assumptions but these are replaced by knowledge gained. The algorithm pipeline is designed to find the simplest items first (segment stains), then interesting subclasses and objects (stroma, lymphocytes), and builds information until it is possible to segment blobs that are normal, DCIS, and the range of benign glands. Once the blobs are found, features can be obtained and DCIS detected. In this work we present the early segmentation results with stains where hematoxylin ranges from blue dominant to red dominant in RGB space.

Differentiating vulvar intraepithelial neoplasia from nonneoplastic epithelial disorders. The toluidine blue test.

PubMed

Joura, E A; Zeisler, H; Lösch, A; Sator, M O; Müllauer-Ertl, S

1998-08-01

To determine the effectiveness of the toluidine blue test in the differentiation of vulvar intraepithelial neoplasia (VIN) and nonneoplastic epithelial disorders (NNEDs). This retrospective clinical study included all women with VIN (n = 24) and NNED (n = 72) referred to a vulvar clinic at a university hospital during a two-year period. Vulvoscopy, staining of vulvar epithelium with 1% toluidine blue and punch biopsy were performed. Vulvar epithelium demonstrated toluidine blue staining in 100% of the patients with VIN 3, in 83% of women with VIN 1-2, in 50% of the women with squamous cell hyperplasia and in 10% of the women with lichen sclerosus. The differences in staining between the groups were statistically significant (P < .001). The sensitivity of toluidine blue staining for the detection of VIN was 92%; the negative predictive value 96% in teh investigated cohort. The specificity for strong staining was 88%. The toluidine blue test is an inexpensive and reliable method of separating VIN from hyperplastic NNED areas and choosing a biopsy site on the vulva.

7 CFR 201.58d - Fungal endophyte test.

Code of Federal Regulations, 2012 CFR

2012-01-01

...) Method of preparation of aniline blue stain for use in testing grass seed and plant material for the presence of fungal endophyte: (1) Prepare a 1 percent aqueous aniline blue solution by dissolving 1 gram aniline blue in 100 ml distilled water. (2) Prepare the endophyte staining solution of one part of 1...

7 CFR 201.58d - Fungal endophyte test.

Code of Federal Regulations, 2013 CFR

2013-01-01

...) Method of preparation of aniline blue stain for use in testing grass seed and plant material for the presence of fungal endophyte: (1) Prepare a 1 percent aqueous aniline blue solution by dissolving 1 gram aniline blue in 100 ml distilled water. (2) Prepare the endophyte staining solution of one part of 1...

7 CFR 201.58d - Fungal endophyte test.

Code of Federal Regulations, 2014 CFR

2014-01-01

...) Method of preparation of aniline blue stain for use in testing grass seed and plant material for the presence of fungal endophyte: (1) Prepare a 1 percent aqueous aniline blue solution by dissolving 1 gram aniline blue in 100 ml distilled water. (2) Prepare the endophyte staining solution of one part of 1...

Platinum blue staining of cells grown in electrospun scaffolds.

PubMed

Yusuf, Mohammed; Millas, Ana Luiza G; Estandarte, Ana Katrina C; Bhella, Gurdeep K; McKean, Robert; Bittencourt, Edison; Robinson, Ian K

2014-01-01

Fibroblast cells grown in electrospun polymer scaffolds were stained with platinum blue, a heavy metal stain, and imaged using scanning electron microscopy. Good contrast on the cells was achieved compared with samples that were gold sputter coated. The cell morphology could be clearly observed, and the cells could be distinguished from the scaffold fibers. Here we optimized the required concentration of platinum blue for imaging cells grown in scaffolds and show that a higher concentration causes platinum aggregation. Overall, platinum blue is a useful stain for imaging cells because of its enhanced contrast using scanning electron microscopy (SEM). In the future it would be useful to investigate cell growth and morphology using three-dimensional imaging methods.

A histological atlas of the tissues and organs of neotenic and metamorphosed axolotl.

PubMed

Demircan, Turan; İlhan, Ayşe Elif; Aytürk, Nilüfer; Yıldırım, Berna; Öztürk, Gürkan; Keskin, İlknur

2016-09-01

Axolotl (Ambystoma Mexicanum) has been emerging as a promising model in stem cell and regeneration researches due to its exceptional regenerative capacity. Although it represents lifelong lasting neoteny, induction to metamorphosis with thyroid hormones (THs) treatment advances the utilization of Axolotl in various studies. It has been reported that amphibians undergo anatomical and histological remodeling during metamorphosis and this transformation is crucial for adaptation to terrestrial conditions. However, there is no comprehensive histological investigation regarding the morphological alterations of Axolotl organs and tissues throughout the metamorphosis. Here, we reveal the histological differences or resemblances between the neotenic and metamorphic axolotl tissues. In order to examine structural features and cellular organization of Axolotl organs, we performed Hematoxylin & Eosin, Luxol-Fast blue, Masson's trichrome, Alcian blue, Orcein and Weigart's staining. Stained samples from brain, gallbladder, heart, intestine, liver, lung, muscle, skin, spleen, stomach, tail, tongue and vessel were analyzed under the light microscope. Our findings contribute to the validation of the link between newly acquired functions and structural changes of tissues and organs as observed in tail, skin, gallbladder and spleen. We believe that this descriptive work provides new insights for a better histological understanding of both neotenic and metamorphic Axolotl tissues. Copyright © 2016 Elsevier GmbH. All rights reserved.

Toluidine blue: rapid and simple malaria parasite screening and species identification.

PubMed

Awale, Rupali; Maji, Ratnaprabha; Patil, Parag; Lingiah, Raghavendra; Mukhopadhyay, Ashok Kumar; Sharma, Subhadra

2017-01-01

Malaria, a febrile illness mostly confined to the tropical countries is transmitted by bite of infected female Anopheles mosquito. In 2015 alone, 88% of the malaria burden and 90% deaths due to malaria were confined to the African and Asian countries. Although number of tests are available for rapid diagnosis and screening for malaria, peripheral blood smear examination remains the gold standard. Leishman stain is recommended by WHO however herein we evaluate one of the alternative methods of staining which is simple and rapid. Fifty patients attending the various outpatient departments of the tertiary care hospital with fever and suspected to have malaria were selected. Two thin-air dried smears prepared from the peripheral venous blood from these subjects were stained by Leishman and Toluidine blue method. The findings of the slides by two independent qualified professionals were noted and the results were analyzed. A total of 14% (7/50) cases were diagnosed to have malaria. All the malaria cases which were positive in Leishman stain were also detected in Toluidine blue stain. Malarial parasites were clearly visible against the homogenously light green background in Toluidine blue. The detection of malarial parasite by Toluidine blue was quick, easy and confirmative. Toluidine blue stained peripheral blood smear allows for easy identification and speciation of malarial parasite at low magnification and in shorter period of time.

Histological observation of a gelatin sponge transplant loaded with bone marrow-derived mesenchymal stem cells combined with platelet-rich plasma in repairing an annulus defect

PubMed Central

Xu, Xiang; Hu, Jianzhong; Lu, Hongbin

2017-01-01

Objective To research the histological characteristics of a gelatin sponge transplant loaded with goat BMSCs (bone marrow-derived mesenchymal stem cells) combined with PRP (platelet-rich plasma) in repairing an annulus defect. Method BMSCs were separated from the iliac crest of goats, sub-cultured and identified after the third generation. Then, PRP was obtained using blood from the jugular vein of goats via two degrees of centrifugation. In the animal experiments, the goats were divided into the following three groups: a sham group, an injury group and a therapeutic group. In the sham group, we decompressed the lamina and exposed the annulus fibrosus. In the injury group, we exposed the annulus fibrosus after decompression of the lamina and created a 1 × 1 cm defect in the annulus using surgical instruments. In the therapeutic group, after decompression of the lamina, we exposed the annulus, created a 1 × 1 cm defect using surgical instruments, and placed a gelatin sponge combined with BMSCs and PRP into the defect for a combined method of repair. Three, six and twelve weeks after the surgery, the previously damaged or undamaged annulus tissue was removed from the three groups. Then, the above tissue was assayed using HE (hematoxylin-eosin) staining, Masson trichrome staining, AB-PAS (Alcian blue-periodic acid Schiff) staining, and type II collagen staining and observed by microscopy. Results From the HE staining, we observed that the number of repair cells gradually increased. Compared to the injury group, the cell density and gross morphology of cells in the therapeutic group were closer to those of the sham group. As observed by Masson trichrome gelatin staining, many of the fibroblast cells or tissues were under repair, and as time progressed, the number of fibroblast cells and amount of tissue gradually increased. The results of the AB-PAS staining suggest that chondrocytes participated in the repair of the annulus. The level of type II collagen gradually increased, as determined by immunohistochemical staining. Conclusion Our results demonstrate that a gelatin sponge transplant loaded with BMSCs and PRP can effectively repair annulus defects. PMID:28178294

Offset-sparsity decomposition for enhancement of color microscopic image of stained specimen in histopathology: further results

NASA Astrophysics Data System (ADS)

Kopriva, Ivica; Popović Hadžija, Marijana; Hadžija, Mirko; Aralica, Gorana

2016-03-01

Recently, novel data-driven offset-sparsity decomposition (OSD) method was proposed by us to increase colorimetric difference between tissue-structures present in the color microscopic image of stained specimen in histopathology. The OSD method performs additive decomposition of vectorized spectral images into image-adapted offset term and sparse term. Thereby, the sparse term represents an enhanced image. The method was tested on images of the histological slides of human liver stained with hematoxylin and eosin, anti-CD34 monoclonal antibody and Sudan III. Herein, we present further results related to increase of colorimetric difference between tissue structures present in the images of human liver specimens with pancreatic carcinoma metastasis stained with Gomori, CK7, CDX2 and LCA, and with colon carcinoma metastasis stained with Gomori, CK20 and PAN CK. Obtained relative increase of colorimetric difference is in the range [19.36%, 103.94%].

Methods of Hematoxylin and Erosin Image Information Acquisition and Optimization in Confocal Microscopy

PubMed Central

Yoon, Woong Bae; Kim, Hyunjin; Kim, Kwang Gi; Choi, Yongdoo; Chang, Hee Jin

2016-01-01

Objectives We produced hematoxylin and eosin (H&E) staining-like color images by using confocal laser scanning microscopy (CLSM), which can obtain the same or more information in comparison to conventional tissue staining. Methods We improved images by using several image converting techniques, including morphological methods, color space conversion methods, and segmentation methods. Results An image obtained after image processing showed coloring very similar to that in images produced by H&E staining, and it is advantageous to conduct analysis through fluorescent dye imaging and microscopy rather than analysis based on single microscopic imaging. Conclusions The colors used in CLSM are different from those seen in H&E staining, which is the method most widely used for pathologic diagnosis and is familiar to pathologists. Computer technology can facilitate the conversion of images by CLSM to be very similar to H&E staining images. We believe that the technique used in this study has great potential for application in clinical tissue analysis. PMID:27525165

Immunohistochemical study of the digestive tract of Oligosarcus hepsetus

PubMed Central

Vieira-Lopes, Danielle A; Pinheiro, Nadja L; Sales, Armando; Ventura, Adriana; Araújo, Francisco G; Gomes, Iracema D; Nascimento, Aparecida A

2013-01-01

AIM: To describe the histology of the digestive tract and to investigate the occurrence of endocrine cells in Oligosarcus hepsetus (O. hepsetus). METHODS: The digestive tract (DT) of O. hepsetus was divided into esophagus, two stomach regions (glandular and non-glandular) and two intestinal regions (anterior and posterior). These specimens were processed by routine histological techniques and stained with hematoxylin-eosin, Gomori’s trichrome, periodic acid Schiff (PAS) and Alcian blue (AB). An immunohistochemical method using avidin-biotin-peroxidase was employed. RESULTS: The esophagus is lined with a non-keratinized stratified squamous epithelium that is reactive to PAS and AB. The stomach has a mucosa lined with a simple columnar epithelium with mucus-secreting cells that are reactive only to PAS. The intestine has a simple columnar epithelium with a brush border and goblet cells that are reactive to PAS and AB. Somatostatin, serotonin and cholecystokinin immunoreactive cells were identified throughout the DT. CONCLUSION: This study revealed adaptations for the species’ diet and showed that the distribution and relative frequency of immunoreactive cells are similar to those of other fish. PMID:23569337

Spectral imaging perspective on cytomics.

PubMed

Levenson, Richard M

2006-07-01

Cytomics involves the analysis of cellular morphology and molecular phenotypes, with reference to tissue architecture and to additional metadata. To this end, a variety of imaging and nonimaging technologies need to be integrated. Spectral imaging is proposed as a tool that can simplify and enrich the extraction of morphological and molecular information. Simple-to-use instrumentation is available that mounts on standard microscopes and can generate spectral image datasets with excellent spatial and spectral resolution; these can be exploited by sophisticated analysis tools. This report focuses on brightfield microscopy-based approaches. Cytological and histological samples were stained using nonspecific standard stains (Giemsa; hematoxylin and eosin (H&E)) or immunohistochemical (IHC) techniques employing three chromogens plus a hematoxylin counterstain. The samples were imaged using the Nuance system, a commercially available, liquid-crystal tunable-filter-based multispectral imaging platform. The resulting data sets were analyzed using spectral unmixing algorithms and/or learn-by-example classification tools. Spectral unmixing of Giemsa-stained guinea-pig blood films readily classified the major blood elements. Machine-learning classifiers were also successful at the same task, as well in distinguishing normal from malignant regions in a colon-cancer example, and in delineating regions of inflammation in an H&E-stained kidney sample. In an example of a multiplexed ICH sample, brown, red, and blue chromogens were isolated into separate images without crosstalk or interference from the (also blue) hematoxylin counterstain. Cytomics requires both accurate architectural segmentation as well as multiplexed molecular imaging to associate molecular phenotypes with relevant cellular and tissue compartments. Multispectral imaging can assist in both these tasks, and conveys new utility to brightfield-based microscopy approaches. Copyright 2006 International Society for Analytical Cytology.

Chronic inflammation in refractory hippocampal sclerosis-related temporal lobe epilepsy.

PubMed

Gales, Jordan M; Prayson, Richard A

2017-10-01

Emerging evidence suggests chronic inflammation may play a role in hippocampal sclerosis-associated temporal lobe epilepsy. We sought to systematically evaluate for its presence in a group of 315 patients who underwent surgery for medically-refractory epilepsy and who had hippocampal sclerosis. Upon histologic review of hematoxylin and eosin stained tissue sections, 95 (41%) cases demonstrated the presence of lymphocytes within the perivascular region and diffusely within the brain parenchyma. Those cases with chronic inflammation evident on hematoxylin and eosin staining were significantly more likely to experience a post-operative seizure recurrence than those without it (p=0.03). In 9 cases of hippocampi with chronic inflammation observed on hematoxylin and eosin stained sections, there was a mixture of both T (CD3+) and B (CD20+) lymphocytes located around blood vessels and interspersed within the brain parenchyma and a predominance of CD4 positive T cells versus CD8 positive cells. Ten hippocampi, apparently devoid of chronic inflammation upon inspection with hematoxylin and eosin stained sections, were stained with the lymphocyte common antigen CD45. In all 10 cases, scattered lymphoid cells were observed in the brain parenchyma, suggesting some level of chronic inflammation may be present in more cases than casual inspection might suggest. This study was the first to evaluate the incidence of chronic inflammation within a large temporal lobe epilepsy population. The study findings suggest chronic inflammation may be a more common component of hippocampal sclerosis -associated temporal lobe epilepsy than previously believed. Copyright © 2017 Elsevier Inc. All rights reserved.

Multi-institutional comparison of whole slide digital imaging and optical microscopy for interpretation of hematoxylin-eosin-stained breast tissue sections.

PubMed

Krishnamurthy, Savitri; Mathews, Kurt; McClure, Steven; Murray, Melissa; Gilcrease, Michael; Albarracin, Constance; Spinosa, John; Chang, Bernard; Ho, James; Holt, John; Cohen, Arthur; Giri, Dilip; Garg, Karuna; Bassett, Roland L; Liang, Kevin

2013-12-01

Whole slide imaging (WSI) is now used for educational purposes, for consultation, and for archiving and quantitation of immunostains. However, it is not routinely used for the primary diagnosis of hematoxylin-eosin-stained tissue sections. To compare WSI using the Aperio digital pathology system (Aperio Technologies, Inc, Vista, California) with optical microscopy (OM) for the interpretation of hematoxylin-eosin-stained tissue sections of breast lesions. The study was conducted at 3 clinical sites; 3 breast pathologists interpreted 150 hematoxylin-eosin-stained slides at each site, 3 times each by WSI and 3 times each by OM. For WSI, slides were scanned using an Aperio ScanScope and interpreted on a computer monitor using Aperio ImageScope software and Aperio Spectrum data management software. Pathologic interpretations were recorded using the College of American Pathologists breast checklist. WSI diagnoses were compared with OM diagnoses for accuracy, precision (interpathologist variation), and reproducibility (intrapathologist variation). Results were considered accurate only if the interpretation matched exactly between WSI and OM. The proportion of accurate results reported by each pathologist was expressed as a percentage for the comparison of the 2 platforms. The accuracy of WSI for classifying lesions as not carcinoma or as noninvasive (ductal or lobular) or invasive (ductal, lobular, or other) carcinoma was 90.5%. The accuracy of OM was 92.1%. The precision and reproducibility of WSI and OM were determined on the basis of pairwise comparisons (3 comparisons for each slide, resulting in 36 possible comparisons). The overall precision of WSI was 90.5% in comparison with 92.1% for OM; reproducibility of WSI was 91.6% in comparison with 94.5% for OM, respectively. In this study, we demonstrated that WSI and OM have similar accuracy, precision, and reproducibility for interpreting hematoxylin-eosin-stained breast tissue sections. Further clinical studies using routine surgical pathology specimens would be useful to confirm these findings and facilitate the incorporation of WSI into diagnostic practice.

Methods of Hematoxylin and Erosin Image Information Acquisition and Optimization in Confocal Microscopy.

PubMed

Yoon, Woong Bae; Kim, Hyunjin; Kim, Kwang Gi; Choi, Yongdoo; Chang, Hee Jin; Sohn, Dae Kyung

2016-07-01

We produced hematoxylin and eosin (H&E) staining-like color images by using confocal laser scanning microscopy (CLSM), which can obtain the same or more information in comparison to conventional tissue staining. We improved images by using several image converting techniques, including morphological methods, color space conversion methods, and segmentation methods. An image obtained after image processing showed coloring very similar to that in images produced by H&E staining, and it is advantageous to conduct analysis through fluorescent dye imaging and microscopy rather than analysis based on single microscopic imaging. The colors used in CLSM are different from those seen in H&E staining, which is the method most widely used for pathologic diagnosis and is familiar to pathologists. Computer technology can facilitate the conversion of images by CLSM to be very similar to H&E staining images. We believe that the technique used in this study has great potential for application in clinical tissue analysis.

Regionalization of surface lipids in insects

PubMed Central

Wang, Yiwen; Yu, Zhitao; Zhang, Jianzhen; Moussian, Bernard

2016-01-01

Cuticular hydrocarbons (CHCs) play a critical role in the establishment of the waterproof barrier that prevents dehydration and wetting in insects. While rich data are available on CHC composition in different species, we know little about their distribution and organization. Here, we report on our studies of the surface barrier of the fruit fly Drosophila melanogaster applying a newly developed Eosin Y staining method. The inert Eosin Y penetrates different regions of the adult body at distinct temperatures. By contrast, the larval body takes up the dye rather uniformly and gradually with increasing temperature. Cooling down specimens to 25°C after incubation at higher temperatures restores impermeability. Eosin Y penetration is also sensitive to lipid solvents such as chloroform indicating that permeability depends on CHCs. As in D. melanogaster adult flies, Eosin Y penetration is regionalized in Tenebrio molitor larvae, whereas it is not in Locusta migratoria nymphs. Regionalization of the fly surface implies tissue-specific variation of the genetic or biochemical programmes of CHC production and deposition. The Eosin Y-based map of CHC distribution may serve to identify the respective factors that are activated to accommodate ecological needs. PMID:27170708

Cell nuclei attributed relational graphs for efficient representation and classification of gastric cancer in digital histopathology

NASA Astrophysics Data System (ADS)

Sharma, Harshita; Zerbe, Norman; Heim, Daniel; Wienert, Stephan; Lohmann, Sebastian; Hellwich, Olaf; Hufnagl, Peter

2016-03-01

This paper describes a novel graph-based method for efficient representation and subsequent classification in histological whole slide images of gastric cancer. Her2/neu immunohistochemically stained and haematoxylin and eosin stained histological sections of gastric carcinoma are digitized. Immunohistochemical staining is used in practice by pathologists to determine extent of malignancy, however, it is laborious to visually discriminate the corresponding malignancy levels in the more commonly used haematoxylin and eosin stain, and this study attempts to solve this problem using a computer-based method. Cell nuclei are first isolated at high magnification using an automatic cell nuclei segmentation strategy, followed by construction of cell nuclei attributed relational graphs of the tissue regions. These graphs represent tissue architecture comprehensively, as they contain information about cell nuclei morphology as vertex attributes, along with knowledge of neighborhood in the form of edge linking and edge attributes. Global graph characteristics are derived and ensemble learning is used to discriminate between three types of malignancy levels, namely, non-tumor, Her2/neu positive tumor and Her2/neu negative tumor. Performance is compared with state of the art methods including four texture feature groups (Haralick, Gabor, Local Binary Patterns and Varma Zisserman features), color and intensity features, and Voronoi diagram and Delaunay triangulation. Texture, color and intensity information is also combined with graph-based knowledge, followed by correlation analysis. Quantitative assessment is performed using two cross validation strategies. On investigating the experimental results, it can be concluded that the proposed method provides a promising way for computer-based analysis of histopathological images of gastric cancer.

Staining Methods for Normal and Regenerative Myelin in the Nervous System.

PubMed

Carriel, Víctor; Campos, Antonio; Alaminos, Miguel; Raimondo, Stefania; Geuna, Stefano

2017-01-01

Histochemical techniques enable the specific identification of myelin by light microscopy. Here we describe three histochemical methods for the staining of myelin suitable for formalin-fixed and paraffin-embedded materials. The first method is conventional luxol fast blue (LFB) method which stains myelin in blue and Nissl bodies and mast cells in purple. The second method is a LBF-based method called MCOLL, which specifically stains the myelin as well the collagen fibers and cells, giving an integrated overview of the histology and myelin content of the tissue. Finally, we describe the osmium tetroxide method, which consist in the osmication of previously fixed tissues. Osmication is performed prior the embedding of tissues in paraffin giving a permanent positive reaction for myelin as well as other lipids present in the tissue.

Interfacial polymerization for colorimetric labeling of protein expression in cells.

PubMed

Lilly, Jacob L; Sheldon, Phillip R; Hoversten, Liv J; Romero, Gabriela; Balasubramaniam, Vivek; Berron, Brad J

2014-01-01

Determining the location of rare proteins in cells typically requires the use of on-sample amplification. Antibody based recognition and enzymatic amplification is used to produce large amounts of visible label at the site of protein expression, but these techniques suffer from the presence of nonspecific reactivity in the biological sample and from poor spatial control over the label. Polymerization based amplification is a recently developed alternative means of creating an on-sample amplification for fluorescence applications, while not suffering from endogenous labels or loss of signal localization. This manuscript builds upon polymerization based amplification by developing a stable, archivable, and colorimetric mode of amplification termed Polymer Dye Labeling. The basic concept involves an interfacial polymer grown at the site of protein expression and subsequent staining of this polymer with an appropriate dye. The dyes Evans Blue and eosin were initially investigated for colorimetric response in a microarray setting, where both specifically stained polymer films on glass. The process was translated to the staining of protein expression in human dermal fibroblast cells, and Polymer Dye Labeling was specific to regions consistent with desired protein expression. The labeling is stable for over 200 days in ambient conditions and is also compatible with modern mounting medium.

Evaluation of staining methods for cytologic diagnosis of oral lesions.

PubMed

Almeida, Janete Dias; Lima, Celina Faig; Brandão, Adriana Aigotti Haberbeck; Cabral, Luiz Antonio Guimarães

2008-01-01

To compare the efficacy ofPapanicolaou, hematoxylin-eosin (H-E), Leishman and periodic acid-Schiff (PAS) staining for cytologic diagnosis of oral lesions. Patients from the Discipline of Stomatology, São José dos Campos Dental School, from the wards of Hosapital Heliópolis and from the dentistry outpatient clinic of the University Hospital, University of São Paulo Medical School, with the following diseases, were selected: erythematous candidiasis (n=9), pseudomembranous candidiasis (n=10), squamous cell carcinoma (n=19), herpes simplex (n=8), paracoccidioidomycosis (n=8) and pemphigus vulgaris (n=1). The different staining methods were compared regarding the quality of definition of cytoplasmic and nuclear morphologic characteristics and the identification of bacteria, fungi, inflammatory cells and secretions. Papanicolaou and H-E staining were considered better methods. In cases of fungal infections, PAS staining is useful and should be applied as a complementary method. Within the limitations of this study, it can be concluded that the cytologic diagnosis of oral lesions along with different staining methods is a useful tool for oral diagnosis.

Performance of new gellan gum hydrogels combined with human articular chondrocytes for cartilage regeneration when subcutaneously implanted in nude mice.

PubMed

Oliveira, J T; Santos, T C; Martins, L; Silva, M A; Marques, A P; Castro, A G; Neves, N M; Reis, R L

2009-10-01

Gellan gum is a polysaccharide that has been recently proposed by our group for cartilage tissue-engineering applications. It is commonly used in the food and pharmaceutical industry and has the ability to form stable gels without the use of harsh reagents. Gellan gum can function as a minimally invasive injectable system, gelling inside the body in situ under physiological conditions and efficiently adapting to the defect site. In this work, gellan gum hydrogels were combined with human articular chondrocytes (hACs) and were subcutaneously implanted in nude mice for 4 weeks. The implants were collected for histological (haematoxylin and eosin and Alcian blue staining), biochemical [dimethylmethylene blue (GAG) assay], molecular (real-time PCR analyses for collagen types I, II and X, aggrecan) and immunological analyses (immunolocalization of collagen types I and II). The results showed a homogeneous cell distribution and the typical round-shaped morphology of the chondrocytes within the matrix upon implantation. Proteoglycans synthesis was detected by Alcian blue staining and a statistically significant increase of proteoglycans content was measured with the GAG assay quantified from 1 to 4 weeks of implantation. Real-time PCR analyses showed a statistically significant upregulation of collagen type II and aggrecan levels in the same periods. The immunological assays suggest deposition of collagen type II along with some collagen type I. The overall data shows that gellan gum hydrogels adequately support the growth and ECM deposition of human articular chondrocytes when implanted subcutaneously in nude mice. Copyright (c) 2009 John Wiley & Sons, Ltd.

Biofilm detection in chronic rhinosinusitis by combined application of hematoxylin-eosin and gram staining.

PubMed

Tóth, László; Csomor, Péter; Sziklai, István; Karosi, Tamás

2011-10-01

The pathomechanism of chronic rhinosinusitis with nasal polyposis (CRS/NP) seems to be unclear. Bacterial-, fungal- and combined biofilms might play a potential role in the pathogenesis of various inflammatory diseases and recently in CRS/NP. A prospective, blinded observational study was performed to confirm that the combination of conventional hematoxylin-eosin (HE) and Gram staining protocols could be used to detect bacterial and fungal biofilms in patients with CRS/NP. A total of 50 patients with CRS/NP undergoing endoscopic sinus surgery (ESS) were analyzed. The negative control group consisted of 12 patients undergoing septoplasty for nasal obstruction without CRS/NP. The nasal polyps and inferior turbinate mucosa specimens applied as negative controls were processed to HE and Gram staining. Biofilm was detected in 44 of 50 patients with CRS/NP and in none of 12 negative controls. In our series, HE method showed an obvious correlation with the results of Gram staining and was allocated to be a good predictor of biofilm existence. It was found that the microscopic structure and thickness of biofilms were strongly associated with the integrity of nasal mucosa and with the characteristics of subepithelial cellular infiltration. This study confirmed the presence of bacterial and fungal biofilms on the surface of NPs obtained from patients with CRS. Since biofilms may affect the severity and recurrence rate of CRS treated by ESS they should be detected histologically. In conclusion, HE staining combined with Gram protocol is a robust and reliable method for the detection of bacterial and fungal biofilms in CRS/NP.

In vivo biocompatibility of new nano-calcium-deficient hydroxyapatite/poly-amino acid complex biomaterials

PubMed Central

Dai, Zhenyu; Li, Yue; Lu, Weizhong; Jiang, Dianming; Li, Hong; Yan, Yonggang; Lv, Guoyu; Yang, Aiping

2015-01-01

Objective To evaluate the compatibility of novel nano-calcium-deficient hydroxyapatite/poly-amino acid (n-CDHA/PAA) complex biomaterials with muscle and bone tissue in an in vivo model. Methods Thirty-two New Zealand white rabbits were used in this study. Biomaterials were surgically implanted into each rabbit in the back erector spinae and in tibia with induced defect. Polyethylene was implanted into rabbits in the control group and n-CDHA/PAA into those of the experimental group. Animals were examined at four different points in time: 2 weeks, 4 weeks, 12 weeks, and 24 weeks after surgery. They were euthanized after embolization. Back erector spinae muscles with the surgical implants were examined after hematoxylin and eosin (HE) staining at these points in time. Tibia bones with the surgical implants were examined by X-ray and scanning electron microscopy (SEM) at these points in time to evaluate the interface of the bone with the implanted biomaterials. Bone tissues were sectioned and subjected to HE, Masson, and toluidine blue staining. Results HE staining of back erector spinae muscles at 4 weeks, 12 weeks, and 24 weeks after implantation of either n-CDHA/PAA or polyethylene showed disappearance of inflammation and normal arrangement in the peripheral tissue of implant biomaterials; no abnormal staining was observed. At 2 weeks after implantation, X-ray imaging of bone tissue samples in both experimental and control groups showed that the peripheral tissues of the implanted biomaterials were continuous and lacked bone osteolysis, absorption, necrosis, or osteomyelitis. The connection between implanted biomaterials and bone tissue was tight. The results of HE, Masson, toluidine blue staining and SEM confirmed that the implanted biomaterials were closely connected to the bone defect and that no rejection had taken place. The n-CDHA/PAA biomaterials induced differentiation of a large number of chondrocytes. New bone trabecula began to form at 4 weeks after implanting n-CDHA/PAA biomaterials, and lamellar bone gradually formed at 12 weeks and 24 weeks after implantation. Routine blood and kidney function tests showed no significant changes at 2 weeks and 24 weeks after implantation of both biomaterials. Conclusion n-CDHA/PAA composites showed good compatibility in in vivo model. In this study, n-CDHA/PAA were found to be safe, nontoxic, and biologically active in bone repair. PMID:26504382

Histological analysis of retraction pocket pars tensa of tympanic membrane in children.

PubMed

Urík, M; Hurník, P; Žiak, D; Machač, J; Šlapák, I; Motyka, O; Vaculová, J; Dvořáčková, J

2016-07-01

Histological and histochemical analysis of retraction pocket of pars tensa of tympanic membrane in children. Identification of morphological abnormalities in comparison with a healthy tympanic membrane as it is described in standard textbook. Identification of signs typical for cholesteatoma and support for a retraction theory of cholesteatoma formation. A prospective study analysing 31 samples of retraction pockets taken during surgery. University Hospital, Children's Medical Centre Samples of retraction pockets were processed by a standard process for light microscopy, stained by haematoxylin-eosin. Van Gieson's stain was used for differential staining of collagen, Verhoeff's stain for elastic fibre tissues, Alcian blue for acidic polysaccharides and PAS (Periodic Acid Schiff) method for basement membrane polysaccharides. The following findings were observed in the samples of retraction pockets: hyperkeratosis (100%), hypervascularisations (100%), subepithelial fragmented elastic fibres (96%), myxoid changes (87%), subepithelial inflammatory infiltration (84%), rete pegs (71%), papilomatosis (71%), intraepithelial inflammatory cellularizations, (48%), intraepithelial spongiosis (16%) and parakeratosis (3%). No basement membrane continuity interruptions were observed. Thickness of retraction pocket, thickness of epidermis, occurrence of rete pegs and frequency of fragmented elastic fibres was higher in a Grade III stage RP than Grade II stage RP (according to Charachon). Morphological abnormalities in the structure of retraction pockets in comparison with a healthy tympanic membrane were described. The changes are typical for a structure of cholesteatoma (these changes are common in matrix and perimatrix), supporting retraction theory of its origin. Our observations show that it is inflammation that probably plays a key role in the pathogenesis of retraction pocket. The frequency of some of the changes increases with the stage of retraction pocket (II-III according to Charachon). Basement membrane continuity interruptions are not typical for retraction pockets. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Distinctive eosinophilic cytoplasmic inclusion bodies in melanocytic nevi: an immunohistochemical and ultrastructural study.

PubMed

Shon, Wonwoo; Wada, David A; Gibson, Lawrence E; Flotte, Thomas J; Scheithauer, Bernd W

2011-11-01

We sought to further determine the histochemical, immunohistochemical and ultrastructural properties of eosinophilic cytoplasmic inclusion bodies in melanocytic nevi. Skin specimens from four patients with a known diagnosis of conventional melanocytic nevus (3) or Spitz nevus (1) and containing intracytoplasmic eosinophilic inclusion bodies were selected. In addition, melanomas (25), Spitz nevi (10) and blue nevi (4) were examined to determine the frequency of the inclusions. Inclusions tended to be located in multinucleated melanocytes with abundant vacuolated cytoplasm. In conventional (hematoxylin and eosin-stained) sections, the degree of density and eosinophilia of intracytoplasmic inclusions varied with size. Periodic acid-Schiff, Fontana and Congo red stains showed no reactivity. All bodies were immunoreactive for ubiquitin but negative for tyrosinase, keratin and vimentin. Ultrastructurally, inclusion bodies were non-membrane bound, ranged from 4 to 7 µm, and were comprised of radiating filamentous structures with or without an electron-dense core. Electron probe x-ray microanalysis revealed no significant peaks. None of additional melanomas, Spitz nevi and blue nevi that were evaluated showed similar inclusions. The inclusion bodies described herein bear no resemblance to other cytoplasmic inclusion bodies previously described in melanocytic lesions. There is no discernible relationship to melanosomes by ultrastructural analysis. We postulate a relationship with dysfunction of ubiquitin-mediated protein degradation occurring in melanocytes. Copyright © 2011 John Wiley & Sons A/S.

[Localizating and Extracting Small Peripheral Nodules of Lung with Simulating 
Radiaotherapy Combining Methylene Blue Staining].

PubMed

Mao, Feng; Zhang, Liang; Gu, Hengle; Zhang, Hui; Lv, Changxing; Shen-Tu, Yang

2016-09-20

With the extensively application of HRCT (high resolution CT) and the popularization of early lung cancer screening, the proportion of small nodullar lung cancer to be operated increases rapidly. Identifying the focus lesions quickly and accurately in operation has shown to be a challenge. We carried out this research trying to make use of and evaluate a new method that localizaes and extracts small peripheral pulmonary nodules by way of simulating radiaotherapy combining methylene blue staining. From February 2012 to January 2015, 97 patients with 100 peripheral pulmonary nodules ≤10 mm in size were simulated puncturing using a radiotherapy planning. When the anaesthesia came into use, methylene blue dye was injected to the virtually identified point corresponding to the surface point, according to the angle and depth previously computed by the radiotherapy planning. The video-assisted thoracoscopic surgery (VATS) wedge resections of the marked lesions were undertaken and the specimens were sent for frozen pathologic examination. The interval time from anesthesia-completing to puncture and injection, The interval time from methylene blue injection to identifying the stained area and the distances between the centre point of the stains and edge of coloured lesion were recorded. Our preoperative localization procedure was successful in 96 of 100 (96%) nodules. The interval time from anesthesia-completing to puncture and injection of methylene blue were (4.85±1.25) min. The interval time from methylene blue injection to identifying the stained area was (16.36±2.36) min. The distances between the centre point of the stains and edge of coloured lesion were (4.78±2.51) mm. No complication was observed in all participants. The new method of locating peripheral pulmonary nodules by simulating simulating radiaotherapy combining methylene blue staining has a high success rate and no complication for localizing small peripheral pulmonary lesions, avoiding the fear and pain of the patients untaken puncture without anaesthesia reducing radial damage.

Nuclear staining with alum hematoxylin.

PubMed

Llewellyn, B D

2009-08-01

The hematoxylin and eosin stain is the most common method used in anatomic pathology, yet it is a method about which technologists ask numerous questions. Hematoxylin is a natural dye obtained from a tree originally found in Central America, and is easily converted into the dye hematein. This dye forms coordination compounds with mordant metals, such as aluminum, and the resulting lake attaches to cell nuclei. Regressive formulations contain a higher concentration of dye than progressive formulations and may also contain a lower concentration of mordant. The presence of an acid increases the life of the solution and in progressive solutions may also affect selectivity of staining. An appendix lists more than 60 hemalum formulations and the ratio of dye to mordant for each.

[Eosin Y-water test for sperm function examination].

PubMed

Zha, Shu-wei; Lü, Nian-qing; Xu, Hao-qin

2015-06-01

Based on the principles of the in vitro staining technique, hypotonic swelling test, and water test, the Eosin Y-water test method was developed to simultaneously detect the integrity of the sperm head and tail and sperm membrane structure and function. As a widely used method in clinical laboratories in China, the Eosin Y-water test is methodologically characterized by three advantages. Firstly, both the sperm head and tail can be detected at the same time, which allows easy and comprehensive assessment of membrane damage in different parts of sperm. Secondly, distilled water is used instead of the usual formula solution to simplify and standardize the test by eliminating any potential effects on the water molecules through the sperm membrane due to different osmotic pressure or different sugar proportions and electrolyte solutions. Thirdly, the test takes less time and thus can be repeated before and after treatment. This article focuses on the fundamental principles and modification of the Eosin Y-water test and its application in sperm function examination and routine semen analysis for male infertility, assessment of the quality of sperm retrieved by testicular fine needle aspiration, semen cryopreservation program development, and evaluation of sperm membrane integrity after microwave radiation.

Immunohistochemical differentiation of reactive from malignant mesothelium as a diagnostic aid in canine pericardial disease.

PubMed

Milne, E; Martinez Pereira, Y; Muir, C; Scase, T; Shaw, D J; McGregor, G; Oldroyd, L; Scurrell, E; Martin, M; Devine, C; Hodgkiss-Geere, H

2018-05-01

To develop a provisional immunohistochemistry panel for distinguishing reactive pericardium, atypical mesothelial proliferation and mesothelioma in dogs. Archived pericardial biopsies were subject to haematoxylin and eosin staining, immunohistochemistry for cytokeratin, vimentin, insulin-like growth factor II mRNA-binding protein 3, glucose transporter 1 and desmin. Samples were scored for intensity and number of cells stained. Ten biopsies of reactive mesothelium, 17 of atypical mesothelial proliferation, 26 of mesothelioma and five of normal pericardium were identified on the basis of haematoxylin and eosin staining. Cytokeratin and vimentin were expressed in all biopsies, confirming mesothelial origin. Normal pericardial samples had the lowest scores for insulin-like growth factor II mRNA-binding protein 3, glucose transporter 1 and desmin. Mesothelioma and atypical proliferative samples were similar to each other, with higher scores for insulin-like growth factor II mRNA-binding protein 3 and glucose transporter 1 than the reactive samples. Desmin staining was variable. Insulin-like growth factor II mRNA-binding protein 3 was the best to distinguish between disease groups. An immunohistochemistry panel of cytokeratin, vimentin, insulin-like growth factor II mRNA-binding protein 3 and glucose transporter 1 could provide superior information compared with haematoxylin and eosin staining alone in the diagnosis of cases of mesothelial proliferation in canine pericardium, but further validation is warranted. © 2018 British Small Animal Veterinary Association.

Regionalization of surface lipids in insects.

PubMed

Wang, Yiwen; Yu, Zhitao; Zhang, Jianzhen; Moussian, Bernard

2016-05-11

Cuticular hydrocarbons (CHCs) play a critical role in the establishment of the waterproof barrier that prevents dehydration and wetting in insects. While rich data are available on CHC composition in different species, we know little about their distribution and organization. Here, we report on our studies of the surface barrier of the fruit fly Drosophila melanogaster applying a newly developed Eosin Y staining method. The inert Eosin Y penetrates different regions of the adult body at distinct temperatures. By contrast, the larval body takes up the dye rather uniformly and gradually with increasing temperature. Cooling down specimens to 25°C after incubation at higher temperatures restores impermeability. Eosin Y penetration is also sensitive to lipid solvents such as chloroform indicating that permeability depends on CHCs. As in D. melanogaster adult flies, Eosin Y penetration is regionalized in Tenebrio molitor larvae, whereas it is not in Locusta migratoria nymphs. Regionalization of the fly surface implies tissue-specific variation of the genetic or biochemical programmes of CHC production and deposition. The Eosin Y-based map of CHC distribution may serve to identify the respective factors that are activated to accommodate ecological needs. © 2016 The Author(s).

Chondrogenesis of Human Adipose-Derived Stem Cells by In Vivo Co-graft with Auricular Chondrocytes from Microtia.

PubMed

Cai, Zhen; Pan, Bo; Jiang, Haiyue; Zhang, Lixia

2015-06-01

To evaluate the efficiency of chondrogenesis of human adipose-derived stem cells (ADSCs) induced by auricular chondrocytes from microtia via subcutaneous co-graft in nude mice. Human ADSCs and auricular chondrocytes were mixed at the ratio of 7:3 and suspended in 0.2 ml of Pluronic F-127 (5.0 × 10(7) cells/ml), and injected into Balb/c nude mice as the experimental group (Exp group). The same quantity of auricular chondrocytes (Ctr.1 group) or ADSCs (Ctr.2 group) in 0.2 ml of Pluronic F-127 was set as positive and negative control groups. The mixture of auricular chondrocytes (1.5 × 10(7) cells/ml) in 0.2 ml of Pluronic F-127 was set as the low concentration of chondrocyte control group (Ctr.3). At 8 weeks after grafting, the newly generated tissue pellets were isolated for morphological examination, haematoxylin and eosin staining, toluidine blue staining and safranin O staining of glycosaminoglycan (GAG), Masson's trichrome staining and immunohistochemical staining of type II collagen, and Verhoeff-iron-hematoxylin staining of elastic fibers. GAG content was determined by Alcian blue colorimetric method, and mRNA expression of type II collagen and aggrecan were examined by real-time PCR. Cartilage-like tissue with a white translucent appearance and good elasticity was generated in the Exp and Ctr.1 groups. The tissue pellets in the Ctr.2 and Ctr.3 groups were much smaller than those in the Ctr.1 group. The mature cartilage lacunas could be observed in the Exp and Ctr.1 groups, while were rarely seen in the Ctr.3 group and not observed in the Ctr.2 group. The expression of cartilage-specific extracellular matrix such as type II collagen, GAG content, aggrecan, and elastic fibers in the Exp group was similar to that in the Ctr.1 group, whereas the expression of these extracellular matrix substances was significantly lower in the Ctr.2 and Ctr.3 groups (both P < 0.01). Auricular chondrocytes from microtia can efficiently promote the chondrogenic differentiation and chondrogenesis of ADSCs by co-grafting in vivo. This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors http://www.springer.com/00266 .

Changes of rabbit meniscus influenced by hyaline cartilage injury of osteoarthritis.

PubMed

Zhao, Jiajun; Huang, Suizhu; Zheng, Jia; Zhong, Chunan; Tang, Chao; Zheng, Lei; Zhang, Zhen; Xu, Jianzhong

2014-01-01

Osteoarthritis (OA) is a common disease in the elderly population. Most of the previous OA-related researches focused on articular cartilage degeneration, osteophyte formation and synovitis etc. However, the role of the meniscus in these pathological changes has not been given enough attention. The goal of our study was to find the pathological changes of the meniscus in OA knee and determine their relationship. 20 months old female Chinese rabbits received either knee damaging operations with articular cartilage scratch method or sham operation randomly on one of their knees. They were sacrificed after 1-6 weeks post-operation. Medial Displacement Index (MDI) for meniscus dislocation, hematoxylin and eosin (HE) for routine histological evaluation, Toluidine blue (TB) stains for evaluating proteoglycans were carried out. Immunohistochemical (IHC) staining was performed with a two-step detection kit. Histological analysis showed chondrocyte clusters around cartilage lesions and moderate loss of proteoglycans in the operation model, as well as MDI increase and all characteristics of OA. High expression of MMP-3 and TIMP-1 also were found in both hyaline cartilage and meniscus. Biomechanical and biochemistry environment around the meniscus is altered when OA occur. If meniscus showed degeneration, subluxation and dysfunction, OA would be more severe. Prompt repair or reconstruction of hyaline cartilage in weight bearing area when it injured could prevent meniscus degeneration and subluxation, then prevent the development of OA.

Safer staining method for acid fast bacilli.

PubMed Central

Ellis, R C; Zabrowarny, L A

1993-01-01

To develop a method for staining acid fast bacilli which excluded highly toxic phenol from the staining solution. A lipophilic agent, a liquid organic detergent, LOC High Studs, distributed by Amway, was substituted. The acid fast bacilli stained red; nuclei, cytoplasm, and cytoplasmic elements stained blue on a clear background. These results compare very favourably with acid fast bacilli stained by the traditional method. Detergents are efficient lipophilic agents and safer to handle than phenol. The method described here stains acid fast bacilli as efficiently as traditional carbol fuchsin methods. LOC High Suds is considerably cheaper than phenol. Images PMID:7687254

Safer staining method for acid fast bacilli.

PubMed

Ellis, R C; Zabrowarny, L A

1993-06-01

To develop a method for staining acid fast bacilli which excluded highly toxic phenol from the staining solution. A lipophilic agent, a liquid organic detergent, LOC High Studs, distributed by Amway, was substituted. The acid fast bacilli stained red; nuclei, cytoplasm, and cytoplasmic elements stained blue on a clear background. These results compare very favourably with acid fast bacilli stained by the traditional method. Detergents are efficient lipophilic agents and safer to handle than phenol. The method described here stains acid fast bacilli as efficiently as traditional carbol fuchsin methods. LOC High Suds is considerably cheaper than phenol.

Digital staining for histopathology multispectral images by the combined application of spectral enhancement and spectral transformation.

PubMed

Bautista, Pinky A; Yagi, Yukako

2011-01-01

In this paper we introduced a digital staining method for histopathology images captured with an n-band multispectral camera. The method consisted of two major processes: enhancement of the original spectral transmittance and the transformation of the enhanced transmittance to its target spectral configuration. Enhancement is accomplished by shifting the original transmittance with the scaled difference between the original transmittance and the transmittance estimated with m dominant principal component (PC) vectors;the m-PC vectors were determined from the transmittance samples of the background image. Transformation of the enhanced transmittance to the target spectral configuration was done using an nxn transformation matrix, which was derived by applying a least square method to the enhanced and target spectral training data samples of the different tissue components. Experimental results on the digital conversion of a hematoxylin and eosin (H&E) stained multispectral image to its Masson's trichrome stained (MT) equivalent shows the viability of the method.

Comparison of structural changes in skin and amnion tissue grafts for transplantation induced by gamma and electron beam irradiation for sterilization.

PubMed

Mrázová, H; Koller, J; Kubišová, K; Fujeríková, G; Klincová, E; Babál, P

2016-06-01

Sterilization is an important step in the preparation of biological material for transplantation. The aim of the study is to compare morphological changes in three types of biological tissues induced by different doses of gamma and electron beam radiation. Frozen biological tissues (porcine skin xenografts, human skin allografts and human amnion) were irradiated with different doses of gamma rays (12.5, 25, 35, 50 kGy) and electron beam (15, 25, 50 kGy). Not irradiated specimens served as controls. The tissue samples were then thawn and fixed in 10 % formalin, processed by routine paraffin technique and stained with hematoxylin and eosin, alcian blue at pH 2.5, orcein, periodic acid Schiff reaction, phosphotungstic acid hematoxylin, Sirius red and silver impregnation. The staining with hematoxylin and eosin showed vacuolar cytoplasmic changes of epidermal cells mainly in the samples of xenografts irradiated by the lowest doses of gamma and electron beam radiation. The staining with orcein revealed damage of fine elastic fibers in the xenograft dermis at the dose of 25 kGy of both radiation types. Disintegration of epithelial basement membrane, especially in the xenografts, was induced by the dose of 15 kGy of electron beam radiation. The silver impregnation disclosed nuclear chromatin condensation mainly in human amnion at the lowest doses of both radiation types and disintegration of the fine collagen fibers in the papillary dermis induced by the lowest dose of electron beam and by the higher doses of gamma radiation. Irradiation by both, gamma rays and the electron beam, causes similar changes on cells and extracellular matrix, with significant damage of the basement membrane and of the fine and elastic and collagen fibers in the papillary dermis, the last caused already by low dose electron beam radiation.

Clinical, Cytological, Histological and Immunohistochemical Features of Cutaneous Mast Cell Tumours in Ferrets (Mustela putorius furo).

PubMed

Vilalta, L; Meléndez-Lazo, A; Doria, G; Ramis, A; Solano-Gallego, L; Pastor, J; Martorell, J

2016-11-01

Cutaneous mast cell tumours (cMCTs) are one of the most common cutaneous tumours in ferrets (Mustela putorius furo). However, limited information is available regarding cytological and histological features of these tumours and studies evaluating KIT expression are lacking in this species. The aims of this prospective study were to describe the most common clinical, cytological and histological features of cMCTs in ferrets and to compare the usefulness of different staining techniques in the diagnosis of these tumours in ferrets as well as evaluating KIT expression in neoplastic mast cells (MCs) by immunohistochemistry. Macroscopically, the tumours were small, round to plaque-like and frequently associated with surface crusting. The most common locations were the extremities and the trunk. MC granules were stained in all cases using toluidine blue (TB) and Wright-Giemsa stains in cytological specimens, but none stained with modified Wright's stain. Haematoxylin and eosin and TB on histological sections failed to stain MC granules in all the cases. Cytological and histological examination revealed low to moderate anisocytosis and anisokaryosis. An infiltrative rather than a delineated or encapsulated growth pattern was noted histologically in all cases. Eosinophilic infiltration was not uncommon and 'collagenolysis' was detected on cytological and histological examination. KIT expression was detected in all cases evaluated. In approximately one third of the cases the MCs exhibited KIT labelling pattern I and in the remaining ferrets, KIT pattern III. No correlation was found between KIT expression pattern and biological behaviour. Copyright © 2016 Elsevier Ltd. All rights reserved.

Identification of immune cell infiltration in hematoxylin-eosin stained breast cancer samples: texture-based classification of tissue morphologies

NASA Astrophysics Data System (ADS)

Turkki, Riku; Linder, Nina; Kovanen, Panu E.; Pellinen, Teijo; Lundin, Johan

2016-03-01

The characteristics of immune cells in the tumor microenvironment of breast cancer capture clinically important information. Despite the heterogeneity of tumor-infiltrating immune cells, it has been shown that the degree of infiltration assessed by visual evaluation of hematoxylin-eosin (H and E) stained samples has prognostic and possibly predictive value. However, quantification of the infiltration in H and E-stained tissue samples is currently dependent on visual scoring by an expert. Computer vision enables automated characterization of the components of the tumor microenvironment, and texture-based methods have successfully been used to discriminate between different tissue morphologies and cell phenotypes. In this study, we evaluate whether local binary pattern texture features with superpixel segmentation and classification with support vector machine can be utilized to identify immune cell infiltration in H and E-stained breast cancer samples. Guided with the pan-leukocyte CD45 marker, we annotated training and test sets from 20 primary breast cancer samples. In the training set of arbitrary sized image regions (n=1,116) a 3-fold cross-validation resulted in 98% accuracy and an area under the receiver-operating characteristic curve (AUC) of 0.98 to discriminate between immune cell -rich and - poor areas. In the test set (n=204), we achieved an accuracy of 96% and AUC of 0.99 to label cropped tissue regions correctly into immune cell -rich and -poor categories. The obtained results demonstrate strong discrimination between immune cell -rich and -poor tissue morphologies. The proposed method can provide a quantitative measurement of the degree of immune cell infiltration and applied to digitally scanned H and E-stained breast cancer samples for diagnostic purposes.

Microscopic morphology and apoptosis of ovarian tissue after cryopreservation using a vitrification method in post-hatching turkey poults, Meleagris gallopavo

USDA-ARS?s Scientific Manuscript database

1. Microscopic morphology of ovarian tissue in post-hatching turkey poults at various ages was investigated. 2. Hematoxylin and eosin staining were used and the diameter of the oocytes and follicles were measured using microphotography. 3. Immediately after hatching, oocytes in one-day turkey pou...

Platinum blue as an alternative to uranyl acetate for staining in transmission electron microscopy.

PubMed

Inaga, Sumire; Katsumoto, Tetsuo; Tanaka, Keiichi; Kameie, Toshio; Nakane, Hironobu; Naguro, Tomonori

2007-04-01

This paper introduces an aqueous solution of platinum blue (Pt-blue) as an alternative to uranyl acetate (UA) for staining in transmission electron microscopy (TEM). Pt-blue was prepared from a reaction of cis-dichlorodiamine-platinum (II) (cis-platin) with thymidine. When Pt-blue was dried on a microgrid and observed by TEM it showed a uniform appearance with tiny particles less than 1 nm in diameter. The effect of Pt-blue as an electron stain was then examined not only for positive staining of conventional ultrathin resin sections and counterstaining of post-embedding immuno-electron microscopy but also for negative staining. In ultrathin sections of the rat liver and renal glomerulus, Pt-blue provided good contrast images, especially in double staining combined with a lead stain (Pb). Almost all cell organelles were clearly observed with high contrast in these sections. Glycogen granules in the hepatic parenchymal cells were particularly electron dense in Pt-blue stained sections compared with those treated with UA. In longitudinal and transverse sections of budding influenza A viruses, a specific arrangement of rod-like structures, which correspond to the ribonucleoprotein complexes, was clearly shown in each virion stained with Pt-blue and Pb. When post-embedding immunoelectron microscopy was performed in ultrathin sections of HeLa cells embedded in Lowicryl K4M, the localization of Ki-67 protein was sufficiently detected even after Pt-blue and Pb staining. The present study also revealed that Pt-blue could be used for the negative staining of E. coli, allowing the visualization of a flagellum. These findings indicate that Pt-blue is a useful, safe, and easily obtainable electron stain that is an alternative to UA for TEM preparations.

Curcuma longa extract – Haldi: A safe, eco-friendly natural cytoplasmic stain

PubMed Central

Suryawanshi, Hema; Naik, Rupali; Kumar, Pramod; Gupta, Rolly

2017-01-01

Background: Eosin is most widely used synthetic dye belonging to the xanthene group. These dyes are efficient but are hazardous to human and animal health. With the increasing awareness of a green earth, it is advisable to use more of eco-friendly and biodegradable material which can be effectively achieved by the use of natural dyes obtained from plants and other natural sources. Turmeric, available as Curcuma longa (domestic), has long been in use in the subcontinent as a spice and flavoring agent in most food preparations. Its health benefit as a natural antibiotic and anti-inflammatory has been successfully established by several researchers. The intense yellow color imparted by turmeric inspired us to explore its efficacy as a potential alternative for eosin in routine histopathological procedures. Aim: The aim of this was to explore the efficacy of turmeric extract as a stand-alone counterstain for hematoxylin and its comparative assessment with routine H and E staining. Materials and Methods: The rhizomes of C. longa were cut into small pieces, dried and milled. This powder was dissolved into alcohol and centrifuged using a centrifugal machine. The supernatant was then collected with the help of micropipette. This supernatant was used as a counterstain for hematoxylin. Results: The data were analyzed using Mann–Whitney U test with Statistical Package for the Social Sciences version 15.0 (SPSS Inc.,). The P value obtained was statistically insignificant (P > 0.05). Conclusion: Although eosin is the most efficient counterstain for hematoxylin, turmeric can also be used as an alternative for eosin. PMID:29391705

Glycol Methacrylate Embedding for the Histochemical Study of the Gastrointestinal Tract of Dogs Naturally Infected with Leishmania Infantum

PubMed Central

Pinto, A.J.W.; de Amorim, I.F.G.; Pinheiro, L.J.; Madeira, I.M.V.M.; Souza, C.C.; Chiarini-Garcia, H.; Caliari, M.V.

2015-01-01

In canine visceral leishmaniasis a diffuse chronic inflammatory exudate and an intense parasite load throughout the gastrointestinal tract (GIT) has been previously reported. However, these studies did not allow a properly description of canine cellular morphology details. The aim of our study was to better characterize these cells in carrying out a qualitative and quantitative histological study in the gastrointestinal tract of dogs naturally infected with Leishmania infantum by examining gut tissues embedded in glycol methacrylate. Twelve infected adult dogs were classified in asymptomatic and symptomatic. Five uninfected dogs were used as controls. After necropsy, three samples of each gut segment, including oesophagus, stomach, duodenum, jejunum, ileum, cecum, colon, and rectum were collected and fixed in Carnoy’s solution for glycol methacrylate protocols. Sections were stained with hematoxylin-eosin, toluidine blue borate, and periodic acid-Schiff stain. Leishmania amastigotes were detected by immunohistochemistry employed in both glycol methacrylate and paraffin embedded tissues. The quantitative histological analysis showed higher numbers of plasma cells, lymphocytes and macrophages in lamina propria of all segments of GIT of infected dogs compared with controls. The parasite load was more intense and cecum and colon, independently of the clinical status of these dogs. Importantly, glycol methacrylate embedded tissue stained with toluidine blue borate clearly revealed mast cell morphology, even after mast cell degranulation. Infected dogs showed lower numbers of mast cells in all gut segments than controls. Despite the glycol methacrylate (GMA) protocol requires more attention and care than the conventional paraffin processing, this embedding procedure proved to be especially suitable for the present histological study, where it allowed to preserve and observe cell morphology in fine detail. PMID:26708180

Eosin fluorescence: A diagnostic tool for quantification of liver injury.

PubMed

Ali, Hamid; Ali, Safdar; Mazhar, Maryam; Ali, Amjad; Jahan, Azra; Ali, Abid

2017-09-01

Hepatitis is one of the most common life threatening diseases. The diagnosis is mainly based on biochemical analysis such as liver function test. However, histopathological evaluation of liver serves far better for more accurate final diagnosis. The goal of our study was to evaluate the eosin fluorescence pattern in CCl 4 -induced liver injury model compared with normal and different treatment groups. For this purpose, liver tissues were stained with H/E and examined under bright field microscope but the fluorescence microscopy of H/E stained slides provided an interesting fluorescence pattern and was quite helpful in identifying different structures. Interesting fluorescence patterns were obtained with FITC, Texas Red and Dual channel filter cubes that were quite helpful in identifying different morphological features of the liver. During the course of hepatic injury, liver cells undergo necrosis, apoptosis and overall cellular microenvironment is altered due to the modification of proteins and other intracellular molecules. Intensified eosin fluorescence was observed around the central vein of injured liver compared to normal indicating enhanced binding of eosin to the more exposed amino acid residues. To conclude, eosin fluorescence pattern varies with the health status of a tissue and can be used further for the diagnosis and quantification of severity of various liver diseases. Copyright © 2017 Elsevier B.V. All rights reserved.

Immunohistochemical assessment of NY-ESO-1 expression in esophageal adenocarcinoma resection specimens

PubMed Central

Hayes, Stephen J; Hng, Keng Ngee; Clark, Peter; Thistlethwaite, Fiona; Hawkins, Robert E; Ang, Yeng

2014-01-01

AIM: To assess NY-ESO-1 expression in a cohort of esophageal adenocarcinomas. METHODS: A retrospective search of our tissue archive for esophageal resection specimens containing esophageal adenocarcinoma was performed, for cases which had previously been reported for diagnostic purposes, using the systematised nomenclature of human and veterinary medicine coding system. Original haematoxylin and eosin stained sections were reviewed, using light microscopy, to confirm classification and tumour differentiation. A total of 27 adenocarcinoma resection specimens were then assessed using immunohistochemistry for NY-ESO-1 expression: 4 well differentiated, 14 moderately differentiated, 4 moderate-poorly differentiated, and 5 poorly differentiated. RESULTS: Four out of a total of 27 cases of esophageal adenocarcinoma examined (15%) displayed diffuse cytoplasmic and nuclear expression for NY-ESO-1. They displayed a heterogeneous and mosaic-type pattern of diffuse staining. Diffuse cytoplasmic staining was not identified in any of these structures: stroma, normal squamous epithelium, normal submucosal gland and duct, Barrett’s esophagus (goblet cell), Barrett’s esophagus (non-goblet cell) and high grade glandular dysplasia. All adenocarcinomas showed an unexpected dot-type pattern of staining at nuclear, paranuclear and cytoplasmic locations. Similar dot-type staining, with varying frequency and size of dots, was observed on examination of Barrett’s metaplasia, esophageal submucosal gland acini and the large bowel negative control, predominantly at the crypt base. Furthermore, a prominent pattern of apical (luminal) cytoplasmic dot-type staining was observed in some cases of Barrett’s metaplasia and also adenocarcinoma. A further morphological finding of interest was noted on examination of haematoxylin and eosin stained sections, as aggregates of lymphocytes were consistently noted to surround submucosal glands. CONCLUSION: We have demonstrated for the first time NY-ESO-1 expression by esophageal adenocarcinomas, Barrett’s metaplasia and normal tissues other than germ cells. PMID:24744590

Primary Cicatricial Alopecias: A Review of Histopathologic Findings in 38 Patients from a Clinical University Hospital in Sao Paulo, Brazil

PubMed Central

Moure, Emanuella Rosyane Duarte; Romiti, Ricardo; Machado, Maria Cecília da Matta Rivitti; Valente, Neusa Yuriko Sakai

2008-01-01

BACKGROUND Scarring alopecias are classified into primary and secondary types according to the initial site of inflammation. In primary scarring alopecias, the hair follicle is the main target of destruction; the term secondary cicatricial alopecia implies that follicular destruction is not the primary pathologic event. AIMS To review the histopathologic diagnoses of cases of cicatricial alopecia in order to classify them according to the North American Hair Research Society. PATIENTS AND METHODS Patients with biopsy specimens diagnosed as cicatricial alopecia seen from 2000 to 2005 at the Dermatologic Department of Hospital das Clinicas, São Paulo University Medical School had hematoxylin and eosin, Periodic acid-Schiff and Weigert stained slides reevaluated and sub-typed into different primary cicatricial alopecias. RESULTS Thirty-eight cases of primary cicatricial alopecias were reclassified as: chronic cutaneous lupus (17), lichen planus pilaris (4), pseudopelade of Brocq (12), folliculitis decalvans (3), dissecting folliculitis (1), and non-specific scarring alopecia (1). In our cases, the methods employed allowed an accurate diagnosis in 12 of 13 cases (92.3%) previously classified as non-specific cicatricial alopecias. CONCLUSIONS Even in the late, pauci or non-inflammatory phases, an approach with systematic evaluation of a constellation of criteria in routine hematoxylin and eosin stain, Periodic acid-Schiff and Weigert stain allowed for a more accurate diagnosis of cicatricial alopecias. PMID:19060995

Interfacial Polymerization for Colorimetric Labeling of Protein Expression in Cells

PubMed Central

Lilly, Jacob L.; Sheldon, Phillip R.; Hoversten, Liv J.; Romero, Gabriela; Balasubramaniam, Vivek; Berron, Brad J.

2014-01-01

Determining the location of rare proteins in cells typically requires the use of on-sample amplification. Antibody based recognition and enzymatic amplification is used to produce large amounts of visible label at the site of protein expression, but these techniques suffer from the presence of nonspecific reactivity in the biological sample and from poor spatial control over the label. Polymerization based amplification is a recently developed alternative means of creating an on-sample amplification for fluorescence applications, while not suffering from endogenous labels or loss of signal localization. This manuscript builds upon polymerization based amplification by developing a stable, archivable, and colorimetric mode of amplification termed Polymer Dye Labeling. The basic concept involves an interfacial polymer grown at the site of protein expression and subsequent staining of this polymer with an appropriate dye. The dyes Evans Blue and eosin were initially investigated for colorimetric response in a microarray setting, where both specifically stained polymer films on glass. The process was translated to the staining of protein expression in human dermal fibroblast cells, and Polymer Dye Labeling was specific to regions consistent with desired protein expression. The labeling is stable for over 200 days in ambient conditions and is also compatible with modern mounting medium. PMID:25536421

Digital simulation of staining in histopathology multispectral images: enhancement and linear transformation of spectral transmittance.

PubMed

Bautista, Pinky A; Yagi, Yukako

2012-05-01

Hematoxylin and eosin (H&E) stain is currently the most popular for routine histopathology staining. Special and/or immuno-histochemical (IHC) staining is often requested to further corroborate the initial diagnosis on H&E stained tissue sections. Digital simulation of staining (or digital staining) can be a very valuable tool to produce the desired stained images from the H&E stained tissue sections instantaneously. We present an approach to digital staining of histopathology multispectral images by combining the effects of spectral enhancement and spectral transformation. Spectral enhancement is accomplished by shifting the N-band original spectrum of the multispectral pixel with the weighted difference between the pixel's original and estimated spectrum; the spectrum is estimated using M < N principal component (PC) vectors. The pixel's enhanced spectrum is transformed to the spectral configuration associated to its reaction to a specific stain by utilizing an N × N transformation matrix, which is derived through application of least mean squares method to the enhanced and target spectral transmittance samples of the different tissue components found in the image. Results of our experiments on the digital conversion of an H&E stained multispectral image to its Masson's trichrome stained equivalent show the viability of the method.

High-resolution, 2- and 3-dimensional imaging of uncut, unembedded tissue biopsy samples.

PubMed

Torres, Richard; Vesuna, Sam; Levene, Michael J

2014-03-01

Despite continuing advances in tissue processing automation, traditional embedding, cutting, and staining methods limit our ability for rapid, comprehensive visual examination. These limitations are particularly relevant to biopsies for which immediate therapeutic decisions are most necessary, faster feedback to the patient is desired, and preservation of tissue for ancillary studies is most important. The recent development of improved tissue clearing techniques has made it possible to consider use of multiphoton microscopy (MPM) tools in clinical settings, which could address difficulties of established methods. To demonstrate the potential of MPM of cleared tissue for the evaluation of unembedded and uncut pathology samples. Human prostate, liver, breast, and kidney specimens were fixed and dehydrated by using traditional histologic techniques, with or without incorporation of nucleic acid fluorescent stains into dehydration steps. A benzyl alcohol/benzyl benzoate clearing protocol was substituted for xylene. Multiphoton microscopy was performed on a home-built system. Excellent morphologic detail was achievable with MPM at depths greater than 500 μm. Pseudocoloring produced images analogous to hematoxylin-eosin-stained images. Concurrent second-harmonic generation detection allowed mapping of collagen. Subsequent traditional section staining with hematoxylin-eosin did not reveal any detrimental morphologic effects. Sample immunostains on renal tissue showed preservation of normal reactivity. Complete reconstructions of 1-mm cubic samples elucidated 3-dimensional architectural organization. Multiphoton microscopy on cleared, unembedded, uncut biopsy specimens shows potential as a practical clinical tool with significant advantages over traditional histology while maintaining compatibility with gold standard techniques. Further investigation to address remaining implementation barriers is warranted.

A Novel Contrast Stain for the Rapid Diagnosis of Pityriasis Versicolor: A Comparison of Chicago Sky Blue 6B Stain, Potassium Hydroxide Mount and Culture

PubMed Central

Lodha, Nikita; Poojary, Shital Amin

2015-01-01

Background: The mycological study of pityriasis versicolor is usually done by potassium hydroxide (KOH) mount and culture. However, KOH mount lacks a color contrast and requires a trained eye to interpret, while culture is difficult to perform, time consuming and has low sensitivity. Chicago Sky Blue 6B (CSB) is a new contrast stain that highlights the fungal hyphae and spores, blue against a purplish background. Aims and Objectives: This study was done to compare the utility of a novel contrast stain (CSB stain) with KOH mount and culture. Materials and Methods: Skin scrapings from the lesions of 100 clinically diagnosed cases of P. versicolor were subjected to (1) KOH mount and CSB stain for direct microscopic examination and (2) culture using Sabouraud's dextrose agar. The statistical analysis of CSB stain and culture was done using KOH mount as the reference method, as it is the most commonly performed and practical diagnostic test available for P. versicolor. An interrater reliability analysis using the Cohen's Kappa statistic was performed to determine consistency (agreement) among the different modalities. Observations and Results: Direct microscopy with CSB stain, KOH mount and mycological culture showed positive results in 98 (98%), 92 (92%) and 56 (56%) patients, respectively. Using KOH mount as the reference method, CSB stain had a sensitivity of 100% which was significantly higher than culture (60.9%). Statistically significant fair agreement was found between CSB stain and KOH mount (94% with κ=0.38, P < 0.001). Negligible agreement was found between CSB stain and culture (66%, κ=0.199, P = 0.001) as well as between KOH mount and culture (64%, κ=0.051, P = 0.107). Conclusion: CSB staining of skin scrapings is the most sensitive method for the diagnosis of pityriasis versicolor. Due to the distinct contrast provided by CSB, it is easy to perform, rapid and qualitatively superior to KOH mount. PMID:26288400

Identification of muscle necrosis in the mdx mouse model of Duchenne muscular dystrophy using three-dimensional optical coherence tomography

NASA Astrophysics Data System (ADS)

Klyen, Blake R.; Shavlakadze, Thea; Radley-Crabb, Hannah G.; Grounds, Miranda D.; Sampson, David D.

2011-07-01

Three-dimensional optical coherence tomography (3D-OCT) was used to image the structure and pathology of skeletal muscle tissue from the treadmill-exercised mdx mouse model of human Duchenne muscular dystrophy. Optical coherence tomography (OCT) images of excised muscle samples were compared with co-registered hematoxylin and eosin-stained and Evans blue dye fluorescence histology. We show, for the first time, structural 3D-OCT images of skeletal muscle dystropathology well correlated with co-located histology. OCT could identify morphological features of interest and necrotic lesions within the muscle tissue samples based on intrinsic optical contrast. These findings demonstrate the utility of 3D-OCT for the evaluation of small-animal skeletal muscle morphology and pathology, particularly for studies of mouse models of muscular dystrophy.

Isolate resistance of Blastocystis hominis to metronidazole.

PubMed

Haresh, K; Suresh, K; Khairul Anus, A; Saminathan, S

1999-04-01

Isolates of Blastocystis hominis from infected immigrant workers from Indonesia, Bangladesh and infected individuals from Singapore and Malaysia were assessed for growth pattern and degree of resistance to different concentrations of metronidazole. Viability of the cells was assessed using eosin-brillian cresyl blue which stained viable cells green and nonviable cells red. The Bangladeshi and Singaporean isolates were nonviable even at the lowest concentration of 0.01 mg/ml, whereas 40% of the initial inoculum of parasites from the Indonesian isolate at day one were still viable in cultures with 1.0 mg/ml metronidazole. The study shows that isolates of B. hominis of different geographical origin have different levels of resistance to metronidazole. The search for more effective drugs to eliminate th parasite appears inevitable, especially since surviving parasites from metronidazole cultures show greater ability to multiply in subcultures than controls.

3D imaging of hematoxylin and eosin stained thick tissues with a sub-femtoliter resolution by using Cr:forsterite-laser-based nonlinear microscopy (Conference Presentation)

NASA Astrophysics Data System (ADS)

Kao, Chien-Ting; Wei, Ming-Liang; Liao, Yi-Hua; Sun, Chi-Kuang

2017-02-01

Intraoperative assessment of excision tissues during cancer surgery is clinically important. The assessment is used to be guided by the examination for residual tumor with frozen pathology, while it is time consuming for preparation and is with low accuracy for diagnosis. Recently, reflection confocal microscopy (RCM) and nonlinear microscopy (NLM) were demonstrated to be promising methods for surgical border assessment. Intraoperative RCM imaging may enable detection of residual tumor directly on skin cancers patients during Mohs surgery. The assessment of benign and malignant breast pathologies in fresh surgical specimens was demonstrated by NLM. Without using hematoxylin and eosin (H and E) that are common dyes for histopathological diagnosis, RCM was proposed to image in vivo by using aluminum chloride for nuclear contrast on surgical wounds directly, while NLM was proposed to detect two photon fluorescence nuclear contrast from acrdine orange staining. In this paper, we propose and demonstrate 3D imaging of H and E stained thick tissues with a sub-femtoliter resolution by using Cr:forsterite-laser-based NLM. With a 1260 nm femtosecond Cr:forsterite laser as the excitation source, the hematoxylin will strongly enhance the third-harmonic generation (THG) signals, while eosin will illuminate strong fluorescence under three photon absorption. Compared with previous works, the 1260 nm excitation light provide high penetration and low photodamage to the exercised tissues so that the possibility to perform other follow-up examination will be preserved. The THG and three-photon process provides high nonlinearity so that the super resolution in 3D is now possible. The staining and the contrast of the imaging is also fully compatible with the current clinical standard on frozen pathology thus facilitate the rapid intraoperative assessment of excision tissues. This work is sponsored by National Health Research Institutes and supported by National Taiwan University Hospital.

Evaluation of efficacy of 1% Crystal Violet & Nuclear Fast Red stain compared to Haematoxyline & Eosin stain for assessing mitotic figures in oral premalignant and malignant lesions.

PubMed

Motiwale, Gauri; Jaiswal, Shradha; Vikey, Ashok; Motiwale, Tejas; Bagulkar, Bhupesh; Bhat, Atul; Kapoor, Prakhar

2016-07-01

Various chromosomal arrangements in cells undergoing division are referred to as Mitotic figure (MF). The abnormal excess of mitotic figures is commonly seen in oral epithelial dysplasia (ED) and oral squamous cell carcinoma (OSCC). In present study, we compared the number of mitotic figures in normal oral mucosa, epithelial dysplasia & OSCC sections with haematoxyline & eosine (H&E) and 1%Crystal Violet & Nuclear Fast Red (CV&NFR) stain, also the efficacy of the CV&NFR stain as compared to H & E stain. We investigated the correlation between the number of mitotic figures & grades of OSCC. Study sample comprised of two serial sections of archival blocks of normal oral mucosa & diagnosed cases of epithelial dysplasia & OSCC. One slide stained with H& E & the other one with 1% CV & NFR. Mitotic figures were counted with the grid eyepiece. There was significant increase in number of MFs in oral ED and OSCC in comparison with normal oral mucosa. There was a highly significant increase in number of MFs in CV&NFR stained tissue sections when compared with H & E stain. Metaphase is the most commonly observed phase of mitosis. In summary, our study proposes the use of Crystal violet & Nuclear fast red stain as a selective stain for better contrast & easy identification MFs. © 2016 Old City Publishing, Inc.

Multiphoton microscopic imaging of histological sections without hematoxylin and eosin staining differentiates carcinoma in situ lesion from normal oesophagus

NASA Astrophysics Data System (ADS)

Chen, Jianxin; Xu, Jian; Kang, Deyong; Xu, Meifang; Zhuo, Shuangmu; Zhu, Xiaoqin; Jiang, Xingshan

2013-10-01

Multiphoton microscopy (MPM) has become a powerful, important tool for tissues imaging at the molecular level. In this paper, this technique was extended to histological investigations, differentiating carcinoma in situ (CIS) lesion from normal oesophagus by imaging histological sections without hematoxylin and eosin (H&E) staining. The results show that the histology procedures of dehydration, paraffin embedding, and de-paraffinizing highlighted two photon excited fluorescence of cytoplasm and nucleolus of epithelial cell and collagen in stroma. MPM has the ability to identify the characteristics of CIS lesion including changes of squamous cells and full epithelium, identification of basement membrane, especially prominent nucleolus. The studies described here show that MPM has the potential for future retrospective studies of tumor staging by employing on histological section specimens without H&E staining.

The Bacterial Endospore Stain on Schaeffer Fulton using Variation of Methylene Blue Solution

NASA Astrophysics Data System (ADS)

Oktari, A.; Supriatin, Y.; Kamal, M.; Syafrullah, H.

2017-02-01

Endospores staining is the type of staining to recognize the presence spore in bacterial vegetative cells. The bacterial endospores need a staining which can penetrate wall thickness of spore bacteria. A method of endospores staining is Schaeffer Fulton method that used Malachite Green. It is an alkaline substance staining that can staining the spore bacteria. In this research, it have found the alternative staining that can replace Malachite Green solution in spore bacterial stain. The alternative staining used is Methylene Blue solution (0,5 %, 0,7%, and 1% concentration) with pH variation (10, 11, and 12), and varyous heating time (3, 4, and 5 minutes). The all treatments staining have been effect on bacterial spores staining results. The warming time greatly affect the dye to penetrate the walls of bacterial spores, this can be seen in the results with various concentration at pH 10, indicates that the not long warm-up time 3 and 4 minutes, bacterial spores are not stained, while in the longer heating time is 5 minutes bacterial spores stained. This is caused because the longer heating time can make the pores of spore wall is open so that can facilitate the dye to get into the bacterial spores.

A field method for making a quantitative estimate of altered tuff in sandstone

USGS Publications Warehouse

Cadigan, R.A.

1954-01-01

The use of benzidine to identify altered tuff in sandstone is practical for field or field laboratory studies associated with stratigraphic correlations, mineral deposit investigations, or paleogeographic interpretations. The method is based on the ability of saturated benzidine (C12H12N2) solution to produce a blue stain on montmorillonite-bearing tuff grains. The method is substantiated by the results of microscopic, X-ray spectrometer, and spectrographic tests which lead to the conclusion that: (1) the benzidine stain test differentiates grains of different composition, (2) the white or gray grains which are stained a uniform blue color are fragments of altered tuff, and (3) white or gray grains which stain in a few small spots are probably silicified tuff. The amount of sand grains taken from a hand specimen or an outcrop which will be held by a penny is spread out on a nonabsorbent white surface and soaked with benzidine for 5 minutes. The approximate number blue grains and the average grain size are used in a chart to determine a reference number which measures relative order of abundance. The chart, based on a volume relationship, corrects for the variation in the number of grains in the sample as the grain size varies. Practical use of the method depends on a knowledge of several precautionary measures as well as an understanding of the limitations of benzidine staining tests.

Angiogenesis and Tissue Engineering Research

DTIC Science & Technology

2010-08-01

grafts were stained with hematoxylin and eosin for general morphology, Masson trichrome stain for collagen organization, and von Geison for elastin...autologous semilunar heart valve. Circulation 111, 2783, 2005. 21. Sodian, R., Hoerstrup, S.P., Sperling , J.S., Daebritz, S., Mar- tin, D.P., Moran, A.M

Quirks of dye nomenclature. 8. Methylene blue, azure and violet.

PubMed

Cooksey, C J

2017-01-01

Methylene blue was synthesized in 1877 and soon found application in medicine, staining for microscopy and as an industrial dye and pigment. An enormous literature has accumulated since its introduction. Early on, it was known that methylene blue could be degraded easily by demethylation; consequently, the purity of commercial samples often was low. Therefore, demethylation products, such as azures and methylene violet, also are considered here. The names and identity of the components, their varying modes of manufacture, analytical methods and their contribution to biological staining are discussed.

A time-dependent degeneration manner of condyle in rat CFA-induced inflamed TMJ.

PubMed

Xu, Liqin; Guo, Huilin; Li, Cheng; Xu, Jie; Fang, Wei; Long, Xing

2016-01-01

Temporomandibular joint (TMJ) inflammation is a potential risk factor of osteoarthritis (OA) but the detailed degenerative changes in the inflamed TMJ remain unclear. In this study, we evaluated the changes of condylar cartilage and subchondral bone in rat inflamed TMJ induced by Freund's complete adjuvant (CFA). Articular cavity was injected with CFA and the TMJ samples were collected 1, 2, 3, and 4-week post-injection. Hematoxylin & Eosin (H&E) staining, toluidine blue (TB) staining, Safranin O (S.O) staining, Masson trichrome staining and micro-CT were used to assess TMJ degeneration during inflammation. Osteoclast and osteoblast activities were analyzed by tartrate-resistant acid phosphatase (TRAP) staining and osteocalcin (OCN) immunohistochemistry staining respectively. The expression of receptor activator of NF-kB ligand (RANKL) and osteoprotegerin (OPG) in condylar cartilage and subchondral bone was also evaluated through immunohistochemistry and RANKL/OPG ratio was evaluated. Reduced cartilage thickness, decreased number of chondrocytes, and down-regulated proteoglycan expression were observed in the condylar cartilage in the inflamed TMJ. Enhanced osteoclast activity, and expanded bone marrow cavity were reached the peak in the 2-week after CFA-injection. Meanwhile the RANKL/OPG ratio in the cartilage and subchondral bone also increased in the 2-week CFA-injection. Immature, unmineralized new bones with irregular trabecular bone structure, atypical condylar shape, up-regulated OCN expression, and decreased bone mineral density (BMD) were found in the inflamed TMJ. The time-dependent degeneration manner of TMJ cartilage and subchondral bone was found in CFA-induced arthritis rat model. The degeneration in the TMJ with inflammation might be a risk factor and should be concerned.

Laparoscopic sentinel lymph node procedure using a combination of patent blue and radioisotope in women with cervical carcinoma.

PubMed

Barranger, Emmanuel; Grahek, Dany; Cortez, Annie; Talbot, Jean Noel; Uzan, Serge; Darai, Emile

2003-06-15

The authors evaluated the feasibility of a laparoscopic sentinel lymph node (SN) procedure with combined radioisotopic and patent blue labeling in patients with cervical carcinoma. Thirteen women (median age, 52.5 years) with cervical carcinoma (Stage Ia2 in 1 patient, Stage Ib1 in 10 patients, Stage Ib2 in 1 patient, and Stage IIa in 1 patient) underwent a laparoscopic SN procedure using an endoscopic gamma probe after both radioactive isotope and patent blue injections. After the procedure, all patients underwent complete laparoscopic pelvic lymphadenectomy and either laparoscopic radical hysterectomy (eight patients) or the Schauta-Amreich operation (five patients). SNs (mean, 1.7 SNs per patient; range, 1-3 SNs per patient) were identified in 12 of 13 patients. A median of 10.5 pelvic lymph nodes per patient (range, 4-17 pelvic lymph nodes per patient) were removed. No lymph node involvement was detected in SNs with hematoxylin and eosin staining. Immunohistochemical studies identified four metastatic SNs in two patients, with micrometastases in two SNs from the first patient and isolated tumor cells in two SNs from the second patient. No false-negative SN results were obtained. The results of this study suggest that SN detection with a combination of radiocolloid and patent blue is feasible in patients with cervical carcinoma. The combination of laparoscopy and the SN procedure permitted minimally invasive management of early-stage disease. Copyright 2003 American Cancer Society.

Advantages of Pure Platelet-Rich Plasma Compared with Leukocyte- and Platelet-Rich Plasma in Treating Rabbit Knee Osteoarthritis

PubMed Central

Yin, Wen-Jing; Xu, Hai-Tao; Sheng, Jia-Gen; An, Zhi-Quan; Guo, Shang-Chun; Xie, Xue-Tao; Zhang, Chang-Qing

2016-01-01

Background Concentrated leukocytes in leukocyte- and platelet-rich plasma (L-PRP) may deliver increased levels of pro-inflammatory cytokines to activate the NF-κB signaling pathway, to counter the beneficial effects of growth factors on osteoarthritic cartilage. However, to date no relevant studies have substantiated that in vivo. Material/Methods Autologous L-PRP and pure platelet-rich plasma (P-PRP) were prepared, measured for componential composition, and injected intra-articularly after 4, 5, and 6 weeks post-anterior cruciate ligament transection. Caffeic acid phenethyl ester (CAPE) was injected intraperitoneally to inhibit NF-κB activation. All rabbits were sacrificed after 8 weeks postoperative. Enzyme-linked immunosorbent assays were performed to determine interleukin 1β (IL-1β) and prostaglandin E2 (PGE2) concentrations in the synovial fluid, Indian ink staining was performed for gross morphological assessment, and hematoxylin and eosin staining and toluidine blue staining were performed for histological assessment. Results Compared with L-PRP, P-PRP injections achieved better outcomes regarding the prevention of cartilage destruction, preservation of cartilaginous matrix, and reduction of IL-1β and PGE2 concentrations. CAPE injections reversed the increased IL-1β and PGE2 concentrations in the synovial fluid after L-PRP injections and improved the outcome of L-PRP injections to a level similar to P-PRP injections, while they had no influence on the therapeutic efficacy of P-PRP injections. Conclusions Concentrated leukocytes in L-PRP may release increased levels of pro-inflammatory cytokines to activate the NF-κB signaling pathway, to counter the beneficial effects of growth factors on osteoarthritic cartilage, and finally, result in a inferior efficacy of L-PRP to P-PRP for the treatment of osteoarthritis. PMID:27086145

Advantages of Pure Platelet-Rich Plasma Compared with Leukocyte- and Platelet-Rich Plasma in Treating Rabbit Knee Osteoarthritis.

PubMed

Yin, Wen-Jing; Xu, Hai-Tao; Sheng, Jia-Gen; An, Zhi-Quan; Guo, Shang-Chun; Xie, Xue-Tao; Zhang, Chang-Qing

2016-04-17

BACKGROUND Concentrated leukocytes in leukocyte- and platelet-rich plasma (L-PRP) may deliver increased levels of pro-inflammatory cytokines to activate the NF-κB signaling pathway, to counter the beneficial effects of growth factors on osteoarthritic cartilage. However, to date no relevant studies have substantiated that in vivo. MATERIAL AND METHODS Autologous L-PRP and pure platelet-rich plasma (P-PRP) were prepared, measured for componential composition, and injected intra-articularly after 4, 5, and 6 weeks post-anterior cruciate ligament transection. Caffeic acid phenethyl ester (CAPE) was injected intraperitoneally to inhibit NF-κB activation. All rabbits were sacrificed after 8 weeks postoperative. Enzyme-linked immunosorbent assays were performed to determine interleukin 1β (IL-1β) and prostaglandin E2 (PGE2) concentrations in the synovial fluid, Indian ink staining was performed for gross morphological assessment, and hematoxylin and eosin staining and toluidine blue staining were performed for histological assessment. RESULTS Compared with L-PRP, P-PRP injections achieved better outcomes regarding the prevention of cartilage destruction, preservation of cartilaginous matrix, and reduction of IL-1β and PGE2 concentrations. CAPE injections reversed the increased IL-1β and PGE2 concentrations in the synovial fluid after L-PRP injections and improved the outcome of L-PRP injections to a level similar to P-PRP injections, while they had no influence on the therapeutic efficacy of P-PRP injections. CONCLUSIONS Concentrated leukocytes in L-PRP may release increased levels of pro-inflammatory cytokines to activate the NF-κB signaling pathway, to counter the beneficial effects of growth factors on osteoarthritic cartilage, and finally, result in a inferior efficacy of L-PRP to P-PRP for the treatment of osteoarthritis.

A Novel Contrast Stain for the Rapid Diagnosis of Pityriasis Versicolor: A Comparison of Chicago Sky Blue 6B Stain, Potassium Hydroxide Mount and Culture.

PubMed

Lodha, Nikita; Poojary, Shital Amin

2015-01-01

The mycological study of pityriasis versicolor is usually done by potassium hydroxide (KOH) mount and culture. However, KOH mount lacks a color contrast and requires a trained eye to interpret, while culture is difficult to perform, time consuming and has low sensitivity. Chicago Sky Blue 6B (CSB) is a new contrast stain that highlights the fungal hyphae and spores, blue against a purplish background. This study was done to compare the utility of a novel contrast stain (CSB stain) with KOH mount and culture. Skin scrapings from the lesions of 100 clinically diagnosed cases of P. versicolor were subjected to (1) KOH mount and CSB stain for direct microscopic examination and (2) culture using Sabouraud's dextrose agar. The statistical analysis of CSB stain and culture was done using KOH mount as the reference method, as it is the most commonly performed and practical diagnostic test available for P. versicolor. An interrater reliability analysis using the Cohen's Kappa statistic was performed to determine consistency (agreement) among the different modalities. Direct microscopy with CSB stain, KOH mount and mycological culture showed positive results in 98 (98%), 92 (92%) and 56 (56%) patients, respectively. Using KOH mount as the reference method, CSB stain had a sensitivity of 100% which was significantly higher than culture (60.9%). Statistically significant fair agreement was found between CSB stain and KOH mount (94% with κ=0.38, P < 0.001). Negligible agreement was found between CSB stain and culture (66%, κ=0.199, P = 0.001) as well as between KOH mount and culture (64%, κ=0.051, P = 0.107). CSB staining of skin scrapings is the most sensitive method for the diagnosis of pityriasis versicolor. Due to the distinct contrast provided by CSB, it is easy to perform, rapid and qualitatively superior to KOH mount.

A Versatile Giemsa Protocol for Permanent Nuclear Staining of Fungi

Treesearch

A. Dan Wilson

1992-01-01

A variety of cytological stains and staining procedures including Giemsa-HCL, acetic-or-cein, propionic-carmine, iron haema-toxylin, safranin O, aniline blue or trypan blue, toluidine blue and basic fuchsin or Feulgen stain have been used to investigate the nuclear condition of reproductive and somatic structures of fungi. Fluorescent stains such as acriflavin acridine...

Can artificial tears prevent Acanthamoeba keratitis? An in vitro approach.

PubMed

Magnet, Angela; Gomes, Thiago Santos; Pardinas, Carmen; Garcia de Blas, Natalia; Sadaba, Cruz; Carrillo, Eugenia; Izquierdo, Fernando; Del Castillo, José Manuel Benítez; Hurtado, Carolina; Del Aguila, Carmen; Fenoy, Soledad

2018-01-22

The use of contact lenses has increased in recent years as has the incidence of Dry Eye Syndrome, partly due to their use. Artificial tears are the most common treatment option. Since these changes can facilitate Acanthamoeba infection, the present study has been designed to evaluate the effect of three artificial tears treatments in the viability of Acanthamoeba genotype T4 trophozoites. Optava Fusion™, Oculotect®, and Artelac® Splash were selected due to their formulation. Viability was assessed using two staining methods, Trypan Blue stain and CTC stain at different time intervals (2, 4, 6, 8 and 24 h). Trypan Blue viability was obtained by manual count with light microscopy while the CTC stain was determined using flow cytometry. Trypan Blue staining results demonstrated a decrease in viability for Optava Fusion™ and Artelac® Splash during the first 4 h of incubation. After, this effect seems to lose strength. In the case of Oculotect®, complete cell death was observed after 2 h. Using flow cytometry analysis, Optava Fusion™ and Oculotect® exhibited the same effect observed with Trypan Blue staining. However, Artelac® Splash revealed decreasing cell respiratory activity after four hours, with no damage to the cell membrane. The present study uses, for the first time, CTC stain analyzed by flow cytometry to establish Acanthamoeba viability demonstrating its usefulness and complementarity with the traditional stain, Trypan Blue. Artelac® Splash, with no preservatives, and Optava Fusion TM, with Purite®, have not shown any useful amoebicidal activity. On the contrary, promising results presented by Ocultect®, with BAK, open up a new possibility for Acanthamoeba keratitis prophylaxis and treatment although in vivo studies should be carried out.

Lineage Analysis in Pulmonary Arterial Hypertension

DTIC Science & Technology

2013-06-01

monocrotaline pyrrole . The fate of lacZ-expressing cells will be correlated with immunofluorescent staining of endothelial marker CD31, mesenchymal marker...A) Pilot study: Time course of development of pulmonary hypertension in pneumonectomized mice injected with monocrotaline pyrrole (P/MCTP, n = 4... pyrrole . A, B) Normal muscular pulmonary artery (PA) adjacent to bronchiole (Br) A) hematoxylin and eosin stain (H&E), B) elastin-van Gieson stain (EVG

[A study on pathology and immunoglobulin of orbital tissues from cases with Grave's ophthalmopathy].

PubMed

Zhou, X; Luo, Q; Xia, R

1999-07-01

To investigate pathological changes and the expression, localization of immunoglobulins (IgA & IgE) in orbital tissues from patients with Grave's ophthalmopathy. Orbital tissues were obtained at surgery from 19 patients with Grave's ophthalmopathy and 17 control subjects. Immunohistochemical studies were carried out by using anti-IgA, anti-IgE antibody and a streptavidin-biotin-peroxidase detection system. In addition, hematoxylin-eosin, alcian blue and Masson trichrome stains were employed to demonstrate the pathologic changes in the tissues. 12 of 17 samples from patients exhibited IgA positive staining for the connective tissue associated with the extraocular muscles and orbicularis oculi, however only 2 control muscles reacted minimally. IgE positive material was found in association with the majority of leukocytes and with muscle fibers in 13 patients, but only 4 control subjects displayed mild reactions. Significant expression of IgE was more often demonstrated in patients with eye disease of short course than in those of longer course. These results support the notion that the eye muscle fiber and fibroblast are important targets of the orbital autoimmune reaction in Graves' ophthalmopathy in which IgA and IgE both play important roles.

Interaction of Bordetella bronchiseptica and Its Lipopolysaccharide with In Vitro Culture of Respiratory Nasal Epithelium

PubMed Central

Middleton, Andrew M.; Martínez, Nhora; Romero, Stefany; Iregui, Carlos

2013-01-01

The nasal septa of fetal rabbits at 26 days of gestation were harvested by cesarean section of the does while under anesthesia and then exposed to Bordetella bronchiseptica or its lipopolysaccharide (LPS) for periods of 2 and 4 hours. A total of 240 explants were used. The tissues were examined using the Hematoxylin & Eosin technique. Then, semithin sections (0.5 μm) were stained with toluidine blue and examined with indirect immunoperoxidase (IPI) and lectin histochemistry. The most frequent and statistically significant findings were as follows: (1) cell death and increased goblet cell activity when exposed to bacteria and (2) cell death, cytoplasmic vacuolation and infiltration of polymorphonuclear leukocytes when exposed to LPS. The lesions induced by the bacterium were more severe than with LPS alone, except for the cytoplasmic vacuolation in epithelial cells. IPI stained the ciliated border of the epithelium with the bacterium more intensely, while LPS lectin histochemistry preferentially labeled the cytoplasm of goblet cell. These data indicate that B. bronchiseptica and its LPS may have an affinity for specific glycoproteins that would act as adhesion receptors in both locations. PMID:23555071

Comparative efficacy of cedarwood oil and xylene in hematoxylin and eosin staining procedures: An experimental study.

PubMed

Indu, Sudip; Ramesh, V; Indu, Priyanka Chakravarty; Prashad, Karthikshree V; Premalatha, B; Ramadoss, K

2014-07-01

Xylene is used as a clearing agent in hematoxylin and eosin (H and E) staining of tissue sections in routine histopathology based diagnosis. However, the hazards associated with exposure to xylene are of concern. Numerous solutions mainly essential oils have been evaluated in the past as clearing agents, which can possibly be substituted for xylene during the routine tissue processing. The aim of this study is to compare the efficacy of essential oil (cedarwood oil), as a possible replacement for xylene in H and E staining procedures. The study was carried out in the Department of Oral Pathology and Microbiology. Thirty paraffin blocks of the routine biopsy specimen were retrieved from the department archives. The cedarwood oil was procured from organic and essential oil dealer in the local market. Two to three paraffin sections of four micron thickness were cut from each of the 30 paraffin blocks of processed tissue specimens, were subjected to different clearing agents: Essential oil (8% cedarwood oil) or xylene and stained with H and E stain. The stained sections were scored based on nuclear and cytoplasmic details, clarity and uniformity of staining. Significant correlation was observed between cedarwood oil and xylene in terms of the three staining quality parameters assessed. We conclude that cedarwood oil can be an effective, eco-friendly and safe alternative to xylene as a clearing agent in the histopathological laboratory.

Mast cells in the sheep, hedgehog and rat forebrain

PubMed Central

MICHALOUDI, HELEN C.; PAPADOPOULOS, GEORGIOS C.

1999-01-01

The study was designed to reveal the distribution of various mast cell types in the forebrain of the adult sheep, hedgehog and rat. Based on their histochemical and immunocytochemical characteristics, mast cells were categorised as (1) connective tissue-type mast cells, staining metachromatically purple with the toluidine blue method, or pale red with the Alcian blue/safranin method, (2) mucosal-type or immature mast cells staining blue with the Alcian blue/safranin method and (3) serotonin immunopositive mast cells. All 3 types of brain mast cells in all species studied were located in both white and grey matter, often associated with intraparenchymal blood vessels. Their distribution pattern exhibited interspecies differences, while their number varied considerably not only between species but also between individuals of each species. A distributional left-right asymmetry, with more cells present on the left side, was observed in all species studied but it was most prominent in the sheep brain. In the sheep, mast cells were abundantly distributed in forebrain areas, while in the hedgehog and the rat forebrain, mast cells were less widely distributed and were relatively or substantially fewer in number respectively. A limited number of brain mast cells, in all 3 species, but primarily in the rat, were found to react both immunocytochemically to 5-HT antibody and histochemically with Alcian blue/safranin staining. PMID:10634696

FURTHER STUDIES ON THE INHIBITION OF CYPRIDINA LUMINESCENCE BY LIGHT, WITH SOME OBSERVATIONS ON METHYLENE BLUE

PubMed Central

Harvey, E. Newton

1926-01-01

1. Eosin, erythrosin, rose bengale, cyanosin, acridine, and methylene blue act photodynamically on the luminescence of a Cypridina luciferin-luciferase solution. In presence of these dyes inhibition of luminescence, which without the dye occurs only in blue-violet light, takes place in green, yellow, orange, or red light, depending on the position of the absorption bands of the dye. 2. Inhibition of Cypridina luminescence without photosensitive dye in blue-violet light, or with photosensitive dye in longer wave-lengths, does not occur in absence of oxygen. Light acts by accelerating the oxidation of luciferin without luminescence. Eosin or methylene blue act by making longer wave-lengths effective, but there is no evidence that these dyes become reduced in the process. 3. The luciferin-oxyluciferin system is similar to the methylene white-methylene blue system in many ways but not exactly similar in respect to photochemical change. Oxidation of the dye is favored in acid solution, reduction in alkaline solution. However, oxidation of luciferin is favored in all pH ranges from 4 to 10 but is much more rapid in alkaline solution, either in light or darkness. There is no evidence that reduction of oxyluciferin is favored in alkaline solution. Clark's observation that oxidation (blueing) of methylene white occurs in complete absence of oxygen has been confirmed for acid solutions. I observed no blueing in light in alkaline solution. PMID:19872301

Methylene Blue-Guided Debridement as an Intraoperative Adjunct for the Surgical Treatment of Periprosthetic Joint Infection.

PubMed

Shaw, Jeremy D; Miller, Steve; Plourde, Anna; Shaw, Daniel L; Wustrack, Rosanna; Hansen, Erik N

2017-12-01

Current methods to identify infected tissue in periprosthetic joint infection (PJI) are inadequate. The purpose of this study was (1) to assess methylene blue-guided surgical debridement as a novel technique in PJI using quantitative microbiology and (2) to evaluate clinical success based on eradication of infection and infection-free survival. Sixteen total knee arthroplasty patients meeting Musculoskeletal Infection Society criteria for PJI undergoing the first stage of 2-stage exchange arthroplasty were included in this prospective study. Dilute methylene blue (0.1%) was instilled in the knee before debridement, residual dye was removed, and stained tissue was debrided. Paired tissue samples, stained and unstained, were collected from the femur, tibia, and capsule during debridement. Samples were analyzed by neutrophil count, semiquantitative culture, and quantitative polymerase chain reaction (PCR). Clinical success was a secondary outcome. The mean age was 64.0 ± 6.0 years, and follow-up was 24.4 ± 3.5 months. More bacteria were found in methylene blue-stained vs unstained tissue-based on semiquantitative culture (P = .001). PCR for staphylococcal species showed 9-fold greater bioburden in methylene blue-stained vs unstained tissue (P = .02). Tissue pathology found 53 ± 46 polymorphonuclear leukocytes per high-power field in methylene blue-stained vs 4 ± 13 in unstained tissue (P = .0001). All subjects cleared their primary infection and underwent reimplantation. At mean 2-year follow-up, 25% of patients failed secondary to new infection with a different organism. These results suggest a role for methylene blue in providing a visual index of surgical debridement in the treatment of PJI. Copyright © 2017 Elsevier Inc. All rights reserved.

Combined histochemical staining, RNA amplification, regional, and single cell cDNA analysis within the hippocampus.

PubMed

Ginsberg, Stephen D; Che, Shaoli

2004-08-01

The use of five histochemical stains (cresyl violet, thionin, hematoxylin & eosin, silver stain, and acridine orange) was evaluated in combination with an expression profiling paradigm that included regional and single cell analyses within the hippocampus of post-mortem human brains and adult mice. Adjacent serial sections of human and mouse hippocampus were labeled by histochemistry or neurofilament immunocytochemistry. These tissue sections were used as starting material for regional and single cell microdissection followed by a newly developed RNA amplification procedure (terminal continuation (TC) RNA amplification) and subsequent hybridization to custom-designed cDNA arrays. Results indicated equivalent levels of global hybridization signal intensity and relative expression levels for individual genes for hippocampi stained by cresyl violet, thionin, and hematoxylin & eosin, and neurofilament immunocytochemistry. Moreover, no significant differences existed between the Nissl stains and neurofilament immunocytochemistry for individual CA1 neurons obtained via laser capture microdissection. In contrast, a marked decrement was observed in adjacent hippocampal sections stained for silver stain and acridine orange, both at the level of the regional dissection and at the CA1 neuron population level. Observations made on the cDNA array platform were validated by real-time qPCR using primers directed against beta-actin and glyceraldehyde-3 phosphate dehydrogenase. Thus, this report demonstrated the utility of using specific Nissl stains, but not stains that bind RNA species directly, in both human and mouse brain tissues at the regional and cellular level for state-of-the-art molecular fingerprinting studies.

AgNOR histochemical expression in benign prostatic hyperplasia and prostatic adenocarcinoma

NASA Astrophysics Data System (ADS)

Rita, R.; Delyuzar; Laksmi, L. I.

2018-03-01

Benign prostatic hyperplasia and prostatic adenocarcinoma were common diseases and usually occurred after the 5th decade of life. The problem in diagnosing using Hematoxylin and Eosin staining was how to differentiate whether it is benign or malignant zone. Therefore, proliferating markers, such as AgNOR, could be helping to over this difficulty. A descriptive study using consecutive sampling as the method of sample recruiting was conducted to describe AgNOR histochemical expression in benign prostatic hyperplasia and prostatic adenocarcinoma. AgNOR staining was done in 13 benign prostatic hyperplasia samples and 7 prostatic adenocarcinoma samples, which have been confirmed using p63 immunohistochemical staining before. Benign prostatic hyperplasia usually showed lower AgNOR proliferating activity while all of theprostatic adenocarcinoma (100%) had high AgNOR proliferating activity.

Issues in using whole slide imaging for diagnostic pathology: "routine" stains, immunohistochemistry and predictive markers.

PubMed

Taylor, C R

2014-08-01

The traditional microscope, together with the "routine" hematoxylin and eosin (H & E) stain, remains the "gold standard" for diagnosis of cancer and other diseases; remarkably, it and the majority of associated biological stains are more than 150 years old. Immunohistochemistry has added to the repertoire of "stains" available. Because of the need for specific identification and even measurement of "biomarkers," immunohistochemistry has increased the demand for consistency of performance and interpretation of staining results. Rapid advances in the capabilities of digital imaging hardware and software now offer a realistic route to improved reproducibility, accuracy and quantification by utilizing whole slide digital images for diagnosis, education and research. There also are potential efficiencies in work flow and the promise of powerful new analytical methods; however, there also are challenges with respect to validation of the quality and fidelity of digital images, including the standard H & E stain, so that diagnostic performance by pathologists is not compromised when they rely on whole slide images instead of traditional stained tissues on glass slides.

Intraoperative imprint cytology for evaluation of sentinel lymph nodes from visceral malignancies.

PubMed

Levine, Edward A; Shen, Perry; Shiver, Stephen A; Waters, Gregory; Brant, Andrew; Geisenger, Kim R

2003-01-01

Although originally described for breast cancer and melanoma, sentinel lymph node (SLN) mapping techniques are being investigated in the treatment of visceral malignancies. There is no literature evaluating intraoperative analysis of SLNs from visceral sites. We evaluated the utility of touch preparation intraoperative imprint cytology (IIC) in evaluating SLNs harvested in the setting of visceral malignancy. SLN mapping procedures involving 50 cases of visceral malignancy (37 colon, 12 gastric, and 1 small bowel), from February 1999 through August 2001, were studied. In each case, subserosal injections of isosulfan blue were used to identify the SLN. The SLNs were then sent fresh to the pathology laboratory for evaluation by IIC. A standard lymphadenectomy was performed in all cases. Postoperatively, the SLNs were evaluated by means of using hematoxylin and eosin staining. If these stains were normal, immunohistochemical analyses using carcinoembryonic antigen and cytokeratin were subsequently performed. SLNs were successfully identified in 46 cases (92%), and a total of 95 SLNs were harvested. The average number of SLNs was 1.9 with a range of one to six. More SLNs were found with gastric than with colonic lesions (2.8 vs. 1.8; P=.017). Evaluable IIC in 41 cases revealed metastatic disease in 10 SLNs, representing seven patients. Of the 34 patients with normal IIC, five were found to have positive SLNs on hematoxylin and eosin staining. An additional three patients were found to have positive SLNs only on immunohistochemical analysis. The overall sensitivity and specificity of IIC was 64% and 100%, respectively. This resulted in a positive predictive value of 100% and a negative predictive value of 86%. The use of IIC to evaluate SLNs from visceral malignancies is clearly feasible. When the IIC of the SLN is positive, the surgeon may feel confident that disease is actually present in the SLN. If there is a negative result, the technique may miss disease that is present on subsequent permanent sections. We do not recommend routine use of IIC; however, it may be of use in clinical trials.

Mast cells distribution and variations in epithelium thickness and basement membrane in oral lichen planus lesion and oral lichenoid reaction

PubMed Central

Jahanshahi, Gholamreza; Ghalayani, Parichehr; Maleki, Laleh

2012-01-01

Background: Oral lichen planus (OLP) is a chronic mucocutaneous lesion with unknown etiology. Oral lichenoid lesions (OLL) comprise a family of lesions with different etiologies. Both lesions have similar clinical and histopathologic characteristics although their management is different. Differential diagnosis between OLP and OLL has always been a major challenge. Materials and Methods: In this prospective analytical study, the role of mast cells in pathogenesis of these lesions was investigated by evaluation of 52 patients with clinical and histopathological diagnosis of OLP (26 cases) and OLL (26 cases) based on WHO criteria, and by applying a more accessible staining methods, Hematoxylin and Eosin, toluidine blue (histochemistry) and Periodic Acid Schiff staining. In order to distinguish these two lesions, number of mast cells and thickness of epithelium and basement membrane were measured using light microscopy. Data were analyzed by SPSS software using t-test method (P<0.001). Results: No significant difference was observed between the total numbers of mast cells of two groups (P=0.148), but a statistically significant difference was detected between degranulated mast cells in two groups (P<0.001). A significant difference was also observed between the thickness of epithelium in two groups (P<0.001), although no difference was seen between basement membrane thickness in these lesions. Conclusion: Number of degranulated mast cells in reticular layer of corium in lichenoid lesions was more than that of OLP. This implies that despite the increase in number of these cells, in both groups of diseases, the role of these cells has not been the same in pathogenesis of the diseases. Moreover, the epithelium thickness was lower in lesions of OLP compared to lesions of oral lichenoid, so this parameter may be a useful criterion together with other histopathological and clinical finding to discriminate these lesions. However, discrepancy of basement membrane thickness can not be a reliable criterion. Finally we suggest more accessible staining methods which are reliable for differentiation of these two lesions. PMID:22623935

Corneal edema and permanent blue discoloration of a silicone intraocular lens by methylene blue.

PubMed

Stevens, Scott; Werner, Liliana; Mamalis, Nick

2007-01-01

To report a silicone intraocular lens (IOL) stained blue by inadvertent intraoperative use of methylene blue instead of trypan blue and the results of experimental staining of various lens materials with different concentrations of the same dye. A "blue dye" was used to enhance visualization during capsulorhexis in a patient undergoing phacoemulsification with implantation of a three-piece silicone lens. Postoperatively, the patient presented with corneal edema and a discolored IOL. Various IOL materials were experimentally stained using methylene blue. Sixteen lenses (4 silicone, 4 hydrophobic acrylic, 4 hydrophilic acrylic, and 4 polymethylmethacrylate) were immersed in 0.5 mL of methylene blue at concentrations of 1%, 0.1%, 0.01%, and 0.001%. These lenses were grossly and microscopically evaluated for discoloration 6 and 24 hours after immersion. The corneal edema resolved within 1 month after the initial surgical procedure. After explantation, gross and microscopic analyses of the explanted silicone lens revealed that its surface and internal substance had been permanently stained blue. In the experimental study, all of the lenses except the polymethylmethacrylate lenses were permanently stained by methylene blue. The hydrophilic acrylic lenses showed the most intense blue staining in all dye concentrations. This is the first clinicopathological report of IOL discoloration due to intraocular use of methylene blue. This and other tissue dyes may be commonly found among surgical supplies in the operating room and due diligence is necessary to avoid mistaking these dyes for those commonly used during ocular surgery.

Propidium iodide (PI) stains Nissl bodies and may serve as a quick marker for total neuronal cell count.

PubMed

Niu, Junfei; Li, Chunman; Wu, Haihui; Feng, Xianling; Su, Qingning; Li, Shihe; Zhang, Lihong; Yew, David Tai Wai; Cho, Eric Yu Pang; Sha, Ou

2015-03-01

Propidium iodide (PI) reacts with both DNA and RNA and is a commonly used fluorescent reagent for nucleic acid staining. The aim of the study was to compare the cellular staining patterns of PI with that of Nissl staining in rat nervous tissues and to report a modified staining method that selectively labels Nissl bodies in neurons. Cryosections and paraffin sections of different tissues of normal Sprague-Dawley rats, including trigeminal ganglia, dorsal root ganglia, spinal cord, liver, and small intestine, were stained by either PI or the hematoxylin and eosin method. Some sections were treated with RNase or DNase before the above staining, and some were double stained with PI and a Nissl stain. The sections were observed by light, fluorescence or confocal microscopy. Results showed strong PI signals detected as patterns of granules in the neuronal cytoplasm of all nervous tissues, whereas the staining of neuronal nuclei was weaker. In contrast, nuclei of neuroglial cells were strongly stained by PI, while the cytoplasm was not obviously stained. Pretreatment of the neural tissue with RNase abolished the PI signals. Furthermore, the PI positive granules in neuronal cytoplasm co-localized with Nissl bodies stained by the fluorescent Nissl stain. When the tissue was pretreated with DNase, PI only stained the cytoplasmic granules of neurons, but not that of glial cells. Our results show that PI stains Nissl bodies and may serve as an economical and convenient neuron marker for neuronal cell counting when specific neural markers such as antibodies are not readily available. Copyright © 2015. Published by Elsevier GmbH.

Functional anatomy of the lacrimal gland in African black ostrich Struthio camelus domesticus in the embryonic and postnatal period.

PubMed

Klećkowska-Nawrot, Joanna; Goździewska-Harłajczuk, Karolina; Nowaczyk, Renata; Krasucki, Krzysztof

2015-03-25

The aim of the present study was morphological and histochemical analysis of the lacrimalgland (LG) in African black ostrich Struthio camelus domesticus in the embryonic and postnatalperiod. Studies were conducted on 50 ostriches aged between the 28th day of incubation until7 months old. Tissue sections were stained with haematoxylin and eosin, Azan trichrome,periodic acid-Schiff, Alcian blue pH 2.5, aldehyde fuchsin and Hale's dialysed iron. The LGin ostrich was classified as a tubulo-acinar type. The primordia of the lobes were determinedin the LG structure on the 28th day of incubation, whilst the weakly visible lobes with aciniand tubules were observed on the 40th day of incubation. Morphometric studies of the LGshowed steady growth, characterised by an increase in both length and width. Histometricmeasurements of lobe size showed little difference between the first, second and third agegroups, whilst in the fourth age group a marked increase in size of lobes was observed.The study showed that, apart from morphological changes, during the growth of the LGthe character of acid mucopolysaccharides changed. Sulphated acid mucopolysaccharideswere indicated, particularly with aldehyde fuchsin (AF) staining in the fourth age group.The Hale's dialysed iron (HDI) staining showed a low concentration of carboxylated acidmucopolysaccharides in the first and second age groups and a higher concentration in thethird and fourth age groups. Periodic acid-Schiff staining (PAS)-positive cells were observedin each age group, but only a small number of cells with a weakly PAS-positive reaction weredemonstrated in the first age group.

Background-free, high sensitivity staining of proteins in one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gels using a luminescent ruthenium complex.

PubMed

Berggren, K; Chernokalskaya, E; Steinberg, T H; Kemper, C; Lopez, M F; Diwu, Z; Haugland, R P; Patton, W F

2000-07-01

SYPRO Ruby dye is a permanent stain comprised of ruthenium as part of an organic complex that interacts noncovalently with proteins. SYPRO Ruby Protein Gel Stain provides a sensitive, gentle, fluorescence-based method for detecting proteins in one-dimensional and two-dimensional sodium dodecyl sulfate-polyacrylamide gels. Proteins are fixed, stained from 3h to overnight and then rinsed in deionized water or dilute methanol/acetic acid solution for 30 min. The stain can be visualized using a wide range of excitation sources commonly used in image analysis systems including a 302 nm UV-B transilluminator, 473 nm second harmonic generation (SHG) laser, 488 nm argon-ion laser, 532 nm yttrium-aluminum-garnet (YAG) laser, xenon arc lamp, blue fluorescent light bulb or blue light-emitting diode (LED). The sensitivity of SYPRO Ruby Protein Gel Stain is superior to colloidal Coomassie Brilliant Blue (CBB) stain or monobromobimane labeling and comparable with the highest sensitivity silver or zinc-imidazole staining procedures available. The linear dynamic range of SYPRO Ruby Protein Gel stain extends over three orders of magnitude, which is vastly superior to silver, zinc-imidazole, monobromobimane and CBB stain. The fluorescent stain does not contain superfluous chemicals (formaldehyde, glutaraldehyde, Tween-20) that frequently interfere with peptide identification in mass spectrometry. While peptide mass profiles are severely altered in protein samples prelabeled with monobromobimane, successful identification of proteins by peptide mass profiling using matrix-assisted laser desorption/ionization mass spectrometry was easily performed after protein detection with SYPRO Ruby Protein Gel stain.

A new, fast and semi-automated size determination method (SASDM) for studying multicellular tumor spheroids

PubMed Central

Monazzam, Azita; Razifar, Pasha; Lindhe, Örjan; Josephsson, Raymond; Långström, Bengt; Bergström, Mats

2005-01-01

Background Considering the width and importance of using Multicellular Tumor Spheroids (MTS) in oncology research, size determination of MTSs by an accurate and fast method is essential. In the present study an effective, fast and semi-automated method, SASDM, was developed to determinate the size of MTSs. The method was applied and tested in MTSs of three different cell-lines. Frozen section autoradiography and Hemotoxylin Eosin (H&E) staining was used for further confirmation. Results SASDM was shown to be effective, user-friendly, and time efficient, and to be more precise than the traditional methods and it was applicable for MTSs of different cell-lines. Furthermore, the results of image analysis showed high correspondence to the results of autoradiography and staining. Conclusion The combination of assessment of metabolic condition and image analysis in MTSs provides a good model to evaluate the effect of various anti-cancer treatments. PMID:16283948

The changes in histopathology and mass in hyperbaric oxygen-treated auricular cartilage grafts in a rabbit model.

PubMed

Bilici, Suat; Yiğit, Özgür; Dönmez, Zehra; Huq, Gülben Erdem; Aktaş, Şamil

2015-04-01

The aim of the study is to investigate the histopathologic and cartilage mass changes in hyperbaric oxygen (HBO)-treated auricular cartilage grafts either crushed or fascia wrapped in a rabbit model. This is a prospective, controlled experimental study. Sixteen rabbits were randomly allocated into control (n = 8) and treatment groups (n = 8). Each group was further grouped as crushed cartilage (n = 4) and fascia wrapped crushed cartilage (n = 4). The eight rabbits in the treatment group had HBO once daily for 10 days as total of 10 sessions. The mass of cartilage, cartilage edge layout, structural layout, staining disorders of the chondroid matrix, necrosis, calcification besides bone metaplasia, chronic inflammation in the surrounding tissues, fibrosis, and increased vascularity were evaluated in the hematoxylin and eosin (H&E)-stained sections. Fibrosis in the surrounding tissue and cartilage matrix was evaluated with Masson's trichrome stain. The toluidine blue staining was used to evaluate loss of metachromasia in matrix. The prevalence of glial fibrillary acidic protein (GFAP) staining in chondrocytes was also evaluated. Although the remaining amount of cartilage mass after implantation does not show a significant difference between the control and the study group (p = 0.322, p <0.05).The difference between control and study group in terms of positive staining with GFAP was statistically significant (p = 0.01, p <0.05). Necrosis and loss of matrix metachromasia were significantly low in the study group compared with control group (p = 0.001, p = 0.006, p <0.05). HBO therapy did not have significant effect on the mass of rabbit auricular cartilage graft. HBO therapy significantly reduced loss of metachromasia, necrosis, and GFAP staining in the auricular cartilage grafts of the animal model. Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.

Assessing the Penetrating Abilities of Experimental Preparation with Dental Infiltrant Features Using Optical Microscope: Preliminary Study.

PubMed

Skucha-Nowak, Małgorzata; Machorowska-Pieniążek, Agnieszka; Tanasiewicz, Marta

2016-01-01

The aim of the infiltration technique is to penetrate demineralized enamel with a low viscosity resin. Icon® (DMG) is the first ever and so far the only dental infiltrant. Bacteriostaticity is one of the properties that should be inherent in dental infiltrants, but Icon lacks this feature. The aim of the preliminary study was to properly choose a dye which would allow us to assess the penetrating abilities of our own, experimental preparation with features of a dental infiltrant with bacteriostatic properties and to compare using an optical microscope the depth of infiltration of the designed experimental preparation with the infiltrant available on the market. The preparation is supposed to infiltrate decalcified human enamel and be assessed with an optical microscope. Eosin, neutral fuchsine and methylene blue were added to experimental preparation with dental infiltrant features and to Icon® (DMG) in order to assess the depth of penetration of the experimental solution into the decalcified layers of enamel. The experimental solution mixes well with eosin, neutral fuchsine, and methylene blue. During the preliminary study, the authors concluded that the experimental solution mixes well with methylene blue, neutral fuchsine, and eosin. An addition of eosin to a preparation which infiltrates inner, demineralized enamel layers, facilitates the assessment of such a preparation with an optical microscope. A designed experimental solution with the main ingredients, i.e., 2-hydroxyethyl methacrylate (HEMA) and tetraethylene glycol dimethacrylate (TEGDMA) with a ratio of 75% to 25% penetrates the demineralized (decalcified) inner parts of the enamel and polymerizes when exposed to light. In order to assess the infiltration of the experimental solution into the demineralized enamel layers, it is required to improve the measurement techniques that utilize optical microscopy.

Digital immunohistochemistry platform for the staining variation monitoring based on integration of image and statistical analyses with laboratory information system

PubMed Central

2014-01-01

Background Digital immunohistochemistry (IHC) is one of the most promising applications brought by new generation image analysis (IA). While conventional IHC staining quality is monitored by semi-quantitative visual evaluation of tissue controls, IA may require more sensitive measurement. We designed an automated system to digitally monitor IHC multi-tissue controls, based on SQL-level integration of laboratory information system with image and statistical analysis tools. Methods Consecutive sections of TMA containing 10 cores of breast cancer tissue were used as tissue controls in routine Ki67 IHC testing. Ventana slide label barcode ID was sent to the LIS to register the serial section sequence. The slides were stained and scanned (Aperio ScanScope XT), IA was performed by the Aperio/Leica Colocalization and Genie Classifier/Nuclear algorithms. SQL-based integration ensured automated statistical analysis of the IA data by the SAS Enterprise Guide project. Factor analysis and plot visualizations were performed to explore slide-to-slide variation of the Ki67 IHC staining results in the control tissue. Results Slide-to-slide intra-core IHC staining analysis revealed rather significant variation of the variables reflecting the sample size, while Brown and Blue Intensity were relatively stable. To further investigate this variation, the IA results from the 10 cores were aggregated to minimize tissue-related variance. Factor analysis revealed association between the variables reflecting the sample size detected by IA and Blue Intensity. Since the main feature to be extracted from the tissue controls was staining intensity, we further explored the variation of the intensity variables in the individual cores. MeanBrownBlue Intensity ((Brown+Blue)/2) and DiffBrownBlue Intensity (Brown-Blue) were introduced to better contrast the absolute intensity and the colour balance variation in each core; relevant factor scores were extracted. Finally, tissue-related factors of IHC staining variance were explored in the individual tissue cores. Conclusions Our solution enabled to monitor staining of IHC multi-tissue controls by the means of IA, followed by automated statistical analysis, integrated into the laboratory workflow. We found that, even in consecutive serial tissue sections, tissue-related factors affected the IHC IA results; meanwhile, less intense blue counterstain was associated with less amount of tissue, detected by the IA tools. PMID:25565007

Detection of the multiphoton signals in stained tissue using nonlinear optical microscopy

NASA Astrophysics Data System (ADS)

Zeng, Yaping; Xu, Jian; Kang, Deyong; Lin, Jiangbo; Chen, Jianxin

2016-10-01

Multiphoton microscopy (MPM) based on two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) imaging, has become a powerful, important tool for tissue imaging at the molecular level. Recently, MPM is also used to image hematoxylin and eosin (H and E)-stained sections in cancer diagnostics. However, several studies have showed that the MPM images of tissue stained with H and E are significantly different from unstained tissue sections. Our aim was to detect of the multiphoton signals in stained tissue by using MPM. In this paper, MPM was used to image histological sections of esophageal invasive carcinoma tissues stained with H, E, H and E and fresh tissue. To detect of the multiphoton signals in stained tissue, the emission spectroscopic of tissue stained with H, E, H and E were obtained. For comparison, the fresh tissues were also investigated. Our results showed that the tissue stained with H, E, H and E could be detected by their TPEF signals. While the tissue stained with H and fresh tissue could be detected by their TPEF and SHG signals. In this work, we detect of the multiphoton signals in stained tissue. These findings will be useful for choosing suitable staining method so to improve the quality of MPM imaging in the future.

Novel methylene blue staining technique for localizing small esophageal leiomyomas during thoracoscopic enucleation.

PubMed

Zhang, Z; Ai, B; Liao, Y; Liu, L; Liu, M

2016-11-01

The treatment of choice for leiomyoma, the most common benign esophageal tumor, is thoracoscopic enucleation. One of the most difficult aspects of thoracoscopic enucleation is the precise localization of small tumors (≤1.5 cm) and tumors without external protrusion. No simple, feasible solutions to this problem are available. We developed a novel methylene blue staining technique to localize small esophageal leiomyomas and evaluated the feasibility of our technique. Between January 2013 and July 2014, eight patients with small esophageal leiomyomas (≤1.5 cm) underwent thoracoscopic enucleation in Tongji Hospital. Preoperative endoscopic ultrasonography was performed in all patients. The leiomyomas were located in the middle (n = 5) and lower (n = 3) thirds of the esophagus. We preoperatively injected 0.5-1.0 mL methylene blue in the submucosa adjacent to the tumors under standard gastroscope guidance. The entire staining process took about 10 minutes. Staining was successful in all patients. The unstained tumor was exposed after the blue-stained mediastinal pleura, and overlying muscle were incised longitudinally. All procedures were successfully completed without conversion to open surgery. No abnormalities were detected in the esophageal mucosa. The median operating time was 60 minutes (range, 40-90 minutes). Postoperative histopathology confirmed leiomyoma in all patients. The median postoperative hospital stay was 6 days (range, 5-7 days). No major complications, such as esophageal leakage or esophageal diverticulum, occurred. Endoscopic methylene blue staining is safe and feasible for localizing small esophageal leiomyomas during thoracoscopic enucleation. This method will enable precise and easy enucleation. © 2015 International Society for Diseases of the Esophagus.

Inadvertent trypan blue posterior capsule staining during cataract surgery.

PubMed

Burkholder, Bryn M; Srikumaran, Divya; Nanji, Afshan; Lee, Bryan; Weinberg, Robert S

2013-04-01

To report 5 cases of inadvertent posterior capsule staining with trypan blue during phacoemulsification. Retrospective, observational case series. Five cases of posterior capsule staining with trypan blue were identified from cataract surgeries performed at an academic institution. All 5 eyes underwent phacoemulsification with use of iris retractors. The surgical videos from each case were reviewed to better understand the mechanisms and risk factors for posterior capsule staining with trypan blue and techniques to avoid this complication. No eyes had clinical evidence of zonular pathology during their preoperative examination. Only 1 patient reported a possible childhood history of trauma to both eyes. One eye had uveitis, requiring posterior synechialysis. All 5 cases involved the use of iris retractors. No other intraoperative complications occurred, and the intraocular lens was successfully placed in the capsular bag in all cases. All eyes had resolution of posterior capsule staining by postoperative day 8. Inadvertent posterior capsule staining with trypan blue can occur in eyes that appear structurally normal. The use of iris retractors may facilitate posterior capsule staining by allowing the posterior flow of trypan blue under the iris and through the zonules to the posterior capsule. Surgeons should consider techniques to minimize the risk of posterior capsule staining, particularly in cases involving the use of iris retractors. Copyright © 2013 Elsevier Inc. All rights reserved.

Assessment of oral cytological features in smokers and nonsmokers after application of toluidine blue.

PubMed

Sharbatdaran, Majid; Abbaszadeh, Hamid; Siadati, Sepideh; Ranaee, Mohammad; Hajian-Tilaki, Karimollah; Rajabi-Moghaddam, Mahdieh

2017-06-01

Smoking is the most important etiologic factor of oral cancer. Exfoliative cytology is the best method for early detection of oral cancer. Toluidine blue staining is used for detection of oral premalignant and malignant lesions. The aim of this study was to enhance the accuracy of oral exfoliative cytology in evaluating dysplastic features using toluidine blue staining. This clinical trials study was performed on 60 male smokers and nonsmokers without clinically oral lesion. Oral exfoliative cytological smears were prepared before and after application of toluidine blue and stained with Papanicolaou and evaluated under light microscope. Cytological features such as cellular clumping nuclear-to-cytoplasmic ratio, cellular and nuclear pleomorphism, micronuclei, binucleation, presence of bacterial colonies, and keratin flakes were assessed and compared before and after application of toluidine blue. Results showed that cellular clumping and micronuclei were significantly decreased after application of toluidine blue and conversely cellular and nuclear pleomorphisms were significantly increased. Frequency of micronuclei and binucleation were greater in smokers than nonsmokers which were insignificant. Cellular and nuclear pleomorphisms were significantly higher in smokers than nonsmokers after application of toluidine blue. Toluidine blue improved cellular, nuclear, and structural features of oral cytological smears and filtered false-positive or false-negative results. Thus, application of toluidine blue in combination with oral exfoliative cytology for early detection of oral cancer is recommended. Diagn. Cytopathol. 2017;45:513-519. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

In vivo biocompatibility of new nano-calcium-deficient hydroxyapatite/poly-amino acid complex biomaterials.

PubMed

Dai, Zhenyu; Li, Yue; Lu, Weizhong; Jiang, Dianming; Li, Hong; Yan, Yonggang; Lv, Guoyu; Yang, Aiping

2015-01-01

To evaluate the compatibility of novel nano-calcium-deficient hydroxyapatite/poly-amino acid (n-CDHA/PAA) complex biomaterials with muscle and bone tissue in an in vivo model. Thirty-two New Zealand white rabbits were used in this study. Biomaterials were surgically implanted into each rabbit in the back erector spinae and in tibia with induced defect. Polyethylene was implanted into rabbits in the control group and n-CDHA/PAA into those of the experimental group. Animals were examined at four different points in time: 2 weeks, 4 weeks, 12 weeks, and 24 weeks after surgery. They were euthanized after embolization. Back erector spinae muscles with the surgical implants were examined after hematoxylin and eosin (HE) staining at these points in time. Tibia bones with the surgical implants were examined by X-ray and scanning electron microscopy (SEM) at these points in time to evaluate the interface of the bone with the implanted biomaterials. Bone tissues were sectioned and subjected to HE, Masson, and toluidine blue staining. HE staining of back erector spinae muscles at 4 weeks, 12 weeks, and 24 weeks after implantation of either n-CDHA/PAA or polyethylene showed disappearance of inflammation and normal arrangement in the peripheral tissue of implant biomaterials; no abnormal staining was observed. At 2 weeks after implantation, X-ray imaging of bone tissue samples in both experimental and control groups showed that the peripheral tissues of the implanted biomaterials were continuous and lacked bone osteolysis, absorption, necrosis, or osteomyelitis. The connection between implanted biomaterials and bone tissue was tight. The results of HE, Masson, toluidine blue staining and SEM confirmed that the implanted biomaterials were closely connected to the bone defect and that no rejection had taken place. The n-CDHA/PAA biomaterials induced differentiation of a large number of chondrocytes. New bone trabecula began to form at 4 weeks after implanting n-CDHA/PAA biomaterials, and lamellar bone gradually formed at 12 weeks and 24 weeks after implantation. Routine blood and kidney function tests showed no significant changes at 2 weeks and 24 weeks after implantation of both biomaterials. n-CDHA/PAA composites showed good compatibility in in vivo model. In this study, n-CDHA/PAA were found to be safe, nontoxic, and biologically active in bone repair.

Fungal Sinusitis

MedlinePlus

... process in which the body's immune system sends eosinophils (white blood cells distinguished by their lobulated nuclei ... orange eosin stain) to attack fungi, and the eosinophils irritate the membranes in the nose. As long ...

Evidence of the gastroprotective and anti- Helicobacter pylori activities of β-mangostin isolated from Cratoxylum arborescens (vahl) blume

PubMed Central

Sidahmed, Heyam Mohamed Ali; Hashim, Najihah Mohd; Mohan, Syam; Abdelwahab, Siddig Ibrahim; Taha, Manal Mohamed Elhassan; Dehghan, Firouzeh; Yahayu, Maizatulakmal; Ee, Gwendoline Cheng Lian; Loke, Mun Fai; Vadivelu, Jamuna

2016-01-01

Purpose β-Mangostin (BM) from Cratoxylum arborescens demonstrated various pharmacological activities such as anticancer and anti-inflammatory. In this study, we aimed to investigate its antiulcer activity against ethanol ulcer model in rats. Materials and methods BM was isolated from C. arborescens. Gastric acid output, ulcer index, gross evaluation, mucus production, histological evaluation using hematoxylin and eosin and periodic acid–Schiff staining and immunohistochemical localization for heat shock protein 70 (HSP70) and Bax proteins were investigated. Possible involvement of reduced glutathione, lipid peroxidation, prostaglandin E2, antioxidant enzymes, superoxide dismutase and catalase enzymes, radical scavenging, nonprotein sulfhydryl compounds, and anti-Helicobacter pylori were investigated. Results BM showed antisecretory activity against the pylorus ligature model. The pretreatment with BM protect gastric mucosa from ethanol damaging effect as seen by the improved gross and histological appearance. BM significantly reduced the ulcer area formation, the submucosal edema, and the leukocytes infiltration compared to the ulcer control. The compound showed intense periodic acid–Schiff staining to the gastric mucus layer and marked amount of alcian blue binding to free gastric mucus. BM significantly increased the gastric homogenate content of prostaglandin E2 glutathione, superoxide dismutase, catalase, and nonprotein sulfhydryl compounds. The compound inhibited the lipid peroxidation revealed by the reduced gastric content of malondialdehyde. Moreover, BM upregulate HSP70 expression and downregulate Bax expression. Furthermore, the compound showed interesting anti-H. pylori activity. Conclusion Thus, it could be concluded that BM possesses gastroprotective activity, which could be attributed to the antisecretory, mucus production, antioxidant, HSP70, antiapoptotic, and anti-H. pylori mechanisms. PMID:26834460

Secondary sea-blue histiocytosis derived from Niemann-Pick disease.

PubMed

Suzuki, Osamu; Abe, Masafumi

2007-04-01

Sea-blue histiocytosis is a rare disorder seen in patients with lipid metabolic or ceroid storage diseases. Sea-blue histiocytes are ceroid-laden macrophages detectable by May-Giemsa staining. We report a case of a 28-year-old woman diagnosed with Niemann-Pick disease at 2 or 3 years of age. To confirm this diagnosis, we examined her bone marrow, which revealed scattered foci containing aggregates of foamy macrophages. May-Giemsa staining identified blue-staining foamy macrophages, referred to as sea-blue histiocytes. In summary, we report the detection of sea-blue histiocytosis in an adult with Niemann-Pick disease.

Colloidal chitin stained with Remazol Brilliant Blue R, a useful substrate to select chitinolytic microorganisms and to evaluate chitinases.

PubMed

Gómez Ramírez, M; Rojas Avelizapa, L I; Rojas Avelizapa, N G; Cruz Camarillo, R

2004-02-01

A simple and sensitive method based on the use of colloidal chitin stained with Remazol Brilliant Blue R (RBB) is proposed to evaluate chitinase activity. If this colloidal-stained substrate is included as a carbon source in a liquid medium, this technique allows the selection or the comparison of chitinolytic microorganisms. The colloidal substrate is proportionally solubilized and the dye released is spectrophotometrically quantified at 595 nm. The procedures used for the staining and fixing of RBB in the colloidal chitin, and a comparison with the commercial substrate chitin-azure, are presented. The influence of several physicochemical and enzymatic parameters on the release of dyes is also shown. Both stained substrates were used for studying the effect of pH, substrate concentration, temperature and time on the chitinase reaction of Bacillus thuringiensis Bt-107.

Cement line staining in undecalcified thin sections of cortical bone

NASA Technical Reports Server (NTRS)

Bain, S. D.; Impeduglia, T. M.; Rubin, C. T.

1990-01-01

A technique for demonstrating cement lines in thin, undecalcified, transverse sections of cortical bone has been developed. Cortical bone samples are processed and embedded undecalcified in methyl methacrylate plastic. After sectioning at 3-5 microns, cross-sections are transferred to a glass slide and flattened for 10 min. Sections of cortical bone are stained for 20 sec free-floating in a fresh solution of 1% toluidine blue dissolved in 0.1% formic acid. The section is dehydrated in t-butyl alcohol, cleared in xylene, and mounted with Eukitt's medium. Reversal lines appear as thin, scalloped, dark blue lines against a light blue matrix, whereas bone formation arrest lines are thicker with a smooth contour. With this technique cellular detail, osteoid differentiation, and fluorochrome labels are retained. Results demonstrate the applicability of a one-step staining method for cement lines which will facilitate the assessment of bone remodeling activity in thin sections of undecalcified cortical bone.

Preparation of colloidal gold for staining proteins electrotransferred onto nitrocellulose membranes.

PubMed

Yamaguchi, K; Asakawa, H

1988-07-01

This paper describes a simple method of preparing colloidal gold for staining protein blots. Colloidal gold was prepared from 0.005 or 0.01% HAuCl4 by the addition of formalin as a reductant and potassium hydroxide. Staining of small cell carcinoma tissue extract blotted onto nitrocellulose membranes with this colloidal gold solution resulted in the appearance of a large number of clear wine-red bands. The sensitivity of gold staining was 60 times higher than that of Coomassie brilliant blue staining and almost comparable to that of silver staining of proteins in polyacrylamide gel. The sensitivity of this method was also satisfactory in comparison with that of enzyme immunoblotting. The colloidal gold prepared by this method is usable for routine work.

Adrenergic nerve fibres and mast cells: correlation in rat thymus.

PubMed

Artico, Marco; Cavallotti, Carlo; Cavallotti, Daniela

2002-10-21

The interactions between adrenergic nerve fibres and mast cells (MCs) were studied in the thymus of adult and old rats by morphological methods and by quantitative analysis of images (QAIs). The whole thymus was drawn in adult (12 months old) rats: normal, sympathectomized or electrostimulated. Thymuses from the above-mentioned animals were weighed, measured and dissected. Thymic slices were stained with eosin orange for detection of microanatomical details and with Bodian's method for identification of the whole nerve fibres. Thymic MCs were stained with Astrablau. Histofluorescence microscopy was used for staining of adrenergic nerve fibres. Finally, all morphological results were submitted to the QAIs and statistical analysis of data. Our results suggest that after surgical sympathectomy, the greater part of adrenergic nerve fibres disappear while related MCs appear to show less evident fluorescence and few granules. On the contrary, electrostimulation of the cervical superior ganglion induced an increase in the fluorescence of adrenergic nerve fibres and of related MCs.

Intraoperative imprint cytology for evaluation of sentinel lymph nodes from Merkel cell carcinoma.

PubMed

Wong, S Lindsey; Young, Yorke D; Geisinger, Kim R; Shen, Perry; Stewart, John H; Sangueza, Omar; Pichardo-Geisinger, Rita; Levine, Edward A

2009-07-01

Merkel cell carcinoma (MCC) is a rare, aggressive cutaneous malignancy. Intraoperative imprint cytology (IIC) can potentially avoid second operations for completion lymphadenectomy when nodal metastases are found during nodal staging with sentinel lymph node biopsy (SLN). This represents the first series of IIC for MCC we are aware of and our initial experience. Patients with biopsy-proven MCC underwent SLN (at the time of wide excision) using a double indicator technique with 99technetium sulfur colloid and isosulfan blue. SLN were identified and bisected and touch imprints of each half were made. One half was air-dried and stained with Diff-Quick and the other was fixed with 95 per cent alcohol and stained with hematoxylin and eosin (H&E). Paraffin-embedded sections were examined by H&E. Eighteen patients underwent successful SLN mapping procedures. IIC was negative in 84.2 per cent (16) cases. Three false-negatives occurred with IIC, but there were no false-positives, making the sensitivity 33 per cent and the specificity 100 per cent. Two of four patients with positive pathology-confirmed SLN also had positive IIC. SLN mapping has usefulness in patients with MCC. IIC is feasible and accurate in evaluating the SLN. IIC is a practical diagnostic tool when intraoperative analysis of SLN biopsy is desired for MCC.

Mast cells and its relation to collagen and VEGF in oral inflammatory lesions.

PubMed

Correia, Kariza V; Gonzalez, Ana C; Viena, Camila S; Wanderley, Flávia G; DE Almeida Reis, Sílvia R; Medrado, Alena R

2016-06-01

The aim of this study was to evaluate the population of intact and degranulated MCs in oral inflammatory lesions. A cross sectional study of 48 samples, including inflammatory fibrous hyperplasia, pyogenic granulomas, periapical granulomas and radicular cysts, was performed. Samples of normal gingival mucosa were used as controls. The degree of edema and lymphoplasmacytic infiltrate was determined by the analysis of hematoxylin-eosin (HE)-stained sections. To determine the collagen fibers contents and correlate it with the MC count, sections stained with Sirius red and Toluidine blue were used. Immunohistochemistry with an antivascular endothelial growth factor (VEGF) was also used to count endothelial cells. Although the total number of intact MCs was higher in the oral inflammatory lesions, these differences were not statistically significant (P=0.33). There were statistically significant differences between the numbers of degranulated MCs from the lesions and those from the normal oral mucosae (P=0.001) and a positive correlation between the number of MCs and the degree of inflammation (P<0.001). The MC count did not correlate with the collagen fibers or VEGF positive cells (P>0.05). The involvement of MCs in the pathogenesis of the oral inflammatory lesions is suggested. However, there was no positive correlation with these cells and collagen fibers or angiogenesis in the lesions studied.

Evaluating the Role of Immunological Cells (Tissue Eosinophils and Mast Cells) in Progression of Oral Squamous Cell Carcinoma.

PubMed

Saxena, S; Singh, A; Singh, P; Sundaragiri, K S; Sankhla, B; Bhargava, A

2018-04-01

Mast cells and eosinophils are increased in oral squamous cell carcinoma. The significance of such an association has been variably thought to be either a potential diagnostic tool for stromal invasion or as a prognostic indicator. The aim of the study was to study the mast cells and eosinophils between normal oral mucosa, leukoplakia and oral squamous cell carcinoma and to study the significance of mast cells in the progression of the lesion. A retrospective study was done on archival tissue received from January 2015 to December 2015, in the Department of Oral Pathology, RUHS College of Dental Sciences, Jaipur, Rajasthan, India. Seventy (70) cases were studied (30 cases each of leukoplakia and carcinoma and 10 cases of control group), sections were stained with toluidine blue solution to reveal mast cells. Eosinophils were studied in Haematoxylin & Eosin stained sections. Mast cell density significantly increased from: normal mucosa to oral leukoplakia to carcinoma, suggesting a role of the mast cells in the development of these lesions. The higher eosinophil counts in carcinoma group compared to dysplasia group proved that they might have a role in stromal invasion. The assessment of these could become, in the future, useful for therapeutic approaches in this subset of the patient.

Methylene blue dyeing of cellular nuclei during salpingoscopy, a new in-vivo method to evaluate vitality of tubal epithelium.

PubMed

Marconi, G; Quintana, R

1998-12-01

The Fallopian tube can be damaged by different noxious substances that may change cellular ultrastructure and function. Alteration of the cell membrane allows the passage of certain aniline dyes, which can stain the nucleus. A total of 310 Fallopian tubes from 163 patients who underwent a surgical or diagnostic laparoscopy during fertility studies was analysed by salpingoscopy. Cellular nuclei were stained by injection of 20 ml of a 10% solution of methylene blue in saline solution (NaCl 10%) through the cervical cannula prior to salpingoscopy. Evaluation of nuclear staining with methylene blue, adhesions, vascular alterations, and the flattening of folds in relation to pregnancy outcome was undertaken. Quantification of salpingoscopic findings was carried out according to a score. Flattening of folds and vascular alterations showed no difference in the pregnant and non-pregnant groups. On the other hand, adhesions and nuclear dyeing were significantly greater in the non-pregnant group (adhesions 13.6 versus 26.8%, P < 0.004, and nuclear dyeing: 25 versus 41.7%, P < 0.009, pregnant versus non-pregnant). Methylene blue dye is a new tool to evaluate in vivo cyto-histological tubal damage, and is a useful and simple method to provide a prognosis of salpingean function.

Urinary and faecal N-methylhistamine concentrations do not serve as markers for mast cell activation or clinical disease activity in dogs with chronic enteropathies.

PubMed

Anfinsen, Kristin P; Berghoff, Nora; Priestnall, Simon L; Suchodolski, Jan S; Steiner, Jörg M; Allenspach, Karin

2014-12-21

This study sought to correlate faecal and urinary N-methylhistamine (NMH) concentrations with resting versus degranulated duodenal mast cell numbers in dogs with chronic enteropathies (CE), and investigate correlations between intestinal mast cell activation and clinical severity of disease as assessed by canine chronic enteropathy clinical activity index (CCECAI), and between urinary and faecal NMH concentrations, mast cell numbers, and histopathological scores. Twenty-eight dogs with CE were included. Duodenal biopsies were stained with haematoxylin and eosin (H&E), toluidine blue, and by immunohistochemical labelling for tryptase. Duodenal biopsies were assigned a histopathological severity score, and duodenal mast cell numbers were counted in five high-power fields after metachromatic and immunohistochemical staining. Faecal and urinary NMH concentrations were measured by gas chromatography-mass spectrometry. There was no correlation between the CCECAI and faecal or urinary NMH concentrations, mast cell numbers, or histopathological score - or between faecal or urinary NMH concentration and mast cell numbers. Post hoc analysis revealed a statistically significant difference in toluidine blue positive mast cells between two treatment groups (exclusion diet with/without metronidazole versus immunosuppression (IS)), with higher numbers among dogs not requiring IS. Faecal and urinary NMH concentrations and duodenal mast cell numbers were not useful indicators of severity of disease as assessed by the CCECAI or histological evaluation. The number of duodenal mast cells was higher in dogs that did not need IS, i.e. in dogs responding to an exclusion diet (with/without metronidazole), than in dogs requiring IS. Further studies comparing the role of mast cells in dogs with different forms of CE are needed.

Preference of Formosan subterranean termites for blue-stained southern yellow pine sapwood.

PubMed

Little, N S; Blount, N A; Londo, A J; Kitchens, S C; Schultz, T P; McConnell, T E; Riggins, J J

2012-10-01

Little research has been conducted to investigate interactions between the invasive Formosan subterranean termite, Coptotermes formosanus Shiraki, and pine bark beetles native to the southeastern United States. Facilitative interactions between these organisms could alter stand dynamics and impact wood utilization strategies. American Wood Protection Association Standard E1-09 choice tests were carried out to determine the feeding preference of Formosan subterranean termites for blue-stained versus unstained southern yellow pine sapwood. Three separate colonies of Formosan subterranean termites consumed on average twice as much air-dried blue-stained southern yellow pine sapwood over unstained air-dried controls. Additionally, Formosan subterranean termites consumed over five-times more kiln-dried blue-stained sapwood than unstained kiln-dried control wafers. The implications of these results are particularly relevant to pine forest ecology, nutrient cycling, and the utilization of blue-stained southern pine building products in the southeastern United States, where Formosan subterranean termites have become established.

SENP1 attenuates the liver fibrosis through down-regulating the expression of SMAD2.

PubMed

Wu, Linshi; Qiu, Weiqing; Sun, Jianhua; Wang, Jian

2018-01-01

To investigate whether SENP1 could play a regulating role in the liver fibrosis process, the Sprague-Dawley (SD) rats were used to establish the liver fibrosis rat models by intraperitoneally injecting with 1 ml/kg of 10% CCl 4 , while the control normal rats were injected with olive oil. Then confirmation experiments to verify the successful establishment of these models were conducted by detecting the cellular and lobular architecture, and liver function indexes using hematoxylin-eosin staining, Masson's trichrome staining and microplate method, respectively. In addition, the expression levels of fibrosis markers including collagen I, collagen III, α-SMA and TGF-β1 were inspected using quantitative real-time PCR (qRT-PCR), as well as SMAD2. Subsequently, the relative mRNA and protein level of SENP1 was also determined via qRT-PCR and western blot analysis. Next, the HSC-T6 cells of SENP1 knock-down were constructed and used to test the relative protein expression levels of α-SMA and SMAD2 in these cells. The results of hematoxylin-eosin staining, Masson's trichrome staining and microplate method turned out that the rat liver fibrosis models were constructed successfully, which was further confirmed by the increased expression of collagen I, collagen III, α-SMA and TGF-β1 in mRNA and protein level, as well as SMAD2. Then the expression of SENP1 was overexpressed in the rat liver fibrosis models induced by CCl 4 and the TGF-β1 treatment could increase the protein expression level of collagen I, collagen III and α-SMA. Lastly, the SENP1 knockdown HSC-T6 cells were successfully constructed, while the silence of SENP1 down-regulated the protein expression of α-SMA and SMAD2. In conclusion, this study provided a new regulation mechanism about the liver fibrosis process. Copyright © 2017 Elsevier Inc. All rights reserved.

[Evaluation of inflammatory cells (tumor infiltrating lymphocytes - TIL) in malignant melanoma].

PubMed

Dundr, Pavel; Němejcová, Kristýna; Bártů, Michaela; Tichá, Ivana; Jakša, Radek

2018-01-01

The evaluation of inflammatory infiltrate (tumor infiltrating lymphocytes - TIL) should be a standard part of biopsy examination for malignant melanoma. Currently, the most commonly used assessment method according to Clark is not optimal and there have been attempts to find an alternative system. Here we present an overview of possible approaches involving five different evaluation methods based on hematoxylin-eosin staining, including the recent suggestion of unified TIL evaluation method for all solid tumors. The issue of methodology, prognostic and predictive significance of TIL determination as well as the importance of immunohistochemical subtyping of inflammatory infiltrate is discussed.

High-Contrast Imaging of Cholesterol Crystals in Rabbit Arteries Ex Vivo Using LED-Based Polarization Microscopy.

PubMed

Cho, Seonghee; Kim, Kyungmin; Kim, Taehoon; Park, Hyoeun; Kim, Jin-Moo; Lee, SeungHoon; Kang, YeonSu; Chang, Kiyuk; Kim, Chulhong

2018-04-19

Detection of cholesterol crystals (Chcs) in atherosclerosis disease is important for understanding the pathophysiology of atherosclerosis. Polarization microscopy (PM) has been in use traditionally for detecting Chcs, but they have difficulty in distinguishing Chcs with other crystalline materials in tissue, such as collagens. Thus, most studies using PM have been limited to studying cell-level samples. Although various methods have been proposed to detect Chcs with high specificity, most of them have low signal-to-noise ratios, a high system construction cost, and are difficult to operate due to a complex protocol. To address these problems, we have developed a simple and inexpensive universal serial bus (USB) PM system equipped with a 5700 K cool-white light-emitting diode (LED). In this system, Chcs are shown in a light blue color while collagen is shown in a yellow color. More importantly, the contrast between Chcs and collagens is improved by a factor of 2.3 under an aqueous condition in these PM images. These imaging results are well-matched with the ones acquired with two-photon microscopy (TPM). The system can visualize the features of atherosclerosis that cannot be visualized by the conventional hematoxylin and eosin and oil-red-o staining methods. Thus, we believe that this simple USB PM system can be widely used to identify Chcs in atherosclerosis.

A comparative analysis of toluidine blue with frozen section in oral squamous cell carcinoma

PubMed Central

2012-01-01

Background Surgical excision of the primary tumor with safe margins remains the mainstay of treatment for oral cavity squamous cell carcinoma (OSCC). The standard of care for assessment of intraoperative margins is frozen section histopathology. Unfortunately the facility is not available at most centers in limited resource countries. Toluidine blue, a metachromatic dye, has been well described in clinical identification of malignant and premalignant lesion in the oral cavity. Considering this we decided to explore intraoperative use of toluidine blue staining, in comparison with frozen sections, for the assessment of tumor-free margins. Methods After obtaining clearance from the in-house ethical review committee, a prospective study was conducted at Aga Khan University Hospital, Karachi, from August 15, 2009 to March 14, 2010. A sample of 56 consenting patients with biopsy-proven OSCC were included in the study, giving us 280 tumor margins. Margins were analyzed using toluidine blue staining and frozen section histopathology. A receiver operator curve (ROC) was then applied to compare assessment of margin status by toluidine blue and frozen section. Results Of the 280 examined margins 11 stained positive with toluidine blue, three were positive on frozen section biopsy, and three were positive on final histopathology. Toluidine blue staining had sensitivity and specificity of 100% and 97%, respectively. The diagnostic accuracy of toluidine blue was found to be 97.1% with a positive predictive value (PPV) of 27.2% and a negative predictive value (NPV) of 100%. Conclusions Toluidine blue can be used as an effective screening modality for the assessment of intraoperative margins in resource limited environments and reducing the number of frozen section biopsies performed. Further by providing real-time clinical information within minutes it can reduce indirect costs such as operating room time. It may also be used as an ad hoc for frozen section biopsies where frozen section facilities are available. PMID:22500814

Optimization of Trichomonas vaginalis Diagnosis during Pregnancy at a University Hospital, Argentina.

PubMed

Testardini, Pamela; Vaulet, María Lucía Gallo; Entrocassi, Andrea Carolina; Menghi, Claudia; Eliseht, Martha Cora; Gatta, Claudia; Losada, Mirta; Touzón, María Sol; Corominas, Ana; Vay, Carlos; Tatti, Silvio; Famiglietti, Angela; Fermepin, Marcelo Rodriguez; Perazzi, Beatriz

2016-04-01

The aim of this study was to evaluate different methods for Trichomonas vaginalis diagnosis during pregnancy in order to prevent maternal and perinatal complications. A total of 386 vaginal exudates from pregnant women were analyzed. T. vaginalis was investigated by 3 types of microscopic examinations direct wet mount with physiologic saline solution, prolonged May-Grunwald Giemsa (MGG) staining, and wet mount with sodium-acetate-formalin (SAF)/methylene blue method. PCR for 18S rRNA gene as well as culture in liquid medium were performed. The sensitivity and specificity of the microscopic examinations were evaluated considering the culture media positivity or the PCR techniques as gold standard. The frequency of T. vaginalis infection was 6.2% by culture and/or PCR, 5.2% by PCR, 4.7% by culture, 3.1% by SAF/methylene blue method and 2.8% by direct wet smear and prolonged MGG staining. The sensitivities were 83.3%, 75.0%, 50.0%, and 45.8% for PCR, culture, SAF/methylene blue method, and direct wet smear-prolonged MGG staining, respectively. The specificity was 100% for all the assessed methods. Microscopic examinations showed low sensitivity, mainly in asymptomatic pregnant patients. It is necessary to improve the detection of T. vaginalis using combined methods providing higher sensitivity, such as culture and PCR, mainly in asymptomatic pregnant patients, in order to prevent maternal and perinatal complications.

Automation-assisted cervical cancer screening in manual liquid-based cytology with hematoxylin and eosin staining.

PubMed

Zhang, Ling; Kong, Hui; Ting Chin, Chien; Liu, Shaoxiong; Fan, Xinmin; Wang, Tianfu; Chen, Siping

2014-03-01

Current automation-assisted technologies for screening cervical cancer mainly rely on automated liquid-based cytology slides with proprietary stain. This is not a cost-efficient approach to be utilized in developing countries. In this article, we propose the first automation-assisted system to screen cervical cancer in manual liquid-based cytology (MLBC) slides with hematoxylin and eosin (H&E) stain, which is inexpensive and more applicable in developing countries. This system consists of three main modules: image acquisition, cell segmentation, and cell classification. First, an autofocusing scheme is proposed to find the global maximum of the focus curve by iteratively comparing image qualities of specific locations. On the autofocused images, the multiway graph cut (GC) is performed globally on the a* channel enhanced image to obtain cytoplasm segmentation. The nuclei, especially abnormal nuclei, are robustly segmented by using GC adaptively and locally. Two concave-based approaches are integrated to split the touching nuclei. To classify the segmented cells, features are selected and preprocessed to improve the sensitivity, and contextual and cytoplasm information are introduced to improve the specificity. Experiments on 26 consecutive image stacks demonstrated that the dynamic autofocusing accuracy was 2.06 μm. On 21 cervical cell images with nonideal imaging condition and pathology, our segmentation method achieved a 93% accuracy for cytoplasm, and a 87.3% F-measure for nuclei, both outperformed state of the art works in terms of accuracy. Additional clinical trials showed that both the sensitivity (88.1%) and the specificity (100%) of our system are satisfyingly high. These results proved the feasibility of automation-assisted cervical cancer screening in MLBC slides with H&E stain, which is highly desirable in community health centers and small hospitals. © 2013 International Society for Advancement of Cytometry.

Methylene blue-aided cholangioscopy in patients with biliary strictures: feasibility and outcome analysis.

PubMed

Hoffman, A; Kiesslich, R; Bittinger, F; Galle, P R; Neurath, M F

2008-07-01

Chromoendoscopy using methylene blue is employed in the gastrointestinal tract to delineate neoplastic lesions. We tested the value of chromoendoscopy during choledochoscopy for characterization of local inflammation, neoplasias, and other alterations in patients with biliary strictures. Patients with suspected biliary lesions were scheduled for endoscopic retrograde cholangiography with subsequent cholangioscopy. After initial inspection of the bile duct, 15 ml methylene blue (0.1 %) was administered via the working channel of the cholangioscope. Newly appearing circumscribed or unstained lesions were judged according to their macroscopic type and staining features. Methylene-blue-aided diagnosis was compared with either clinical follow-up of the patients or, in some cases, with the results of targeted biopsies. A total of 55 patients [biliary stenosis/cholestasis of unknown origin (n = 24), stenosis after orthotopic liver transplantation (n = 11), primary sclerosing cholangitis (n = 20)] were included. Methylene blue unmasked subtle mucosal changes and permitted macroscopic characterization of circumscribed lesions. Characteristic surface staining patterns were seen in chronic inflammation, dysplasia, and ischemic-type biliary lesions. Nondysplastic mucosa appeared homogeneously stained, whereas scarred strictures showed a weak uptake of methylene blue. In this prospective feasibility study, methylene-blue-aided cholangioscopy was used for the first time to define different staining patterns of the bile duct. The differences in staining patterns identified normal, dysplastic, and inflamed mucosa of the bile duct, as was proved by follow-up or, in some cases, histology. Whereas homogeneous staining predicted the presence of normal mucosa, absence of staining of circumscribed lesions, or diffused staining of such lesions, represented neoplastic changes or inflammation.

Using Perls Staining to Trace the Iron Uptake Pathway in Leaves of a Prunus Rootstock Treated with Iron Foliar Fertilizers

PubMed Central

Rios, Juan J.; Carrasco-Gil, Sandra; Abadía, Anunciación; Abadía, Javier

2016-01-01

The aim of this study was to trace the Fe uptake pathway in leaves of Prunus rootstock (GF 677; Prunus dulcis × Prunus persica) plants treated with foliar Fe compounds using the Perls blue method, which detects labile Fe pools. Young expanded leaves of Fe-deficient plants grown in nutrient solution were treated with Fe-compounds using a brush. Iron compounds used were the ferrous salt FeSO4, the ferric salts Fe2(SO4)3 and FeCl3, and the chelate Fe(III)-EDTA, all of them at concentrations of 9 mM Fe. Leaf Fe concentration increases were measured at 30, 60, 90 min, and 24 h, and 70 μm-thick leaf transversal sections were obtained with a vibrating microtome and stained with Perls blue. In vitro results show that the Perls blue method is a good tool to trace the Fe uptake pathway in leaves when using Fe salts, but is not sensitive enough when using synthetic Fe(III)-chelates such as Fe(III)-EDTA and Fe(III)-IDHA. Foliar Fe fertilization increased leaf Fe concentrations with all Fe compounds used, with inorganic Fe salts causing larger leaf Fe concentration increases than Fe(III)-EDTA. Results show that Perls blue stain appeared within 30 min in the stomatal areas, indicating that Fe applied as inorganic salts was taken up rapidly via stomata. In the case of using FeSO4 a progression of the stain was seen with time toward vascular areas in the leaf blade and the central vein, whereas in the case of Fe(III) salts the stain mainly remained in the stomatal areas. Perls stain was never observed in the mesophyll areas, possibly due to the low concentration of labile Fe pools. PMID:27446123

Automated image analysis method for detecting and quantifying macrovesicular steatosis in hematoxylin and eosin-stained histology images of human livers.

PubMed

Nativ, Nir I; Chen, Alvin I; Yarmush, Gabriel; Henry, Scot D; Lefkowitch, Jay H; Klein, Kenneth M; Maguire, Timothy J; Schloss, Rene; Guarrera, James V; Berthiaume, Francois; Yarmush, Martin L

2014-02-01

Large-droplet macrovesicular steatosis (ld-MaS) in more than 30% of liver graft hepatocytes is a major risk factor for liver transplantation. An accurate assessment of the ld-MaS percentage is crucial for determining liver graft transplantability, which is currently based on pathologists' evaluations of hematoxylin and eosin (H&E)-stained liver histology specimens, with the predominant criteria being the relative size of the lipid droplets (LDs) and their propensity to displace a hepatocyte's nucleus to the cell periphery. Automated image analysis systems aimed at objectively and reproducibly quantifying ld-MaS do not accurately differentiate large LDs from small-droplet macrovesicular steatosis and do not take into account LD-mediated nuclear displacement; this leads to a poor correlation with pathologists' assessments. Here we present an improved image analysis method that incorporates nuclear displacement as a key image feature for segmenting and classifying ld-MaS from H&E-stained liver histology slides. 52,000 LDs in 54 digital images from 9 patients were analyzed, and the performance of the proposed method was compared against the performance of current image analysis methods and the ld-MaS percentage evaluations of 2 trained pathologists from different centers. We show that combining nuclear displacement and LD size information significantly improves the separation between large and small macrovesicular LDs (specificity = 93.7%, sensitivity = 99.3%) and the correlation with pathologists' ld-MaS percentage assessments (linear regression coefficient of determination = 0.97). This performance vastly exceeds that of other automated image analyzers, which typically underestimate or overestimate pathologists' ld-MaS scores. This work demonstrates the potential of automated ld-MaS analysis in monitoring the steatotic state of livers. The image analysis principles demonstrated here may help to standardize ld-MaS scores among centers and ultimately help in the process of determining liver graft transplantability. © 2013 American Association for the Study of Liver Diseases.

Histological staining methods preparatory to laser capture microdissection significantly affect the integrity of the cellular RNA.

PubMed

Wang, Hongyang; Owens, James D; Shih, Joanna H; Li, Ming-Chung; Bonner, Robert F; Mushinski, J Frederic

2006-04-27

Gene expression profiling by microarray analysis of cells enriched by laser capture microdissection (LCM) faces several technical challenges. Frozen sections yield higher quality RNA than paraffin-imbedded sections, but even with frozen sections, the staining methods used for histological identification of cells of interest could still damage the mRNA in the cells. To study the contribution of staining methods to degradation of results from gene expression profiling of LCM samples, we subjected pellets of the mouse plasma cell tumor cell line TEPC 1165 to direct RNA extraction and to parallel frozen sectioning for LCM and subsequent RNA extraction. We used microarray hybridization analysis to compare gene expression profiles of RNA from cell pellets with gene expression profiles of RNA from frozen sections that had been stained with hematoxylin and eosin (H&E), Nissl Stain (NS), and for immunofluorescence (IF) as well as with the plasma cell-revealing methyl green pyronin (MGP) stain. All RNAs were amplified with two rounds of T7-based in vitro transcription and analyzed by two-color expression analysis on 10-K cDNA microarrays. The MGP-stained samples showed the least introduction of mRNA loss, followed by H&E and immunofluorescence. Nissl staining was significantly more detrimental to gene expression profiles, presumably owing to an aqueous step in which RNA may have been damaged by endogenous or exogenous RNAases. RNA damage can occur during the staining steps preparatory to laser capture microdissection, with the consequence of loss of representation of certain genes in microarray hybridization analysis. Inclusion of RNAase inhibitor in aqueous staining solutions appears to be important in protecting RNA from loss of gene transcripts.

Histological staining methods preparatory to laser capture microdissection significantly affect the integrity of the cellular RNA

PubMed Central

Wang, Hongyang; Owens, James D; Shih, Joanna H; Li, Ming-Chung; Bonner, Robert F; Mushinski, J Frederic

2006-01-01

Background Gene expression profiling by microarray analysis of cells enriched by laser capture microdissection (LCM) faces several technical challenges. Frozen sections yield higher quality RNA than paraffin-imbedded sections, but even with frozen sections, the staining methods used for histological identification of cells of interest could still damage the mRNA in the cells. To study the contribution of staining methods to degradation of results from gene expression profiling of LCM samples, we subjected pellets of the mouse plasma cell tumor cell line TEPC 1165 to direct RNA extraction and to parallel frozen sectioning for LCM and subsequent RNA extraction. We used microarray hybridization analysis to compare gene expression profiles of RNA from cell pellets with gene expression profiles of RNA from frozen sections that had been stained with hematoxylin and eosin (H&E), Nissl Stain (NS), and for immunofluorescence (IF) as well as with the plasma cell-revealing methyl green pyronin (MGP) stain. All RNAs were amplified with two rounds of T7-based in vitro transcription and analyzed by two-color expression analysis on 10-K cDNA microarrays. Results The MGP-stained samples showed the least introduction of mRNA loss, followed by H&E and immunofluorescence. Nissl staining was significantly more detrimental to gene expression profiles, presumably owing to an aqueous step in which RNA may have been damaged by endogenous or exogenous RNAases. Conclusion RNA damage can occur during the staining steps preparatory to laser capture microdissection, with the consequence of loss of representation of certain genes in microarray hybridization analysis. Inclusion of RNAase inhibitor in aqueous staining solutions appears to be important in protecting RNA from loss of gene transcripts. PMID:16643667

The distribution patterns of COMP and matrilin-3 in septal, alar and triangular cartilages of the human nose.

PubMed

Wiggenhauser, Paul Severin; Schwarz, Silke; Rotter, Nicole

2018-05-02

The biomechanical characteristics of septal cartilage depend strongly on the distinct extracellular matrix of cartilage tissue; therefore, it is essential that the components of this matrix are identified and understood. Cartilage oligomeric matrix protein (COMP) and matrilin-3 are localised in articular cartilage. This study was the first to examine all subtypes of mature human nasal cartilages (alar, triangular and septal) with specific attention to the distribution of COMP and matrilin-3. Three whole fresh-frozen noses from human donors were dissected, and exemplary biopsies were examined using histochemical staining (haematoxylin and eosin and Alcian blue) and immunohistochemistry (collagen II, COMP and matrilin-3). The following three zones within the nasal cartilage were identified: superficial, intermediate and central. COMP was detected as highest in the intermediate zones in all three subtypes of nasal cartilage, whereas matrilin-3 was detected with pericellular deposition mainly within septal cartilage predominantly in the superficial zones. The distinct staining patterns of COMP and matrilin-3 underscore the different functional roles of both proteins in nasal cartilage. According to the literature, COMP might be involved with collagen II in the formation of networks, whereas matrilin-3 is reported to prevent ossification or regulate mechanosensitivity. The predominant staining observed in septal cartilage suggests matrilin-3's modulatory role because of its presence in the osteochondral junctional zone and given that the biomechanical load in septal cartilage is different from that in alar or triangular cartilage. In conclusion, COMP and matrilin-3 were detected in mature human nasal cartilage but displayed different staining patterns that might be explained by the functional roles of the respective matrix protein; however, further research is necessary to identify and define the functional aspects of this morphological difference.

Fabrication and characterization of nanowalls CdS/dye sensitized solar cells

NASA Astrophysics Data System (ADS)

Abdulelah, Haider; Ali, Basil; Mahdi, M. A.; Hassan, J. J.; Al-Taay, H. F.; Jennings, P.

2017-06-01

A microwave assisted chemical bath deposition (MA-CBD) was adopted to fabricate nanowalls CdS nanocrystalline thin film. Nanomaterials (such as nanowalls structure) have attracted significant attention due to their fascinating properties and unique applications, especially in optoelectronic nanodevices. Here we describe the fabrication of dye sensitized solar cells (DSSCs) based nanowalls cadmium sulfide (CdS) nanocrystalline thin films. The surface morphology, crystalline structure, and optical properties of the prepared nanocrystalline thin films are investigated. Rhodamine B, Malachite green, Eosin methylene blue, and Cresyl violet perchlorate dyes are used to fabricate the DSSCS devices. Current-voltage (I-V) characteristics show that the nanowall CdS/Eosin methylene blue device is the highest conversion efficiency of 0.89% under 100 mW/cm2. However, heat treatment of the fabricated solar cells causes significant enhancement in the output of all devices.

Cell wall staining with Trypan blue enables quantitative analysis of morphological changes in yeast cells.

PubMed

Liesche, Johannes; Marek, Magdalena; Günther-Pomorski, Thomas

2015-01-01

Yeast cells are protected by a cell wall that plays an important role in the exchange of substances with the environment. The cell wall structure is dynamic and can adapt to different physiological states or environmental conditions. For the investigation of morphological changes, selective staining with fluorescent dyes is a valuable tool. Furthermore, cell wall staining is used to facilitate sub-cellular localization experiments with fluorescently-labeled proteins and the detection of yeast cells in non-fungal host tissues. Here, we report staining of Saccharomyces cerevisiae cell wall with Trypan Blue, which emits strong red fluorescence upon binding to chitin and yeast glucan; thereby, it facilitates cell wall analysis by confocal and super-resolution microscopy. The staining pattern of Trypan Blue was similar to that of the widely used UV-excitable, blue fluorescent cell wall stain Calcofluor White. Trypan Blue staining facilitated quantification of cell size and cell wall volume when utilizing the optical sectioning capacity of a confocal microscope. This enabled the quantification of morphological changes during growth under anaerobic conditions and in the presence of chemicals, demonstrating the potential of this approach for morphological investigations or screening assays.

Effect of sex on histological and histochemical structures of different parts of the kidney in Japanese quail.

PubMed

Mobini, Behzad; Abdollahi, MohammadHossein

2016-09-01

The aim of the present study was to investigate the effect of gender on the histological and histochemical structures of different anatomical regions of the kidney in Japanese quail (Coturnix japonica). Tissue samples from cranial, middle and caudal divisions of each kidney were obtained from 20 male and 20 female adult, healthy Japanese quail. The sections stained with hematoxylin & eosin ( H & E: ), Masson's trichrome, Verhoeff's, Alcian blue (pH 2.5), Periodic acid-Schiff, and Gomori's method for reticulum. Unusual findings of the kidney in Japanese quail were the presence of three types of nephrons, all the connective tissue fibers in capsule and interlobular septa and AB-reactions of the proximal convoluted cells. No significant sex-based differences were found. The various histological structures of the kidney showed no significant differences among different divisions of the left and right kidneys. It is concluded that the general histological and histochemical properties of the kidney in Japanese quail were similar to those of chickens and some other species, but that there were also some differences. One of the major differences was brush border of interdigitating microvilli on luminal surface of collecting ducts in Japanese quail. © 2016 Poultry Science Association Inc.

Pneumocystis PCR: It Is Time to Make PCR the Test of Choice

PubMed Central

Doyle, Laura; Vogel, Sherilynn

2017-01-01

Abstract Background The testing strategy for Pneumocystis at the Cleveland Clinic changed from toluidine blue staining to polymerase chain reaction (PCR). We studied the differences in positivity rates for these assays and compared each with the detection of Pneumocystis in companion specimens by cytology and surgical pathology. Methods We reviewed the results of all Pneumocystis test orders 1 year before and 1 year after the implementation of a Pneumocystis-specific PCR. We also reviewed the corresponding cytology and surgical pathology results, if performed. Finally, we reviewed the medical records of patients with rare Pneumocystis detected by PCR in an effort to differentiate colonization vs true disease. Results Toluidine blue staining and surgical pathology had similar sensitivities and negative predictive values, both of which were superior to cytology. There was a >4-fold increase in the annual detection of Pneumocystis by PCR compared with toluidine blue staining (toluidine blue staining: 11/1583 [0.69%] vs PCR: 44/1457 [3.0%]; chi-square P < .001). PCR detected 1 more case than surgical pathology and was far more sensitive than cytology. Chart review demonstrated that the vast majority of patients with rare Pneumocystis detected were immunosuppressed, had radiologic findings supportive of this infection, had no other pathogens detected, and were treated for pneumocystosis by the clinical team. Conclusion PCR was the most sensitive method for the detection of Pneumocystis and should be considered the diagnostic test of choice. Correlation with clinical and radiologic findings affords discrimination of early true disease from the far rarer instances of colonization. PMID:29062861

Curcuma longa extract - Haldi: A safe, eco-friendly natural cytoplasmic stain.

PubMed

Suryawanshi, Hema; Naik, Rupali; Kumar, Pramod; Gupta, Rolly

2017-01-01

Eosin is most widely used synthetic dye belonging to the xanthene group. These dyes are efficient but are hazardous to human and animal health. With the increasing awareness of a green earth, it is advisable to use more of eco-friendly and biodegradable material which can be effectively achieved by the use of natural dyes obtained from plants and other natural sources. Turmeric, available as Curcuma longa (domestic), has long been in use in the subcontinent as a spice and flavoring agent in most food preparations. Its health benefit as a natural antibiotic and anti-inflammatory has been successfully established by several researchers. The intense yellow color imparted by turmeric inspired us to explore its efficacy as a potential alternative for eosin in routine histopathological procedures. The aim of this was to explore the efficacy of turmeric extract as a stand-alone counterstain for hematoxylin and its comparative assessment with routine H and E staining. The rhizomes of C. longa were cut into small pieces, dried and milled. This powder was dissolved into alcohol and centrifuged using a centrifugal machine. The supernatant was then collected with the help of micropipette. This supernatant was used as a counterstain for hematoxylin. The data were analyzed using Mann-Whitney U test with Statistical Package for the Social Sciences version 15.0 (SPSS Inc.,). The P value obtained was statistically insignificant ( P > 0.05). Although eosin is the most efficient counterstain for hematoxylin, turmeric can also be used as an alternative for eosin.

Glycosaminoglycan blotting on nitrocellulose membranes treated with cetylpyridinium chloride after agarose-gel electrophoretic separation.

PubMed

Maccari, Francesca; Volpi, Nicola

2002-09-01

We describe a method for blotting and immobilizing several nonsulfated and sulfated complex polysaccharides on membranes made hydrophilic and positively charged by a cationic detergent after their separation by conventional agarose gel electrophoresis. Nitrocellulose membranes were derivatized with the cationic detergent cetylpyridinium chloride (CPC) and mixtures of glycosaminoglycans (GAGs) were capillary-blotted after their separation in agarose gel electrophoresis in barium acetate/1,2-diaminopropane. Single purified species of variously sulfated polysaccharides were transferred onto the derivatized membranes after electrophoresis with an efficiency of 100% and stained with alcian blue (irreversible staining) and toluidine blue (reversible staining) permitting about 0.1 nug threshold of detection. Nonsulfated polyanions, hyaluronic acid, a fructose-containing polysaccharide with a chondroitin backbone purified from Escherichia coli U1-41, and its defructosylated product, were also electrophoretically separated and transferred onto membranes. The limit of detection for desulfated GAGs was about 0.1-0.5 nug after irreversible or reversible staining. GAG extracts from bovine, lung and aorta, and human aorta and urine were separated by agarose gel electrophoresis and blotted on CPC-treated nitrocellulose membranes. The polysaccharide composition of these extracts was determined. The membrane stained with toluidine blue (reversible staining) was destained and the same lanes used for immunological detection or other applications. Reversible staining was also applied to recover single species of polysaccharides after electrophoretic separation of mixtures of GAGs and their transfer onto membranes. Single bands were released from the membrane with an efficiency of 70-100% for further biochemical characterization.

Functional Regeneration of Recurrent Laryngeal Nerve Injury During Thyroid Surgery Using an Asymmetrically Porous Nerve Guide Conduit in an Animal Model

PubMed Central

Choi, Jeong-Seok; Oh, Se Heang; An, Hye-Young; Kim, Young-Mo; Lee, Jin Ho

2014-01-01

Background: Vocal cord paralysis (VCP) caused by recurrent laryngeal nerve (RLN) damage during thyroidectomy commonly results in serious medico-legal problems. The purpose of this study was to evaluate the usefulness of an asymmetrically porous polycaprolactone (PCL)/Pluronic F127 nerve guide conduit (NGC) for functional regeneration in a RLN injury animal model. Methods: A biodegradable, asymmetrically porous PCL/F127 NGC with selective permeability was fabricated for use in this study. A 10-mm segment of left RLN was resected in 28 New Zealand white rabbits, and then an asymmetrically porous NGC or a nonporous silicone tube was interposed between both stumps and securely fixed. Vocal cord mobility was endoscopically evaluated at one, four, and eight weeks postoperatively. Nerve growth through NGCs was assessed by toluidine blue staining, and thyroarytenoid (TA) muscle atrophy was evaluated by hematoxylin and eosin staining. Immunohistochemical stainings for acetylcholinesterase (AchE), anti-neurofilament (NF), and anti-S100 protein were also conducted, and transmission electron microscopy (TEM) was used to evaluate functional nerve regeneration. Results: At eight weeks postoperatively, endoscopic evaluations showed significantly better recovery from VCP in the asymmetrically porous PCL/F127 NGC group (6 of 10 rabbits) than in the silicone tube group (1 of 10 rabbits). Continued nerve growth on the damaged nerve endings was observed with time in the asymmetrically porous PCL/F127 NGC-interposed RLNs. TA muscle dimensions and AchE expressions in TA muscle were significantly greater in the asymmetrically porous PCL/F127 NGC group than in the silicone tube group. Furthermore, immunohistochemical staining revealed the expression of NF and S100 protein in the regenerated nerves in the asymmetrically porous PCL/F127 NGC group at eight weeks postoperatively, and at this time, TEM imaging showed myelinated axons in the regenerated RLNs. Conclusion: The study shows that asymmetrically porous PCL/F127 NGC provides a favorable environment for RLN regeneration and that it has therapeutic potential for the regeneration of RLN damage. PMID:24015805

Microvessel prediction in H&E Stained Pathology Images using fully convolutional neural networks.

PubMed

Yi, Faliu; Yang, Lin; Wang, Shidan; Guo, Lei; Huang, Chenglong; Xie, Yang; Xiao, Guanghua

2018-02-27

Pathological angiogenesis has been identified in many malignancies as a potential prognostic factor and target for therapy. In most cases, angiogenic analysis is based on the measurement of microvessel density (MVD) detected by immunostaining of CD31 or CD34. However, most retrievable public data is generally composed of Hematoxylin and Eosin (H&E)-stained pathology images, for which is difficult to get the corresponding immunohistochemistry images. The role of microvessels in H&E stained images has not been widely studied due to their complexity and heterogeneity. Furthermore, identifying microvessels manually for study is a labor-intensive task for pathologists, with high inter- and intra-observer variation. Therefore, it is important to develop automated microvessel-detection algorithms in H&E stained pathology images for clinical association analysis. In this paper, we propose a microvessel prediction method using fully convolutional neural networks. The feasibility of our proposed algorithm is demonstrated through experimental results on H&E stained images. Furthermore, the identified microvessel features were significantly associated with the patient clinical outcomes. This is the first study to develop an algorithm for automated microvessel detection in H&E stained pathology images.

A brief review of other notable protein detection methods on acrylamide gels.

PubMed

Kurien, Biji T; Scofield, R Hal

2012-01-01

Several methods have been described to stain proteins analyzed on acrylamide gels. These include ultrasensitive protein detection in one-dimensional and two-dimensional gel electrophoresis using a fluorescent product from the fungus Epicoccum nigrum; a fluorescence-based Coomassie Blue protein staining; visualization of proteins in acrylamide gels using ultraviolet illumination; fluorescence visualization of proteins in sodium dodecyl sulfate-polyacrylamide gels using environmentally benign, nonfixative, saline solution; and increasing the sensitivity four- to sixfold for detecting trace proteins in dye or silver stained polyacrylamide gels using polyethylene glycol 6000. All these methods are reviewed briefly in this chapter.

Multiphoton microscopy as a diagnostic imaging modality for pancreatic neoplasms without hematoxylin and eosin stains

NASA Astrophysics Data System (ADS)

Chen, Youting; Chen, Jing; Chen, Hong; Hong, Zhipeng; Zhu, Xiaoqin; Zhuo, Shuangmu; Chen, Yanling; Chen, Jianxin

2014-09-01

Hematoxylin and eosin (H&E) staining of tissue samples is the standard approach in histopathology for imaging and diagnosing cancer. Recent reports have shown that multiphoton microscopy (MPM) provides better sample interface with single-cell resolution, which enhances traditional H&E staining and offers a powerful diagnostic tool with potential applications in oncology. The purpose of this study was to further expand the versatility of MPM by establishing the optical parameters required for imaging unstained histological sections of pancreatic neoplasms, thereby providing an efficient and environmentally sustainable alternative to H&E staining while improving the accuracy of pancreatic cancer diagnoses. We found that the high-resolution MPM images clearly distinguish between the structure of normal pancreatic tissues compared with pancreatic neoplasms in unstained histological sections, and discernable differences in tissue architecture and cell morphology between normal versus tumorigenic cells led to enhanced optical diagnosis of cancerous tissue. Moreover, quantitative assessment of the cytomorphological features visualized from MPM images showed significant differences in the nuclear-cytoplasmic ratios of pancreatic neoplasms compared with normal pancreas, as well as further distinguished pancreatic malignant tumors from benign tumors. These results indicate that the MPM could potentially serve as an optical tool for the diagnosis of pancreatic neoplasms in unstained histological sections.

Experience with a cholinesterase histochemical technique for rectal suction biopsies in the diagnosis of Hirschsprung's disease

PubMed Central

Trigg, P. H.; Belin, R.; Haberkorn, S.; Long, W. J.; Nixon, H. H.; Plaschkes, J.; Spitz, L.; Willital, G. H.

1974-01-01

Cryostat sections from 160 rectal suction biopsies were stained for cholinesterases by the method of Karnovsky and Roots (1964) in an attempt to facilitate the diagnosis of Hirschsprung's disease. The method proved at least as reliable as experienced assessment of paraffin haematoxylin-eosin sections, and appeared to offer the advantages of reduced scanning fatigue and superior demonstration of the increased cholinesterase-positive nerves in Hirschprung's disease. Contrary to the findings of Meier-Ruge (1971) it was not possible to base a diagnosis on mucosal cholinesterase activity. Images PMID:4832300

Methodology of aniline blue staining of chromatin and the assessment of the associated nuclear and cytoplasmic attributes in human sperm.

PubMed

Sati, Leyla; Huszar, Gabor

2013-01-01

In this chapter, the laboratory methods for detection of sperm biomarkers that are aimed at identifying arrested sperm development are summarized. These probes include sperm staining with aniline blue for persistent histones, representing a break in the histone-transition protein-protamine sequence, immunocytochemistry with cytoplasmic sperm proteins, highlighting cytoplasmic retention during spermiogenesis, DNA nick translation testing for DNA chain fragmentation due to various reasons, for instance low HspA2 chaperone protein levels, and consequential diminished DNA repair. Finally, we briefly provide references on our work on sperm hyaluronan binding, abnormal Tybergerg sperm morphology, and the increased levels of chromosomal aneuploidies in sperm with developmental arrest. A very interesting aspect of the biomarker field is the discovery (Sati et al, Reprod Biomed Online 16:570-579, 2008) that the various nuclear and cytoplasmic defects detected by the biomarkers are related, and may simultaneously occur within the same spermatozoa as evidenced by a combination of biomarkers, such as aniline blue staining (persistent histones) coupled with cytoplasmic retention, DNA fragmentation, Caspase-3, Tygerberg abnormal morphology, and increased levels of chromosomal aneuploidies. We show examples of this >80% overlap in staining patterns within the same spermatozoa.

Decellularized amnion scaffold with activated PRP: a new paradigm dressing material for burn wound healing.

PubMed

Kshersagar, Jeevitaa; Kshirsagar, Ravi; Desai, Shashikant; Bohara, Raghvendra; Joshi, Meghnad

2018-03-05

Direct application of amnion has greater risk of immunological rejection and infection. Decellularization is an effective method to lower the risk of immune complications and infections. The bioreactor assembly with multiple cassettes was designed for decellurization of multiple amnions with different cell types simultaneously in single run. A detergent-based protocol was modified to remove all cellular components from amnion and diminish the DNA content to render it non-immunogenic. Amnion (n = 10) were treated with 2% sodium dodecyl sulphate (SDS), 5% dimethyl sulfoxide (DMSO) and 2% sodium deoxycholeate (SD). Decellularized amnion samples were analyzed by haematoxylin-eosin staining (HE), Alcian blue pH 1 (AB-pH-1), 4,6-diamnionidino-2-phenylindol (DAPI), Massion's trichrome stain, DNA quantification, mechanical testing and scanning electron microscopy (SEM). Histological analysis showed complete removal of cellular components and the histoarchitecture of scaffold remained intact. Amnion scaffold activated with platelet rich plasma (PRP) and calcium chloride composition supported better adherence to the wound than amnion alone. Only single application showed good healing. In vivo assessment of activated amnion revealed stable dressing. It has good promising outcome. At day 7, histologically the wounds treated with activated amnion were almost closed without scarring and showed well differentiated epidermis, proliferation of keratinocytes, hair follicles and basement membrane as compared to controls and silver nitrate gel dressings in a mouse (Mus musculus). Cryopreservation had no adverse effect on the mechanical properties of the amnion scaffold. Cryopreservation of decellularized amnion by Dulbecco's modified eagle medium (DMEM) was expected to prepare off-the-shelf skin substitutes and preserve them to be immediately available upon request of patients' needs.

Effect of Photo-Fenton Bleaching on Tetracycline-stained Dentin in vitro.

PubMed

Bennett, Zackary Yale; Walsh, Laurence James

2015-02-01

Tetracycline-stained tooth structure is difficult to bleach using nightguard tray methods. The possible benefits of in-office light-accelerated bleaching systems based on the photo-Fenton reaction are of interest as possible adjunctive treatments. This study was a proof of concept for possible benefits of this approach, using dentine slabs from human tooth roots stained in a reproducible manner with the tetracycline antibiotic demeclocycline hydrochloride. Color changes overtime in tetra-cycline stained roots from single rooted teeth treated using gel (Zoom! WhiteSpeed(®)) alone, blue LED light alone, or gel plus light in combination were tracked using standardized digital photography. Controls received no treatment. Changes in color channel data were tracked overtime, for each treatment group (N = 20 per group). Dentin was lighter after bleaching, with significant improvements in the dentin color for the blue channel (yellow shade) followed by the green channel and luminosity. The greatest changes occurred with gel activated by light (p < 0.0001), which was superior to effects seen with gel alone. Use of the light alone did not significantly alter shade. This proof of concept study demonstrates that bleaching using the photo-Fenton chemistry is capable of lightening tetracycline-stained dentine. Further investigation of the use of this method for treating tetracycline-stained teeth in clinical settings appears warranted. Because tetracycline staining may respond to bleaching treatments based on the photo-Fenton reaction, systems, such as Zoom! WhiteSpeed, may have benefits as adjuncts to home bleaching for patients with tetracycline-staining.

Free radicals generated by electrolysis reduces nitro blue tetrazolium in isolated rat heart.

PubMed

Chahine, R; Huet, M P; Oliva, L; Nadeau, R

1997-02-01

Oxygen free radicals (OFR) are highly cytotoxic when produced in the myocardium under certain pathological conditions. In isolated rat hearts perfused retrogradely, OFR were generated by electrolysis of the Krebs-Henseleit buffer (two platinum electrodes, DC current, 10 mA, 1 min). In order to find evidence that OFR are produced, we used nitro blue tetrazolium (NBT) a soluble compound which yields a dark blue formazan pigment in the presence of reducing agents. Hearts were subdivided into: control, electrolysed, NBT (3.3 mg/ml) perfusion during electrolysis in the presence or absence of scavengers. The xanthine-xanthine oxidase (XXO) system known to produce superoxide radical was used as a reference. Specimens were fixed with formaldehyde and stained with eosine or Kernechtrot in preparation for light microscopical examination. Several areas of acute necrosis expressed by hyalinisation and loss of striation were observed in electrolysed hearts which present a pattern of wavy disrupted myofibers and an increase in interstitial spaces. A very faint deposition of formazan was observed in some rare areas of NBT perfused heart. Only the electrolysed group perfused with NBT and the one perfused with XXO plus NBT presented an extensive formazan deposition, mostly in the areas of fibre necrosis. Formazan was barely detectable when superoxide dismutase plus catalase were perfused in the XXO system, while it was still apparent when perfused in electrolysed hearts. These results support the hypothesis that electrolysis can be used to generate different species of OFR and to evaluate the protective action of scavenger and antioxidants against OFR-induced myocardial damage.

Surgical Marking Pen Dye Inhibits Saphenous Vein Cell Proliferation and Migration in Saphenous Vein Graft Tissue

PubMed Central

Kikuchi, Shinsuke; Kenagy, Richard D; Gao, Lu; Wight, Thomas N; Azuma, Nobuyoshi; Sobel, Michael; Clowes, Alexander W

2014-01-01

Objective Markers containing dyes such as crystal violet (CAS 548-62-9) are routinely used on the adventitia of vein bypass grafts to avoid twisting during placement. Since little is known about how these dyes affect vein graft healing and function, we determined the effect of crystal violet on cell migration and proliferation, which are responses to injury after grafting. Methods Fresh human saphenous veins were obtained as residual specimens from leg bypass surgeries. Portions of the vein that had been surgically marked with crystal violet were analyzed separately from those that had no dye marking. In the laboratory, they were split into easily dissected inner and outer layers after removal of endothelium. This f cleavage plane was within the circular muscle layer of the media. Cell migration from explants was measured daily as either 1) % migration positive explants, which exclusively measures migration, or 2) the number of cells on the plastic surrounding each explant, which measures migration plus proliferation. Cell proliferation and apoptosis (Ki67 and TUNEL staining, respectively) were determined in dye-marked and unmarked areas of cultured vein rings. The dose-dependent effects of crystal violet were measured for cell migration from explants as well as proliferation, migration, and death of cultured outer layer cells. Dye was extracted from explants with ethanol and quantified by spectrophotometry. Results There was significantly less cell migration from visibly blue, compared to unstained, outer layer explants by both methods. There was no significant difference in migration from inner layer explants adjacent to blue-stained or unstained sections of vein, because dye did not penetrate to the inner layer. Ki67 staining of vein in organ culture, which is a measure of proliferation, progressively increased up to 6 days in non-blue outer layer and was abolished in the blue outer layer. Evidence of apoptosis (TUNEL staining) was present throughout the wall and not different in blue-stained and unstained vein wall segments. Blue outer layer explants had 65.9±8.0 ng dye/explant compared to 2.1±1.3 for non-blue outer layer explants. Dye applied in vitro to either outer or inner layer explants dose-dependently inhibited migration (IC50=8.5 ng/explant). The IC50s of crystal violet for outer layer cell proliferation and migration were 0.1 and 1.2 μg/ml, while the EC50 for death was between 1 and 10 μg/ml. Conclusion Crystal violet inhibits venous cell migration and proliferation indicating that alternative methods should be considered for marking vein grafts. PMID:25935273

Comparison of different diagnostic techniques for the detection of cryptosporidiosis in bovines

PubMed Central

Rekha, K. M. H.; Puttalakshmamma, G. C.; D’Souza, Placid E.

2016-01-01

Aim: Aim of the present study was to compare different methods, viz., Sheather's sugar flotation (SSF), Ziehl-Neelsen (ZN), Kinyoun's acid-fast method (KAF), safranin-methylene blue staining (SMB), and negative staining techniques such as nigrosin staining, light green staining, and malachite green staining for the detection of Cryptosporidium spp. oocysts in bovines. Materials and Methods: A total of 455 fecal samples from bovines were collected from private, government farms and from the clinical cases presented to Department of Medicine, Veterinary College, Bengaluru. They were subjected for SSF, ZN, KAF, SMB and negative staining methods. Results: Out of 455 animal fecal samples screened 5.71% were found positive for Cryptosporidium spp. oocysts. The species were identified as Cryptosporidium parvum in calves and Cryptosporidium andersoni in adults based on the morphological characterization and micrometry of the oocysts. Conclusions: Of all the techniques, fecal flotation with sheather's was found to be more specific and sensitive method for the detection of Cryptosporidium spp. oocysts. Among the conventional staining methods, the SMB gives better differentiation between oocysts and yeast. Among the three negative staining methods, malachite green was found sensitive over the other methods. PMID:27051211

Sustained methylene blue staining to guide anatomic hepatectomy for hepatocellular carcinoma: Initial experience and technical details.

PubMed

Shou-wang, Cai; Shi-zhong, Yang; Wen-ping, Lv; Geng, Chen; Wan-qing, Gu; Wei-dong, Duan; Wei-yi, Wang; Zhi-qiang, Huang; Jia-hong, Dong

2015-07-01

The boundary of the target hepatic segment within the liver parenchyma cannot be marked by the use of a conventional anatomic hepatectomy approach. This study describes a novel methylene blue staining technique for guiding the anatomic resection of hepatocellular carcinoma (HCC). Between February 2009 and February 2012, anatomic hepatectomy was performed in 106 patients with HCC via a novel, sustained methylene blue staining technique. Sustained staining was achieved by injecting methylene blue into the distal aspect of the portal vein after exposing Glisson's sheath. The hepatic pedicle was immediately ligated, and the hepatic parenchymal transection was performed along the interface between methylene blue stained tissue and unstained tissue. Anatomic hepatectomies included subsegmentectomy (n = 16), monosegmentectomy (n = 57), multisegmentectomy (n = 27), and hemihepatectomy (n = 6). The portal vein was injected successfully with methylene blue in 100% of cases, and complete staining of the target hepatic segment was achieved in 98 of 106 (92.5%) cases. Mean intraoperative bleeding was 360 ± 90 mL, and the postoperative complication rate was 24.5% (26/106). No perioperative mortality occurred. Operative margins were all negative on pathologic examination. Mean duration of postoperative follow-up was 40 months (range, 24-60). No local recurrence (around the operative margin) occurred. This novel technique of achieving sustained staining by injecting methylene blue then immediately ligating the hepatic pedicle is simple and feasible. It can guide the selection of the operative margin during hepatic parenchyma transection to improve the accuracy of anatomic hepatectomy for the treatment of HCC. Copyright © 2015 Elsevier Inc. All rights reserved.

Immunohistochemical assessment of NY-ESO-1 expression in esophageal adenocarcinoma resection specimens.

PubMed

Hayes, Stephen J; Hng, Keng Ngee; Clark, Peter; Thistlethwaite, Fiona; Hawkins, Robert E; Ang, Yeng

2014-04-14

To assess NY-ESO-1 expression in a cohort of esophageal adenocarcinomas. A retrospective search of our tissue archive for esophageal resection specimens containing esophageal adenocarcinoma was performed, for cases which had previously been reported for diagnostic purposes, using the systematised nomenclature of human and veterinary medicine coding system. Original haematoxylin and eosin stained sections were reviewed, using light microscopy, to confirm classification and tumour differentiation. A total of 27 adenocarcinoma resection specimens were then assessed using immunohistochemistry for NY-ESO-1 expression: 4 well differentiated, 14 moderately differentiated, 4 moderate-poorly differentiated, and 5 poorly differentiated. Four out of a total of 27 cases of esophageal adenocarcinoma examined (15%) displayed diffuse cytoplasmic and nuclear expression for NY-ESO-1. They displayed a heterogeneous and mosaic-type pattern of diffuse staining. Diffuse cytoplasmic staining was not identified in any of these structures: stroma, normal squamous epithelium, normal submucosal gland and duct, Barrett's esophagus (goblet cell), Barrett's esophagus (non-goblet cell) and high grade glandular dysplasia. All adenocarcinomas showed an unexpected dot-type pattern of staining at nuclear, paranuclear and cytoplasmic locations. Similar dot-type staining, with varying frequency and size of dots, was observed on examination of Barrett's metaplasia, esophageal submucosal gland acini and the large bowel negative control, predominantly at the crypt base. Furthermore, a prominent pattern of apical (luminal) cytoplasmic dot-type staining was observed in some cases of Barrett's metaplasia and also adenocarcinoma. A further morphological finding of interest was noted on examination of haematoxylin and eosin stained sections, as aggregates of lymphocytes were consistently noted to surround submucosal glands. We have demonstrated for the first time NY-ESO-1 expression by esophageal adenocarcinomas, Barrett's metaplasia and normal tissues other than germ cells.

Quantitative comparison of MiTF, Melan-A, HMB-45 and Mel-5 in solar lentigines and melanoma in situ.

PubMed

Kim, Jinah; Taube, Janis M; McCalmont, Timothy H; Glusac, Earl J

2011-10-01

It is often challenging to reliably assess the number of lesional melanocytes in intraepidermal melanocytic proliferations involving sun-damaged skin. Therefore, dermatopathologists routinely use immunostains to help differentiate melanocytes from surrounding keratinocytes. Forty-three cases of solar lentigo or melanoma in situ (of the lentigo maligna type) were retrospectively chosen (20 melanomas in situ and 23 solar lentigo). Microphthalmia transcription factor (MiTF), HMB-45, Melan-A and Mel-5 immunostains were performed with an Azure blue counterstain, and the mean melanocyte counts were calculated within a 1-mm segment of epidermis. In solar lentigines, the mean melanocyte counts were 27 (MiTF), 23 (HMB-45 and Mel-5) and 41 (Melan-A), as compared to hematoxylin and eosin (H&E) (25). In melanoma in situ, the mean melanocyte counts were 112 (MiTF), 149 (Melan-A), 111 (HMB-45) and 80 (Mel-5), as compared to H&E (109). These results show that Melan-A significantly overestimates the density of melanocytes within dermatoheliotic skin. Compared to other tested stains, nuclear staining MiTF allowed greater distinction of melanocytes from keratinocytes with melanized cytoplasm. These findings indicate that MiTF is a superior marker for quantification of melanocytes in the evaluation of subtle intraepidermal melanocytic proliferations and in the differential diagnosis of solar lentigo. Copyright © 2011 John Wiley & Sons A/S.

Age-related changes in the macula. A histopathological study of fifty Indian donor eyes.

PubMed

Biswas, Jyotirmay; Raman, Rajiv

2002-09-01

Age-related macular degeneration (ARMD) is clinically less common in India compared to the West. Therefore, clinicians are unfamiliar with histopathologic evidence of age-related macular changes in the Indian population. Fifty consecutive human donor eyes removed for corneal grafting were studied for gross, microscopic and histochemical features of age-related changes in the macula in the Indian population. A horizontal block was cut from the globe including the optic disc, and the macula. Six sections, 6 microns thick, were cut from three levels in the macula at a distance of 140 microns. These were stained with haemotoxylin-eosin, periodic acid-Schiff, Mallory, Masson trichrome, alcian blue and von Kossa stains. The presence of basal laminar deposits, drusen and thickening and calcification of Bruch's membrane in the macula were assessed at 400 x magnification using a modified version of Sark's classification. Twenty-four donor eyes (48%) had some form of age-related macular change. These included basal laminar deposits, hard drusen, soft drusen, extensive retinal pigment epithelium atrophy of the macula, and disciform degeneration of macula. A combination of changes was often seen. Age-related changes were more common in the seventh and eighth decade. Our study shows that histological changes characteristic of the early stages of age-related macular degeneration are fairly common in the Indian population. However, advanced macular changes are significantly rare.

Achilles tendinosis – a morphometrical study in a rat model

PubMed Central

Silva, Rafael Duarte; Glazebrook, Mark Anthony; Campos, Vinicius Castro; Vasconcelos, Anilton Cesar

2011-01-01

This study addresses the morphopathogenesis of Achilles tendinosis, using a rat model and presenting quantitative analysis of time-dependent histological changes. Thirty Wistar rats were used, randomly split in experimental and control groups. Animals of the experimental group were submitted to a treadmill running scheme. Five animals of each group were euthanized at four, eight and sixteen weeks. Achilles tendons were collected and processed routinely for histopath sections. Slides were stained by Hematoxylin-Eosin, Picrosirius Red, Alcian Blue, AgNOR, TUNEL and evaluated morphometrically. Cellular density decreased slightly along the time and was higher in the experimental group than in controls at fourth, eighth and sixteenth weeks. Fiber microtearing, percentual of reticular fibers and glycosaminoglycans content increased along the time and were higher in experimental group than in controls at all-time intervals. AgNOR labeling here interpreted as a marker of transcription activity was higher in the experimental groups than in controls at all-time intervals. Apoptotic cells were more frequent and diffusely distributed in tendinosis samples than in control groups. These results suggest that as mechanical overload is becoming chronic, cellular turnover and matrix deposition increases leading to tendinosis. The combination of staining techniques and morphometry used here to describe the evolution of lesions occurring in a rat model system has proved to be suited for the study of induced Achilles tendinosis. PMID:22076169

Alginate Bead-Encapsulated PEDF Induces Ectopic Bone Formation In Vivo in the Absence of Co-Administered Mesenchymal Stem Cells.

PubMed

Elahy, Mina; Doschak, Michael R; Hughes, Jeffery D; Baindur-Hudson, Swati; Dass, Crispin R

2018-01-01

Bone defects can be severely debilitating and reduce quality of life. Osteoregeneration can alleviate some of the complications in bony defects. For therapeutic use in future, a single factor that can cause potent bone regeneration is highly preferred as it will be more costeffective, any off-target effects will be more easily monitored and potentially managed, and for ease of administration which would lead to better patient compliance and satisfaction. We demonstrate that pigment epithelium-derived factor (PEDF), one such factor that is known to be potent against angiogenesis, promotes osteoblastogenesis in mesenchymal stem cells in vitro, but does not need co-encapsulation of cells in alginate bead scaffolds for osteogeneration in vivo. Osteogenic differentiation by PEDF in vitro was confirmed with immunoblotting and immunocytochemical staining for bone markers (alkaline phosphatase, osteocalcin, osteopontin, collagen I), calcified mineral deposition, and assay for alkaline phosphatase activity. PEDF-mediated bone formation in a muscle pocket in vivo model was confirmed by microcomputed tomography (microCT), histology (haematoxylin and eosin, Alcian blue staining), immunostaining for bone markers and for collagen I-processing proteins (heat shock protein 47 and membrane type I matrix metalloproteinase). PEDF therefore presents itself as a promising biological for osteogeneration. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Traumatic brain injury caused by laser-induced shock wave in rats: a novel laboratory model for studying blast-induced traumatic brain injury

NASA Astrophysics Data System (ADS)

Hatano, Ben; Matsumoto, Yoshihisa; Otani, Naoki; Saitoh, Daizoh; Tokuno, Shinichi; Satoh, Yasushi; Nawashiro, Hiroshi; Matsushita, Yoshitaro; Sato, Shunichi

2011-03-01

The detailed mechanism of blast-induced traumatic brain injury (bTBI) has not been revealed yet. Thus, reliable laboratory animal models for bTBI are needed to investigate the possible diagnosis and treatment for bTBI. In this study, we used laser-induced shock wave (LISW) to induce TBI in rats and investigated the histopathological similarities to actual bTBI. After craniotomy, the rat brain was exposed to a single shot of LISW with a diameter of 3 mm at various laser fluences. At 24 h after LISW exposure, perfusion fixation was performed and the extracted brain was sectioned; the sections were stained with hematoxylin-eosin. Evans blue (EB) staining was also used to evaluate disruption of the blood brain barrier. At certain laser fluence levels, neural cell injury and hemorrhagic lesions were observed in the cortex and subcortical region. However, injury was limited in the tissue region that interacted with the LISW. The severity of injury increased with increasing laser fluence and hence peak pressure of the LISW. Fluorescence originating from EB was diffusively observed in the injuries at high fluence levels. Due to the grade and spatial controllability of injuries and the histological observations similar to those in actual bTBI, brain injuries caused by LISWs would be useful models to study bTBI.

A Histopathological Exploration of the Madurella mycetomatis Grain

PubMed Central

Ibrahim, Anahid Izzat; El Hassan, Ahmed Mohammed; Fahal, Ahmed; van de Sande, Wendy W.

2013-01-01

Although the Madurella mycetomatis grains seem to interfere with the host defense mechanisms and impede the antifungal drugs penetration, yet their histological features are not fully known and hence this study was set out to determine that. The study included 80 patients with confirmed M. mycetomatis eumycetoma. After informed written consent, surgical biopsies were obtained from the excised tissues during the patients’ surgical treatment. All sections were stained with haematoxylin and eosin, Grocott’s hexamine silver, Periodic Acid-Schiff’s, Masson-Fontana, Perl’s Prussian Blue, Von-kossa’s, Formalin Inducing Fluorescence and Schmorl’s stains. Modified bleaching technique was used. The concentrations of Zinc, Copper, Calcium, Iron, Lead, Cobalt and Nickel were determined by Atomic Absorption Spectrophotometer. The M. Mycetomatis grains appeared to consist of lipid, protein and melanin. The melanin was located on the hyphal wall as thick layers. The Zinc, Copper and Calcium concentrations in the grains were four, six, and sixteen folds higher than in normal tissue respectively, the other metals were found in the same concentrations as in normal tissue. In the grains, calcium was located in the melanin vicinity. From this study, it can be concluded that, the grains contain melanin, heavy metals, proteins, lipids and they contribute in the formation of the grain cement matrix. These elements seem to contribute in the organism pathogenicity and might impede the penetration of various anti-fungal agents. PMID:23483927

In vivo lymph node mapping by Cadmium Tellurium quantum dots in rats.

PubMed

Si, Chengshuai; Zhang, Yunpeng; Lv, Xianbo; Yang, Wuli; Ran, Zhipeng; Sun, Peng

2014-12-01

Intraoperative lymph node mapping (LNM) is highly significant for many surgeries in patients with cancer. Many types of tracers are currently used, but the ideal method has not yet been identified. We aimed to identify a stable lymphatic drainage pathway in an animal model and compared the effects of quantum dots (QD), a new fluorescent tracer, with those of methylene blue in intraoperative LNM. Indian ink (0.2 mL) was subcutaneously injected into the plantar metatarsal regions of six Sprague-Dawley rats. After 2 wk of incubation and subsequent dissection, the potentially stained LNs were examined pathologically to identify the lymphatic drainage pathway. After applying anesthesia, 0.1 mL methylene blue (2%) and QD (1 mg/mL) were injected into the plantar metatarsal regions of six rats for intraoperative LNM. The QD group was observed with a near-infrared imaging system, and the methylene blue group was directly observed. Drainages were recorded at 5, 10, 30, 60, and 120 min and at 1 d. Two three-level drainage pathways, that is, a peripheral drainage (popliteal LNs, inguinal LNs, and axillary LNs) and a central drainage (popliteal lymph node [LN], iliac LN, and renal LN) pathways were identified. Both methylene blue and QD stained the sentinel lymph node (SLNs) quickly, but methylene blue was difficult to identify in the deep tissues and the LNs beyond the SLN. Furthermore, the blue-stained LNs remain dyed for only 2 h. In contrast, the QDs exhibited high target-to-background ratios in both the SLNs and the following LNs. Additionally, the fluorescence lasted from 5 min-1 d after injection. An ideal lymphatic drainage model was found. QDs are excellent tracers for intraoperative LNM compared with methylene blue. Near infrared fluorescent imaging is a promising LNM method for clinical practice. Copyright © 2014 Elsevier Inc. All rights reserved.

Curcuma longa extract as a histological dye for collagen fibres and red blood cells

PubMed Central

Avwioro, O G; Onwuka, S K; Moody, J O; Agbedahunsi, J M; Oduola, T; Ekpo, O E; Oladele, A A

2007-01-01

Crude ethanolic extract and column chromatographic fractions of the Allepey cultivar of Curcuma longa Roxb, commonly called turmeric (tumeric) in commerce, were used as a stain for tissue sections. Staining was carried out under basic, acidic and neutral media conditions. Inorganic and organic dissolution solvents were used. The stain was used as a counterstain after alum and iron haematoxylins. C. longa stained collagen fibres, cytoplasm, red blood cells and muscle cells yellow. It also stained in a fashion similar to eosin, except for its intense yellow colour. Preliminary phytochemical evaluation of the active column fraction revealed that it contained flavonoids, free anthraquinone and deoxy sugar. A cheap, natural dye can thus be obtained from C. longa. PMID:17451535

Methylene blue intra-arterial staining of resected colorectal cancer specimens improves accuracy of nodal staging: A randomized controlled trial.

PubMed

Reima, H; Saar, H; Innos, K; Soplepmann, J

2016-11-01

Metastatic involvement of regional lymph nodes is a major prognostic factor of colorectal cancer, which influences also its treatment strategy. International consensus foresees retrieval of ≥12 lymph nodes from colorectal specimens. The aim of the study was to assess the effect of intra-arterial staining of colorectal specimens with methylene blue on lymph node harvest. A total of 266 radically operated colorectal cancer patients were randomized into the methylene blue staining and non-staining groups. In the staining group, methylene blue solution was injected into the colorectal specimen's artery after its removal. The specimens were analysed for lymph node count, diameter and metastatic involvement. The median number of lymph nodes was higher in the staining group, 27 (95% CI 23-31%), compared with the control group, 16 (95% CI 14-19, p < 0.001). The number of examined lymph-nodes was ≥12 in 86% of the cases in the staining group and in 69% of the cases in the control group (p = 0.001). In the staining group more small-diameter (≤4 mm) lymph nodes were examined (median number 20.5 vs. 10, p < 0.001). The proportion of patients with metatatic lymph nodes was 42% in the staining group and 43% in the control group (NS). Methylene blue staining improves significantly staging accuracy through finding more small-diameter lymph nodes. It enables to detect ≥12 lymph nodes in the majority of cases. We recommend routine use of this technique in all colorectal resections with curative intent. Copyright © 2016 Elsevier Ltd, BASO ~ the Association for Cancer Surgery, and the European Society of Surgical Oncology. All rights reserved.

Effects of Blue Light Emitting Diode Irradiation On the Proliferation, Apoptosis and Differentiation of Bone Marrow-Derived Mesenchymal Stem Cells.

PubMed

Yuan, Ye; Yan, Gege; Gong, Rui; Zhang, Lai; Liu, Tianyi; Feng, Chao; Du, Weijie; Wang, Ying; Yang, Fan; Li, Yuan; Guo, Shuyuan; Ding, Fengzhi; Ma, Wenya; Idiiatullina, Elina; Pavlov, Valentin; Han, Zhenbo; Cai, Benzhi; Yang, Lei

2017-01-01

Blue light emitting diodes (LEDs) have been proven to affect the growth of several types of cells. The effects of blue LEDs have not been tested on bone marrow-derived mesenchymal stem cells (BMSCs), which are important for cell-based therapy in various medical fields. Therefore, the aim of this study was to determine the effects of blue LED on the proliferation, apoptosis and osteogenic differentiation of BMSCs. BMSCs were irradiated with a blue LED light at 470 nm for 1 min, 5 min, 10 min, 30 min and 60 min or not irradiated. Cell proliferation was measured by performing cell counting and EdU staining assays. Cell apoptosis was detected by TUNEL staining. Osteogenic differentiation was evaluated by ALP and ARS staining. DCFH-DA staining and γ-H2A.X immunostaining were used to measure intracellular levels of ROS production and DNA damage. Both cell counting and EdU staining assays showed that cell proliferation of BMSCs was significantly reduced upon blue LED irradiation. Furthermore, treatment of BMSCs with LED irradiation was followed by a remarkable increase in apoptosis, indicating that blue LED light induced toxic effects on BMSCs. Likewise, BMSC osteogenic differentiation was inhibited after exposure to blue LED irradiation. Further, blue LED irradiation was followed by the accumulation of ROS production and DNA damage. Taken together, our study demonstrated that blue LED light inhibited cell proliferation, inhibited osteogenic differentiation, and induced apoptosis in BMSCs, which are associated with increased ROS production and DNA damage. These findings may provide important insights for the application of LEDs in future BMSC-based therapies. © 2017 The Author(s). Published by S. Karger AG, Basel.

Quantitative assessment of silver-stained nucleolar organizer region in odontogenic cysts to correlate the growth and malignant potentiality

PubMed Central

Biswas, Sailendra Nath; Paul, R R; Ray, Jay Gopal; Majumdar, Sumit; Uppala, Divya

2017-01-01

Context: The most common and important odontogenic cyst involving jaws is the odontogenic keratocyst (OKC) or primordial cyst, the dentigerous cyst and the radicular cyst. These cysts all though do not show similar behavior, they all have the potentiality to recur. Silver nitrate staining of the nucleolar organizer regions (AgNORs) of the benign and malignant lesions is becoming very useful as a diagnostic indicator. Thus, the aim of this study is to assess the diagnostic potential of AgNORs in the cystic epithelium of common odontogenic cysts. Materials and Methods: Archived specimens of odontogenic cysts were stained with hematoxylin and eosin stain and AgNOR stain. Results: The comparative evaluation of the AgNOR counts was done among the three varieties of odontogenic cysts, i.e., radicular cysts, dentigerous cysts and OKC and were observed that the mean for OKC was significantly higher than that of radicular cyst. Conclusion: Therefore, AgNor could be used as an efficient tool for comparative evaluation of microscopic features such as epithelial thickness, surface keratinization and mural proliferation in dentigerous cyst to that of the AgNOR count. PMID:29391734

Comparison of the Cellient(™) automated cell block system and agar cell block method.

PubMed

Kruger, A M; Stevens, M W; Kerley, K J; Carter, C D

2014-12-01

To compare the Cellient(TM) automated cell block system with the agar cell block method in terms of quantity and quality of diagnostic material and morphological, histochemical and immunocytochemical features. Cell blocks were prepared from 100 effusion samples using the agar method and Cellient system, and routinely sectioned and stained for haematoxylin and eosin and periodic acid-Schiff with diastase (PASD). A preliminary immunocytochemical study was performed on selected cases (27/100 cases). Sections were evaluated using a three-point grading system to compare a set of morphological parameters. Statistical analysis was performed using Fisher's exact test. Parameters assessing cellularity, presence of single cells and definition of nuclear membrane, nucleoli, chromatin and cytoplasm showed a statistically significant improvement on Cellient cell blocks compared with agar cell blocks (P < 0.05). No significant difference was seen for definition of cell groups, PASD staining or the intensity or clarity of immunocytochemical staining. A discrepant immunocytochemistry (ICC) result was seen in 21% (13/63) of immunostains. The Cellient technique is comparable with the agar method, with statistically significant results achieved for important morphological features. It demonstrates potential as an alternative cell block preparation method which is relevant for the rapid processing of fine needle aspiration samples, malignant effusions and low-cellularity specimens, where optimal cell morphology and architecture are essential. Further investigation is required to optimize immunocytochemical staining using the Cellient method. © 2014 John Wiley & Sons Ltd.

Brilliant Blue G double staining enhances successful internal limiting membrane peeling with minimal adverse effect by low cellular permeability into live cells.

PubMed

Hisatomi, Toshio; Notomi, Shoji; Tachibana, Takashi; Oishi, Seiichiro; Asato, Ryo; Yamashita, Takehiro; Murakami, Yusuke; Ikeda, Yasuhiro; Enaida, Hiroshi; Sakamoto, Taiji; Ishibashi, Tatsuro

2015-02-01

Brilliant Blue G is used as a surgical adjuvant for retinal surgery. Although BBG double or multiple staining was reported, the effectiveness and safety of repeated staining is still elusive. To further examine the effectiveness and safety, we examined BBG in clinical cases in vivo, primary cell culture in vitro, and surgically resected specimen ex vivo. A retrospective interventional case series with in vitro and ex vivo studies were performed. Vitrectomy was performed in 28 cases of epiretinal membrane with BBG single to multiple staining. The surgically resected membranes were stained by BBG with or without cellular fixation. Primary cell cultures were examined with BBG and live/death cell markers, such as Calcein AM and TUNEL. Single staining provided satisfactory staining in seven cases. Double or multiple staining substantially visualized internal limiting membrane (21 cases), especially the edges of remaining internal limiting membrane (11 cases). Adverse retinal staining was not noted and the final visual acuity showed no difference with multiple staining. The live cells barely stained with BBG, while some dead cells were stained. Brilliant Blue G multiple staining substantially enhanced the visualization of internal limiting membrane. The absence of abnormal staining supports the safety of repeated BBG staining.

Counterion dye staining of proteins in one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and tryptic gel digestion of stained protein for mass spectrometry.

PubMed

Cong, Wei-Tao; Wang, Xu; Hwang, Sun-Young; Jin, Li-Tai; Choi, Jung-Kap

2012-01-01

A fast and matrix-assisted laser desorption/ionization mass spectrometry compatible protein staining method in one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis is described. It is based on the counterion dye staining method that employs oppositely charged two dyes, zincon and ethyl violet, to form an ion-pair complex. The protocol, including fixing, staining, and quick washing steps, can be completed in 1-1.5 h, depending upon gel thickness. It has the sensitivity comparable to the colloidal Coomassie Brilliant Blue G stain using phosphoric acid as a component of staining solution (4-8 ng). The counterion dye stain does not induce protein modifications that complicate interpretation of peptide mapping data from mass spectrometry. Considering the speed, sensitivity, and compatibility with mass spectrometry, the counterion dye stain may be more practical than any other dye-based protein stains for routine proteomic researches.

Natural Intestinal Protozoa in Rodents (Rodentia: Gerbillinae, Murinae, Cricetinae) in Northwestern Iran

PubMed Central

MOHEBALI, Mehdi; ZAREI, Zabiholah; Khanaliha, Khadijeh; KIA, Eshrat Beigom; MOTAVALLI-HAGHI, Afsaneh; DAVOODI, Jaber; REZAEIAN, Tahereh; TARIGHI, Fathemeh; REZAEIAN, Mostafa

2017-01-01

Background: Majority of parasitic infections in rodents have zoonotic importance. This study aimed to determine the frequency and intensity of intestinal protozoa infections of rodents including Meriones persicus, Mus musculus and, Cricetulus migratorius. Methods: This survey was conducted in Meshkin Shahr district in northwestern Iran from Mar. to Dec. of 2014. Intestinal samples of 204 rodents including M. persicus (n=117), M. musculus (n=63) and C. migratorius (n=24) were parasitologically examined. Formalin-ether concentration method was done for all of rodents stool samples and observed with light microscope. All of suspected cases were stained with trichorome staining Method. Cultivation in dichromate potassium 2.5% was carried out for all of coccidian positive samples. Acid fast and aniline blue staining methods were used for detecting of coccidian oocysts and intestinal microsporidial spores, respectively. Results: About 121(59.3%) of the caught rodents were generally infected with intestinal protozoa. Entamoeba muris 14(6.9%), Trichomonas muris 55(27.0%), Chilomastix betencourtti 17 (8.3%), Giardia muris 19(9.3%), Eimeria spp. 46(22.5%), Isospora spp. 4(2%) and Cryptosporidium spp. 1(0.5%) were found from the collected rodents. Microsporidian spores were identified in 63 (31%) out of the 204 collected rodents using aniline blue staining method. Conclusion: Since some of the infections are zoonotic importance thus, control of rodents can be decreased new cases of the parasitic zoonoses in humans. PMID:28979348

Comparison of modified Chicago sky blue stain and potassium hydroxide mount for the diagnosis of dermatomycoses and onychomycoses.

PubMed

Liu, Zhong; Sheng, Ping; Yang, Yan-Ping; Li, Wen; Huang, Wen-Ming; Wang, Jie-Di; Fan, Yi-Ming

2015-05-01

The diagnostic value of modified Chicago sky blue (CSB) stain and potassium hydroxide (KOH) mount for superficial mycoses was compared using fungal culture as gold standard. The sensitivity and screening time of the CSB stain were superior to the KOH mount. The CBS stain is simple, quick and reliable for diagnosing superficial mycoses. Copyright © 2015. Published by Elsevier B.V.

Toluidine blue-O is a Nissl bright-field counterstain for lipophilic fluorescent tracers Di-ASP, DiI and DiO.

PubMed

Chelvanayagam, D K; Beazley, L D

1997-03-01

The stain toluidine blue-O (tol blue), applied to sections of neural tissue, is shown to be compatible with the vivid fluorescent lipophilic neural tracers 4-(4-dihexadecylaminostyryl)-N-methylpyridinium iodide (Di-ASP), 3,3'-dioctadecyloxacarbocyanine perchlorate (DiO) and 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI). As with other Nissl stains, toluidine blue-O fluoresces in the red end of the spectrum but such fluorescence quenches upon binding with tissue. Moreover, progressive staining occurs at concentrations low enough to minimise any background fluorescence attributable to non-specific residence of the stain. The bright yellow Di-ASP and vivid green DiO signals are spectrally removed from the red fluorescence of toluidine blue-O. With toluidine blue-O counterstaining, Di-ASP generally offers contrast superior to that with DiI, however, the latter is improved by viewing in a polarised green bright field. Visible Di-ASP emission, although broad, peaks at a more film-sensitive region of the spectrum than that for DiI, thus reducing the photographic exposure required.

Automated selection of the most epithelium-rich areas in gynecologic tumor sections.

PubMed

Schipper, N W; Baak, J P; Smeulders, A W

1991-12-01

The paper describes an image analysis technique for automated selection of the epithelium-rich areas in standard paraffin tissue sections of ovarian and endometrial premalignancies and malignancies. Two staining procedures were evaluated, Feulgen (pararosanilin) and CAM 5.2, demonstrating the presence of cytokeratin 8 and 18; both were counterstained with naphthol yellow. The technique is based on the corresponding image processing method of automated estimation of the percentage of epithelium in interactively selected microscope fields. With the technique, one image is recorded with a filter to demonstrate where epithelium and stroma lie. This filter is chosen according to the type of staining: it is yellow (lambda = 552 nm) for Feulgen and blue (lambda = 470 nm) for anticytokeratin CAM 5.2. When stroma cannot be distinguished from lumina with the green filter or from epithelium with the blue filter, a second image is recorded from the same microscope field, with a blue filter (lambda = 420 nm) for Feulgen and a yellow filter (lambda = 576 nm) for anticytokeratin CAM 5.2. Discrimination between epithelium and stroma is based on the image contrast range and the packing of nuclei in the yellow image and on the automated classification of the gray value histogram peaks in the blue image. For Feulgen stain the method was evaluated on 30 ovarian tumors of the common epithelial types (8 borderline tumors and 22 carcinomas with various degrees of differentiation) and 30 endometrial carcinomas of different grades.(ABSTRACT TRUNCATED AT 250 WORDS)

Pneumocystis PCR: It Is Time to Make PCR the Test of Choice.

PubMed

Doyle, Laura; Vogel, Sherilynn; Procop, Gary W

2017-01-01

The testing strategy for Pneumocystis at the Cleveland Clinic changed from toluidine blue staining to polymerase chain reaction (PCR). We studied the differences in positivity rates for these assays and compared each with the detection of Pneumocystis in companion specimens by cytology and surgical pathology. We reviewed the results of all Pneumocystis test orders 1 year before and 1 year after the implementation of a Pneumocystis -specific PCR. We also reviewed the corresponding cytology and surgical pathology results, if performed. Finally, we reviewed the medical records of patients with rare Pneumocystis detected by PCR in an effort to differentiate colonization vs true disease. Toluidine blue staining and surgical pathology had similar sensitivities and negative predictive values, both of which were superior to cytology. There was a >4-fold increase in the annual detection of Pneumocystis by PCR compared with toluidine blue staining (toluidine blue staining: 11/1583 [0.69%] vs PCR: 44/1457 [3.0%]; chi-square P < .001). PCR detected 1 more case than surgical pathology and was far more sensitive than cytology. Chart review demonstrated that the vast majority of patients with rare Pneumocystis detected were immunosuppressed, had radiologic findings supportive of this infection, had no other pathogens detected, and were treated for pneumocystosis by the clinical team. PCR was the most sensitive method for the detection of Pneumocystis and should be considered the diagnostic test of choice. Correlation with clinical and radiologic findings affords discrimination of early true disease from the far rarer instances of colonization.

CUTANEOUS ZYGOMYCOSIS DUE TO SAKSENAEA VASIFORMIS IN AN IMMUNOCOMPETENT HOST

PubMed Central

Baradkar, V P; Kumar, S

2009-01-01

Saksenaea vasiformis is an emerging zygomycete species, most often associated with cutaneous, subcutaneous and rhino- orbito-cerebral infections. Herein, we report a case of cutaneous zygomycosis of face caused by Saksenaea vasiformis in a 54-year-old immunocompetent female. The diagnosis was carried out by microscopy using KOH mount, Gram staining, Gomori's methenamine silver staining, hemotoxylin and eosin staining and culture on Sabouraud's Dextrose agar without actidione. Slide cultures were put up on Czapek Dox agar, which showed typical flask-shaped sporangium with rhizoids. The patient was treated successfully with intravenous amphotericin B. PMID:20101346

Detection of Pneumocystis jirovecii by nested PCR in HIV-negative patients with pulmonary disease.

PubMed

Santos, Cristina Rodrigues; de Assis, Ângela M; Luz, Edson A; Lyra, Luzia; Toro, Ivan F; Seabra, José Claudio C; Daldin, Dira H; Marcalto, Tathiane U; Galasso, Marcos T; Macedo, Ronaldo F; Schreiber, Angélica Z; Aoki, Francisco H

Nested PCR can be used to determine the status of Pneumocystis jirovecii infection in other lung diseases. This study sought to detect a target DNA fragment (mitochondrial large subunit rRNA or mtL SUrRNA) of P. jirovecii in patients with lung disease who underwent bronchoscopy with collection of bronchoalveolar lavage (BAL). The results from toluidine blue staining were compared with those obtained using molecular methods that included an "in house" DNA extraction procedure, PCR and nested PCR. Fifty-five BAL samples from patients with atypical chest X-rays were screened for P. jirovecii. None of the samples was positive for P. jirovecii using toluidine blue staining. In contrast, P. jirovecii DNA was detected by nested PCR in BAL samples from 36 of 55 patients (65.5%). The lung diseases in the patients included cancer, pneumonia, tuberculosis, and chronic obstructive pulmonary disease (COPD). Other chronic problems in the patients included hypertension, diabetes, smoking, and alcoholism. Nested PCR showed high sensitivity for detecting P. jirovecii, especially when compared with toluidine blue staining. Using this method, P. jirovecii infection was detected in HIV-negative patients with lung disease. Copyright © 2016 Asociación Española de Micología. Publicado por Elsevier España, S.L.U. All rights reserved.

Immunoperoxidase staining for identification of Aspergillus species in routinely processed tissue sections.

PubMed Central

Verweij, P E; Smedts, F; Poot, T; Bult, P; Hoogkamp-Korstanje, J A; Meis, J F

1996-01-01

AIMS: To evaluate the performance of an immunoperoxidase stain using the monoclonal antibody EB-A1 to detect Aspergillus species in formalin fixed, paraffin wax embedded tissue. METHODS: The monoclonal antibody EB-A1 directed against galactomannan was used to detect Aspergillus species in 23 patients with suspected or confirmed invasive aspergillosis. Immunostaining was performed on formalin fixed, paraffin wax embedded tissue using the streptavidin-biotin method and compared with conventional haematoxylin and eosin, periodic acid-Schiff, and Gomori-Grocott stains. Results of immunostaining were semiquantitatively analysed with regard to both intensity of staining and number of positively staining micro-organisms. Tissue sections from 16 patients with confirmed invasive mycoses due to Candida species, Apophysomyces elegans, Rhizopus oryzae, Pseudallescheria boydii and Histoplasma capsulatum were used as controls. RESULTS: In 19 (83%) of 23 cases invasive aspergillosis was confirmed by both histological examination and culture (18 Aspergillus fumigatus and one A flavus). Immunoperoxidase stains were positive in 17 (89%) of 19 cases including one case of disseminated infection due to A flavus. Furthermore, the immunoperoxidase stain was positive in a culture negative tissue section with histological evidence of mycelial development, indicating the presence of Aspergillus species. Some cross-reactivity was observed with the highly related fungus P boydii, although the number of mycelial elements that stained was low. CONCLUSIONS: Immunoperoxidase staining using the monoclonal antibody EB-A1 performs well on routinely processed tissue sections and permits detection and generic identification of Aspergillus species, although it was no better than conventional histopathology in identifying the presence of an infection. An additional advantage is that the immunostain may help to provide an aetiological diagnosis when cultures remain negative. Images PMID:8943743

Evaluation of hydrogels for soft tissue adhesives in vitro and in vivo analyses

NASA Astrophysics Data System (ADS)

Yuan, Liu; Fan, Wenshuai; Han, Linyingjun; Guo, Changan; Yan, Zuoqin; Zhu, Meifang; Mo, Xiumei

2018-03-01

In this study, natural materials (sodium alginate, dextran, gelatin and carboxymethyl chitosan) were modified to get aldehyde components and amino components. Upon mixing the two-component solutions together, four kinds of Schiff base hydrogels formed successfully within 5-300 s and could seal the wound tissue. The cytotoxicity tests of hydrogel extraction solution confirmed that the hydrogels are nontoxic materials. The adhesive ability was evaluated in vivo by measuring the adhesive strength after sealing the skin incisions on the back of rats. All the hydrogels showed higher adhesive strength than that of commercial fibrin glue and the blank control. The histological staining observation by hematoxylin and eosin staining (HE) and Masson's trichrome staining (MTC) methods suggested that the hydrogels had good biocompatibility and biodegradation in vivo. They have only normal initial inflammation to skin tissue and could improve the formation of new collagen in the incision section. So, the prepared hydrogels were both safe and effective tissue adhesive, which had the great potentials to be used as skin tissue adhesive.

Comparison between Malassezia Folliculitis and Non-Malassezia Folliculitis

PubMed Central

Song, Hyo Sang; Kim, Sue Kyung

2014-01-01

Background Among the various types of folliculitis, differentiation of Malassezia folliculitis (MF) from other forms of folliculitis is important because it is usually treated with antifungal agents. Objective We attempted to find a method to enhance the detection rate of MF, and examined the differences in the clinical manifestation between MF and non-MF (NMF). Methods We performed a retrospective study involving patients with folliculitis who were previously diagnosed with MF or NMF on the basis of serial tissue sectioning and diastase-Periodic acid-Schiff (d-PAS) staining findings. The clinical features of MF and NMF were compared. Results Among a total of 100 folliculitis patients, 20 were diagnosed with MF and 80 with NMF. Tissues from the 80 patients with NMF were sectioned serially into 10 slices and stained with hematoxylin and eosin stain; among these, 10 had many round-to-oval yeast organisms in the hair follicles that confirmed MF. Finally, d-PAS staining was used to detect the presence of yeast in the NMF slides. Notably, among the 70 d-PAS-stained samples, yeast organisms were found in 6 samples, confirming MF. As a result, the diagnosis of 16 patients changed from NMF to MF. Compared with NMF, MF showed major involvement of the trunk and low involvement of the face and legs as well as male predilection. Conclusion Physicians should consider serial sectioning and/or d-PAS staining of folliculitis lesions, particularly of those on the trunk of male patients, even if no yeast organisms are detected initially. PMID:25324652

Evaluation of trypan blue stain in a haemocytometer for rapid detection of cerebrospinal fluid sterility in HIV patients with cryptococcal meningitis.

PubMed

Kwizera, Richard; Akampurira, Andrew; Kandole, Tadeo K; Nielsen, Kirsten; Kambugu, Andrew; Meya, David B; Boulware, David R; Rhein, Joshua

2017-08-22

Quantitative culture is the most common method to determine the fungal burden and sterility of cerebrospinal fluid (CSF) among persons with cryptococcal meningitis. A major drawback of cultures is a long turnaround-time. Recent evidence demonstrates that live and dead Cryptococcus yeasts can be distinguished using trypan blue staining. We hypothesized that trypan blue staining combined with haemocytometer counting may provide a rapid estimation of quantitative culture count and detection of CSF sterility. To test this, we evaluated 194 CSF specimens from 96 HIV-infected participants with cryptococcal meningitis in Kampala, Uganda. Cryptococcal meningitis was diagnosed by CSF cryptococcal antigen (CRAG). We stained CSF with trypan blue and quantified yeasts using a haemocytometer. We compared the haemocytometer readings versus quantitative Cryptococcus CSF cultures. Haemocytometer counting with trypan blue staining had a sensitivity of 98% (64/65), while CSF cultures had a sensitivity of 95% (62/65) with reference to CSF CRAG for diagnostic CSF specimens. For samples that were positive in both tests, the haemocytometer had higher readings compared to culture. For diagnostic specimens, the median of log 10 transformed counts were 5.59 (n = 64, IQR = 5.09 to 6.05) for haemocytometer and 4.98 (n = 62, IQR = 3.75 to 5.79) for culture; while the overall median counts were 5.35 (n = 189, IQR = 4.78-5.84) for haemocytometer and 3.99 (n = 151, IQR = 2.59-5.14) for cultures. The percentage agreement with culture sterility was 2.4% (1/42). Counts among non-sterile follow-up specimens had a median of 5.38 (n = 86, IQR = 4.74 to 6.03) for haemocytometer and 2.89 (n = 89, IQR = 2.11 to 4.38) for culture. At diagnosis, CSF quantitative cultures correlated with haemocytometer counts (R 2  = 0.59, P < 0.001). At 7-14 days, quantitative cultures did not correlate with haemocytometer counts (R 2  = 0.43, P = 0.4). Despite a positive correlation, the haemocytometer counts with trypan blue staining did not predict the outcome of quantitative cultures in patients receiving antifungal therapy.

Desmosomes: A light microscopic and ultrastructural analysis of desmosomes in odontogenic cysts.

PubMed

Raju, Pratima; Wadhwan, Vijay; Chaudhary, Minal S

2014-01-01

Desmosomes together with adherens junctions represent the major adhesive cell-cell junctions of epithelial cells. Any damage to these junctions leads to loss of structural balance. The present study was designed to analyze the desmosomal junctions in different odontogenic cysts and compare them with their corresponding hematoxylin and eosin (H and E)   stained sections. Ten cases each of odontogenic keratocyst (OKC), dentigerous cysts (DCs), radicular cysts (RCs) and normal mucosa were stained with hematoxylin and eosin. Scanning electron microscopy (SEM) analysis of the sections was then carried out of all the sections. The area of interest on H and E stained section was marked and this marking was later superimposed onto the corresponding unstained sections and were subjected to SEM analysis. OKC at ×1000 magnification showed many prominent desmosomes. However, an increase in the intercellular space was also noted. SEM analysis demonstrated similar findings with the presence of many desmosomes, though they were seen to be damaged and fragile. H and E stained DC under oil immersion did not show any prominent desmosomes. SEM analysis of the same confirmed the observation and very minimal number were seen with a very condense arrangement of the epithelial cells. RC at ×1000 magnification revealed plenty of desmosomes, which were again confirmed by SEM. The number and quality of desmosomal junctions in all the cysts has a role in the clinical behavior of the cyst.

Safety evaluation of poly(lactic-co-glycolic acid)/poly(lactic-acid) microspheres through intravitreal injection in rabbits.

PubMed

Rong, Xianfang; Yuan, Weien; Lu, Yi; Mo, Xiaofen

2014-01-01

Poly(lactic-co-glycolic acid) (PLGA) and/or poly(lactic-acid) (PLA) microspheres are important drug delivery systems. This study investigated eye biocompatibility and safety of PLGA/PLA microspheres through intravitreal injection in rabbits. Normal New Zealand rabbits were randomly selected and received intravitreal administration of different doses (low, medium, or high) of PLGA/PLA microspheres and erythropoietin-loaded PLGA/PLA microspheres. The animals were clinically examined and sacrificed at 1, 2, 4, 8, and 12 weeks postadministration, and retinal tissues were prepared for analysis. Retinal reactions to the microspheres were evaluated by terminal deoxynucleotidyl transferase-mediated dUTP nick end staining and glial fibrillary acidic protein immunohistochemistry. Retinal structure changes were assessed by hematoxylin and eosin staining and transmission electron microscopy. Finally, retinal function influences were explored by the electroretinography test. Terminal deoxynucleotidyl transferase-mediated dUTP nick end staining revealed no apoptotic cells in the injected retinas; immunohistochemistry did not detect any increased glial fibrillary acidic protein expression. Hematoxylin and eosin staining and transmission electron microscopy revealed no micro- or ultrastructure changes in the retinas at different time points postintravitreal injection. The electroretinography test showed no significant influence of scotopic or photopic amplitudes. The results demonstrated that PLGA/PLA microspheres did not cause retinal histological changes or functional damage and were biocompatible and safe enough for intravitreal injection in rabbits for controlled drug delivery.

A comparative study for selectivity of micronuclei in oral exfoliated epithelial cells

PubMed Central

Grover, S; Mujib, ABR; Jahagirdar, A; Telagi, N; Kulkarni, PG

2012-01-01

Background: Micronucleus (MN) represents small, additional nuclei formed by the exclusion of chromosome fragments or whole chromosomes lagging at mitosis. MN rates, therefore, indirectly reflect chromosome breakage or impairment of the mitotic apparatus. During the last few decades, micronuclei (“MNi”) in oral exfoliated epithelial cells are widely used as biomarkers of chromosomal damage, genome instability and cancer risk in humans. However, until now only little attention has been given to the effect of different staining procedures on the results of these MN assays. Aim: To compare the MNi frequencies in oral exfoliated epithelial cells using three different stains, i.e.,Feulgen stain, Papanicolaou stain (Pap) and hemotoxylin and eosin stain (H and E). Materials and Methods: Oral exfoliated cells from 45 cases of potentially malignant disorders (15 oral submucous fibrosis, 15 lichen planus and 15 leukoplakia) and 15 controls with healthy mucosa, were taken and MNi frequencies (No. of MNi/1000 cells) were compared using three different stains. Results: Mean MNi frequency in cases was found to be 3.8 with Feulgen stain, 16.8 with PAP and 25.9 with H and E. In controls, mean MNi frequency was 1.6 with Feulgen stain, 7.7 with PAP and 9.6 with H and E stain. Statistically significant value (P < 0.01) were observed when the three stains were compared together using Kruskal Walli's ANOVA test. Conclusion: Feulgen being a DNA-specific stain gave the least counts, although statistically significant results from the comparison of MNi frequency between cases and controls were obtained with all the three stains. PMID:23326025

Examination of West Nile Virus Neuroinvasion and Neuropathogenesis in the Central Nervous System of a Murine Model.

PubMed

Sultana, Hameeda

2016-01-01

West Nile virus (WNV) is a neurotropic virus that causes inflammation and neuronal loss in the Central Nervous System leading to encephalitis and death. In this chapter, detailed methods to detect WNV in the murine brain tissue by quantitative real-time polymerase chain reaction and viral plaque assays are described. Determination of WNV neuropathogenesis by Hematoxylin and Eosin staining and immunohistochemical procedures are provided. In addition, TUNEL assays to determine neuronal loss during WNV neuropathogenesis are discussed in detail. Collectively, the methods mentioned in this chapter provide an overview to understand neuroinvasion and neuropathogenesis in a murine model of WNV infection.

Staining paraffin embedded sections of scald of barley before paraffin removal.

PubMed

Xi, K; Burnett, P A

1997-07-01

Staining of paraffin embedded sections with periodic acid-Schiff reagent and fast green before paraffin removal resulted in differentiation of barley seed and leaf tissue from fungal structures of Rhynchosporium secalis. Crystal violet, toluidine blue O and antiline blue also successfully stained fungal structures of R. secalis in barley leaf tissues. Staining of embedded sections before paraffin removal allows simple processing of a series of sections, saves time and reduces solvent consumption.

Extracorporeal human bone-like tissue generation

PubMed Central

Rosenberg, N.; Rosenberg, O.

2012-01-01

Objectives The need for bone tissue supplementation exists in a wide range of clinical conditions involving surgical reconstruction in limbs, the spine and skull. The bone supplementation materials currently used include autografts, allografts and inorganic matrix components; but these pose potentially serious side-effects. In particular the availability of the autografts is usually limited and their harvesting causes surgical morbidity. Therefore for the purpose of supplementation of autologous bone graft, we have developed a method for autologous extracorporeal bone generation. Methods Human osteoblast-like cells were seeded on porous granules of tricalcium phosphate and incubated in osteogenic media while exposed to mechanical stimulation by vibration in the infrasonic range of frequencies. The generated tissue was examined microscopically following haematoxylin eosin, trichrome and immunohistochemical staining. Results Following 14 days of incubation the generated tissue showed histological characteristics of bone-like material due to the characteristic eosinophilic staining, a positive staining for collagen trichrome and a positive specific staining for osteocalcin and collagen 1. Macroscopically, this tissue appeared in aggregates of between 0.5 cm and 2 cm. Conclusions We present evidence that the interaction of the cellular, inorganic and mechanical components in vitro can rapidly generate three-dimensional bone-like tissue that might be used as an autologous bone graft. PMID:23610651

An optimized color transformation for the analysis of digital images of hematoxylin & eosin stained slides.

PubMed

Zarella, Mark D; Breen, David E; Plagov, Andrei; Garcia, Fernando U

2015-01-01

Hematoxylin and eosin (H&E) staining is ubiquitous in pathology practice and research. As digital pathology has evolved, the reliance of quantitative methods that make use of H&E images has similarly expanded. For example, cell counting and nuclear morphometry rely on the accurate demarcation of nuclei from other structures and each other. One of the major obstacles to quantitative analysis of H&E images is the high degree of variability observed between different samples and different laboratories. In an effort to characterize this variability, as well as to provide a substrate that can potentially mitigate this factor in quantitative image analysis, we developed a technique to project H&E images into an optimized space more appropriate for many image analysis procedures. We used a decision tree-based support vector machine learning algorithm to classify 44 H&E stained whole slide images of resected breast tumors according to the histological structures that are present. This procedure takes an H&E image as an input and produces a classification map of the image that predicts the likelihood of a pixel belonging to any one of a set of user-defined structures (e.g., cytoplasm, stroma). By reducing these maps into their constituent pixels in color space, an optimal reference vector is obtained for each structure, which identifies the color attributes that maximally distinguish one structure from other elements in the image. We show that tissue structures can be identified using this semi-automated technique. By comparing structure centroids across different images, we obtained a quantitative depiction of H&E variability for each structure. This measurement can potentially be utilized in the laboratory to help calibrate daily staining or identify troublesome slides. Moreover, by aligning reference vectors derived from this technique, images can be transformed in a way that standardizes their color properties and makes them more amenable to image processing.

Segmentation of epidermal tissue with histopathological damage in images of haematoxylin and eosin stained human skin

PubMed Central

2014-01-01

Background Digital image analysis has the potential to address issues surrounding traditional histological techniques including a lack of objectivity and high variability, through the application of quantitative analysis. A key initial step in image analysis is the identification of regions of interest. A widely applied methodology is that of segmentation. This paper proposes the application of image analysis techniques to segment skin tissue with varying degrees of histopathological damage. The segmentation of human tissue is challenging as a consequence of the complexity of the tissue structures and inconsistencies in tissue preparation, hence there is a need for a new robust method with the capability to handle the additional challenges materialising from histopathological damage. Methods A new algorithm has been developed which combines enhanced colour information, created following a transformation to the L*a*b* colourspace, with general image intensity information. A colour normalisation step is included to enhance the algorithm’s robustness to variations in the lighting and staining of the input images. The resulting optimised image is subjected to thresholding and the segmentation is fine-tuned using a combination of morphological processing and object classification rules. The segmentation algorithm was tested on 40 digital images of haematoxylin & eosin (H&E) stained skin biopsies. Accuracy, sensitivity and specificity of the algorithmic procedure were assessed through the comparison of the proposed methodology against manual methods. Results Experimental results show the proposed fully automated methodology segments the epidermis with a mean specificity of 97.7%, a mean sensitivity of 89.4% and a mean accuracy of 96.5%. When a simple user interaction step is included, the specificity increases to 98.0%, the sensitivity to 91.0% and the accuracy to 96.8%. The algorithm segments effectively for different severities of tissue damage. Conclusions Epidermal segmentation is a crucial first step in a range of applications including melanoma detection and the assessment of histopathological damage in skin. The proposed methodology is able to segment the epidermis with different levels of histological damage. The basic method framework could be applied to segmentation of other epithelial tissues. PMID:24521154

Chromatic shifts in the fluorescence emitted by murine thymocytes stained with Hoechst 33342.

PubMed

Petersen, Timothy W; Ibrahim, Sherrif F; Diercks, Alan H; van den Engh, Ger

2004-08-01

Many methods in flow cytometry rely on staining DNA with a fluorescent dye to gauge DNA content. From the relative intensity of the fluorescence signature, one can then infer position in cell cycle, amount of DNA (i.e., for sperm selection), or, as in the case of flow karyotyping, to distinguish individual chromosomes. This work examines the staining of murine thymocytes with a common DNA dye, Hoechst 33342, to investigate nonlinearities in the florescence intensity as well as chromatic shifts. Murine thymocytes were stained with Hoechst 33342 and measured in a flow cytometer at two fluorescence emission bands. In other measurements, cells were stained at different dye concentrations, and then centrifuged. The supernatant was then used for a second round of staining to test the amount of dye uptake. Finally, to test for resonant energy transfer, we measured fluorescence anisotropy at two different wavelengths. The fluorescence of cells stained with Hoechst 33342 is a nonlinear process that shows an overall decrease in intensity with increased dye uptake, and spectral shift to the red. Along with the spectral shift of the fluorescence to the longer wavelengths, we document decreases in the fluorescence anisotropy that may indicate resonant energy transfer. At low concentrations, Hoechst 33342 binds to the minor groove of DNA and shows an increase in fluorescence and a blue shift upon binding. At higher concentrations, at which the dye molecules can no longer bind without overlapping, the blue fluorescence decreases and the red fluorescence increases until there is approximately one dye molecule per DNA base pair. The ratio of the blue fluorescence to the red fluorescence is an accurate indicator of the cellular dye concentration.

Alternative to xylene as a clearing agent in histopathology

PubMed Central

Alwahaibi, Nasar; Aljaradi, Shaima; Alazri, Horiyah

2018-01-01

INTRODUCTION: Clearing is an essential step in processing tissue for light microscopy. Xylene is the clearing agent used most commonly worldwide. Xylene is toxic and therefore a threat to personnel working in histopathology laboratories. We evaluated a safer alternative clearing agent for use in the histopathology laboratory. MATERIALS AND METHODS: We used 230 formalin-fixed, paraffin-embedded tissue blocks from 19 different tissues. Half of the specimens were processed using xylene and half were processed using UltraClear™. Tissues were evaluated for eight parameters: sectioning, nuclear staining, cytoplasmic staining, overall cell morphology, clarity of staining, uniformity of staining, quality of immunohistochemistry (IHC), and cost. RESULTS: Both UltraClear™ and xylene processed sections scored 100% for IHC. Sections processed using UltraClear™ were easy to cut (81.7%) as were xylene processed sections (96.5%). UltraClear™ processed sections showed 67%, 60.9%, 52.2%, 63.5%, and 67% for nuclear staining, cytoplasmic staining, cell morphology, clarity of staining, and uniformity of staining, respectively. UltraClear™ is twice as expensive as xylene. We found that tissues processed using UltraClear™ were easy to cut and worked well for both hematoxylin and eosin and IHC staining. CONCLUSION: UltraClear™ is less toxic, less flammable, friendlier to the environment, and easy to handle, but it is two times expensive than xylene. The findings of this study recommend the use of UltraClear™ solution as a routine clearing agent in histopathology laboratories. However, further studies are required. PMID:29692586

Natural Intestinal Protozoa in Rodents (Rodentia: Gerbillinae, Murinae, Cricetinae) in Northwestern Iran.

PubMed

Mohebali, Mehdi; Zarei, Zabiholah; Khanaliha, Khadijeh; Kia, Eshrat Beigom; Motavalli-Haghi, Afsaneh; Davoodi, Jaber; Rezaeian, Tahereh; Tarighi, Fathemeh; Rezaeian, Mostafa

2017-01-01

Majority of parasitic infections in rodents have zoonotic importance. This study aimed to determine the frequency and intensity of intestinal protozoa infections of rodents including Meriones persicus, Mus musculus and, C ricetulus migratorius . This survey was conducted in Meshkin Shahr district in northwestern Iran from Mar. to Dec. of 2014. Intestinal samples of 204 rodents including M. persicus (n=117), M. musculus (n=63) and C. migratorius (n=24) were parasitologically examined. Formalin-ether concentration method was done for all of rodents stool samples and observed with light microscope. All of suspected cases were stained with trichorome staining Method. Cultivation in dichromate potassium 2.5% was carried out for all of coccidian positive samples. Acid fast and aniline blue staining methods were used for detecting of coccidian oocysts and intestinal microsporidial spores, respectively. About 121(59.3%) of the caught rodents were generally infected with intestinal protozoa. Entamoeba muris 14(6.9%), Trichomonas muris 55(27.0%), Chilomastix betencourtti 17 (8.3%), Giardia muris 19(9.3%), Eimeria spp. 46(22.5%) , Isospora spp. 4(2%) and Cryptosporidium spp. 1(0.5%) were found from the collected rodents. Microsporidian spores were identified in 63 (31%) out of the 204 collected rodents using aniline blue staining method. Since some of the infections are zoonotic importance thus, control of rodents can be decreased new cases of the parasitic zoonoses in humans.

[Seven cases of parathyroidectomy for secondary hyperparathyroidism using methylene blue: suggestion for the method of methylene blue infusion].

PubMed

Kadoya, Tatsuo; Kinoshita, Yuki; Shiraishi, Munehiro; Uehara, Hirofumi; Yamamoto, Toshinori; Suetsugu, Keiko

2014-08-01

Intraoperative staining of the parathyroid glands with intravenously administered methylene blue is well described and has been demonstrated as an effective and safe method to facilitate parathyroidectomy. However, there have been several literatures of the development of postoperative neurological toxicity in patients who received methylene blue infusion during parathyroidectomy. We report the method of methylene blue infusion during parathyroidectomy at our institution. Seven adult patients who had undergone parathyroidectomy for secondary hyperparathyroidism associated with chronic renal failure were included in this study. Methylene blue was administered at a constant rate of 4 mg x kg(-1) x hr(-1) with a 1% solution just before the start of operation. The infusion was stopped after the first parathyroid gland was identified. The mean dose of methylene blue used was 2.2 +/- 0.8 mg x kg(-1). Consequently, the dose of methylene blue by this method could be decreased to less than half of the previously administered dose (6 mg x kg(-1)) at our institution. The dose of methylene blue used should be kept to the minimum required to identify the parathyroid glands in each case.

Dimethylformamide interferes with Coomassie dye staining of proteins on blue native gel electrophoresis.

PubMed

Raghupathy, V; Oommen, Anna; Ramachandran, Anup

2014-06-15

Blue native gel electrophoresis (BN-PAGE) is used extensively for characterization of mitochondrial respiratory complexes and uses the binding of Coomassie brilliant blue G-250 to visualize proteins. Oxidative modification of sulfhydryl groups of such proteins can be evaluated by labeling with iodoacetamide conjugated to biotin (BIAM) and detected with streptavidin peroxidase on Western blots following BN-PAGE. However, dissolving BIAM in dimethylformamide, a recommended solvent, reduces Coomassie blue G staining to proteins during BN-PAGE. This interference is prevented by dissolving BIAM in dimethyl sulfoxide. Precautions in the use of the dye for protein staining subsequent to BIAM labeling are discussed. Copyright © 2014 Elsevier Inc. All rights reserved.

Nanocomposites of high-density polyethylene with amorphous calcium phosphate: in vitro biomineralization and cytocompatibility of human mesenchymal stem cells.

PubMed

Hild, Nora; Fuhrer, Roland; Mohn, Dirk; Bubenhofer, Stephanie B; Grass, Robert N; Luechinger, Norman A; Feldman, Kirill; Dora, Claudio; Stark, Wendelin J

2012-10-01

Polyethylene is widely used as a component of implants in medicine. Composites made of high-density polyethylene (HDPE) containing different amounts of amorphous calcium phosphate nanoparticles were investigated concerning their in vitro biomedical performance. The nanoparticles were produced by flame spray synthesis and extruded with HDPE, the latter complying with Food and Drug Administration regulations. Mechanical properties such as Young's modulus and contact angle as well as in vitro biomineralization of the nanocomposites hot-pressed into thin films were evaluated. The deposition of a hydroxyapatite layer occurred upon immersion in simulated body fluid. Additionally, a cell culture study with human mesenchymal stem cells for six weeks allowed a primary assessment of the cytocompatibility. Viability assays (alamarBlue and lactate dehydrogenase detection) proved the absence of cytotoxic effects of the scaffolds. Microscopic images after hematoxylin and eosin staining confirmed typical growth and morphology. A preliminary experiment analyzed the alkaline phosphatase activity after two weeks. These findings motivate further investigations on bioactive HDPE in bone tissue engineering.

Anti-inflammatory effects of tilmicosin in a noninfectious mouse model of allergic asthma.

PubMed

Ci, Xinxin; Chu, Xiao; Xiang, Hua; Li, Xiangchao; Deng, Xuming

2011-12-01

Tilmicosin, a semi-synthetic tylosin-derived macrolide antibiotic commonly used by veterinarians, has been shown to possess anti-inflammatory activity. However, possible use in asthma treatment has not yet been studied. In this study, we investigated the anti-inflammatory properties of tilmicosin using a murine asthma model. BALB/c mice were sensitized and challenged by intraperitoneal (i.p.) or nasal administration of ovalbumin. Tilmicosin (10 and 20 mg/kg) treatment resulted in a marked reduction in the presence of several types of immune cells and cytokines in the bronchoalveolar lavage fluids of mice. Levels of ovalbumin-specific Immunoglobulin E (IgE) were significantly decreased following treatment with tilmicosin (10 and 20 mg/kg). Histological studies using H&E (haematoxylin and eosin) and AB-PAS (alcian blue-periodic acid-Schiff) staining demonstrated that tilmicosin substantially inhibited both ovalbumin-induced inflammatory cells in lung tissues and goblet cell hyperplasia in the airway. These findings provided new insight into the immunopharmacological role of tilmicosin in terms of its effects in a murine model of asthma.

Transitional metaplasia in intestinal epithelium of rats submitted to intestinal cystoplasty and treatment with L -lysine.

PubMed

Santos, Alessandra Marques Dos; Coelho, Joao Paulo Ferreira; Juanes, Camila de Carvalho; Azevedo, Rafael Barbosa de; Diniz, Clara Araujo; Jamacaru, Francisco Vagnaldo Fechine; Dornelas, Conceição Aparecida

2017-04-01

To evaluated the effects of L-lysine on the intestinal and urothelial epithelia in cystoplasty in rats. Twenty-eight 9-week-old rats were assigned to 4 groups: Group A (n=8) cystoplasty followed by administration of L-lysine (150 mg/kg body weight by gavage) for 30 weeks; Group B (n=8) cystoplasty + water for 30 weeks; Group C (n=6) L-lysine for 30 weeks; Group D (n=6) water for 30 weeks. On histopathology with hematoxylin and eosin, mild to moderate hyperplasia transitional was observed in at the site of anastomosis in all animals submitted to cystoplasty (Groups A and B), but "transitional metaplasia" of the intestinal glandular epithelium was more accentuated in Group A (p=0.045). No inflammatory cells, dysplasia or abnormalities were observed. Staining with Alcian blue revealed a substantial reduction of goblet cells and mucins in the colon segment (Groups A and B). The administration of L-lysine to rats accelerated the development of transitional metaplasia in the epithelium of the colon segment in cystoplasty.

Beneficial effects of grape seed proanthocyanidin extract on arterial remodeling in spontaneously hypertensive rats via protecting against oxidative stress.

PubMed

Liang, Ying; Wang, Jian; Gao, Haiqing; Wang, Quanzhen; Zhang, Jun; Qiu, Jie

2016-10-01

Arterial remodeling is a pathogenic occurrence during hypertension and, in turn, is closely associated with the development and complications of hypertension. Grape seed proanthocyanidin extract (GSPE) has been reported to exhibit a protective effect on cardiovascular disease, however its effect on arterial remodeling remains to be fully elucidated. In the present study, the effects of GSPE on arterial remodeling were analyzed by treating spontaneously hypertensive rats (SHRs) with GSPE (250 mg/kg·day). Arterial remodeling was quantified through morphological methods; thoracic aortas were stained with hematoxylin-eosin or sirius red‑victoria blue. The arterial ultrastructure was imaged using transmission electron microscopy. The content of nitric oxide (NO) and endothelin‑1 (ET‑1) were examined to determine endothelial function. Oxidative stress was assessed by malondialdehyde (MDA) levels and the activities of the antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT). Administration of GSPE markedly alleviated hypertension‑induced arterial remodeling, which was not associated with blood pressure control. ET‑1 production was reduced, while NO production was increased in the GSPE group, which exhibited improved endothelial function. In addition, treatment with GSPE significantly ameliorated oxidative stress by improving SOD and CAT activities and reducing MDA formation. In conclusion, GSPE may attenuate hypertension‑induced arterial remodeling by repressing oxidative stress and is recommended as a potential anti‑arterial remodeling agent for patients with hypertensive vascular diseases.

Comparison of aerobic conjunctival bacterial flora in pregnant, reproductive-aged and postmenopausal women

PubMed Central

Balikoglu-Yilmaz, Melike; Sen, Emine; Sevket, Osman; Polat, Yusuf; Karabulut, Aysun; Uysal, Omer

2012-01-01

AIM To evaluate the effect of hormonal status on aerobic conjunctival flora in women. METHODS One hundred fifty-eight women [reproductive-aged (n=55), pregnant (n=51), and postmenopausal (n=52)] who admitted to outpatient clinic of Obstetrics and Gynecology Department of Denizli State Hospital were enrolled. Age, body-mass index (BMI), obstetric history, cigarette smoking, drug usage, presence of systemic disease, and intraocular pressure (IOP) were recorded for each patient. The samples were taken from the lower fornix with two culture swabs and directly incubated in culture containing 5% sheep blood, eosin-methylene blue and chocolate agar. The other swab specimen was Gram stained. All growths and microscopic results were analyzed. RESULTS The coagulase-negative Staphylococcus was the predominant organism isolated in the conjunctival samples in both three groups. The aerobic microorganism growth rate for all isolated aerobic organisms revealed no significant change in the three groups (P >0.05). The conjunctival culture positivity rates were similar in the three groups (49% in reproductive-aged, 57% in pregnant and 58% in postmenopausal women) (P >0.05). Age, IOP, BMI, gravidity, parity, cigarette smoking, drug usage, and presence of systemic diseases did not have an effect on culture positivity in three groups. CONCLUSION Results of this study showed that conjunctival aerobic flora and bacterial colonization did not differ between reproductive-aged, pregnant and postmenopausal women. PMID:23275909

Automated classification of immunostaining patterns in breast tissue from the human protein atlas.

PubMed

Swamidoss, Issac Niwas; Kårsnäs, Andreas; Uhlmann, Virginie; Ponnusamy, Palanisamy; Kampf, Caroline; Simonsson, Martin; Wählby, Carolina; Strand, Robin

2013-01-01

The Human Protein Atlas (HPA) is an effort to map the location of all human proteins (http://www.proteinatlas.org/). It contains a large number of histological images of sections from human tissue. Tissue micro arrays (TMA) are imaged by a slide scanning microscope, and each image represents a thin slice of a tissue core with a dark brown antibody specific stain and a blue counter stain. When generating antibodies for protein profiling of the human proteome, an important step in the quality control is to compare staining patterns of different antibodies directed towards the same protein. This comparison is an ultimate control that the antibody recognizes the right protein. In this paper, we propose and evaluate different approaches for classifying sub-cellular antibody staining patterns in breast tissue samples. The proposed methods include the computation of various features including gray level co-occurrence matrix (GLCM) features, complex wavelet co-occurrence matrix (CWCM) features, and weighted neighbor distance using compound hierarchy of algorithms representing morphology (WND-CHARM)-inspired features. The extracted features are used into two different multivariate classifiers (support vector machine (SVM) and linear discriminant analysis (LDA) classifier). Before extracting features, we use color deconvolution to separate different tissue components, such as the brownly stained positive regions and the blue cellular regions, in the immuno-stained TMA images of breast tissue. We present classification results based on combinations of feature measurements. The proposed complex wavelet features and the WND-CHARM features have accuracy similar to that of a human expert. Both human experts and the proposed automated methods have difficulties discriminating between nuclear and cytoplasmic staining patterns. This is to a large extent due to mixed staining of nucleus and cytoplasm. Methods for quantification of staining patterns in histopathology have many applications, ranging from antibody quality control to tumor grading.

Comparison of two modalities: a novel technique, 'chromohysteroscopy', and blind endometrial sampling for the evaluation of abnormal uterine bleeding.

PubMed

Alay, Asli; Usta, Taner A; Ozay, Pinar; Karadugan, Ozgur; Ates, Ugur

2014-05-01

The objective of this study was to compare classical blind endometrial tissue sampling with hysteroscopic biopsy sampling following methylene blue dyeing in premenopausal and postmenopausal patients with abnormal uterine bleeding. A prospective case-control study was carried out in the Office Hysteroscopy Unit. Fifty-four patients with complaints of abnormal uterine bleeding were evaluated. Data of 38 patients were included in the statistical analysis. Three groups were compared by examining samples obtained through hysteroscopic biopsy before and after methylene blue dyeing, and classical blind endometrial tissue sampling. First, uterine cavity was evaluated with office hysteroscopy. Methylene blue dye was administered through the hysteroscopic inlet. Tissue samples were obtained from stained and non-stained areas. Blind endometrial sampling was performed in the same patients immediately after the hysteroscopy procedure. The results of hysteroscopic biopsy from methylene blue stained and non-stained areas and blind biopsy were compared. No statistically significant differences were determined in the comparison of biopsy samples obtained from methylene-blue stained, non-stained areas and blind biopsy (P > 0.05). We suggest that chromohysteroscopy is not superior to endometrial sampling in cases of abnormal uterine bleeding. Further studies with greater sample sizes should be performed to assess the validity of routine use of endometrial dyeing. © 2014 The Authors. Journal of Obstetrics and Gynaecology Research © 2014 Japan Society of Obstetrics and Gynecology.

A novel gelatin hydrogel carrier sheet for corneal endothelial transplantation.

PubMed

Watanabe, Ryou; Hayashi, Ryuhei; Kimura, Yu; Tanaka, Yuji; Kageyama, Tomofumi; Hara, Susumu; Tabata, Yasuhiko; Nishida, Kohji

2011-09-01

We examined the feasibility of using gelatin hydrogels as carrier sheets for the transplantation of cultivated corneal endothelial cells. The mechanical properties, transparency, and permeability of gelatin hydrogel sheets were compared with those of atelocollagen sheets. Immunohistochemistry (ZO-1, Na(+)/K(+)-ATPase, and N-cadherin), hematoxylin and eosin staining, and scanning electron microscopy were performed to assess the integrity of corneal endothelial cells that were cultured on gelatin hydrogel sheets. The gelatin hydrogel sheets displayed greater transparency, elastic modulus, and albumin permeability compared to those of atelocollagen sheets. The corneal endothelial cells on gelatin hydrogel sheets showed normal expression levels of ZO-1, Na(+)/K(+)-ATPase, and N-cadherin. Hematoxylin and eosin staining revealed the formation of a continuous monolayer of cells attached to the gelatin hydrogel sheet. Scanning electron microscopy observations showed that the corneal endothelial cells were arranged in a regular, mosaic, and polygonal pattern with normal cilia. These results indicate that the gelatin hydrogel sheet is a promising material to transport corneal endothelial cells during transplantation.

Combining platelet-rich plasma and tissue-engineered skin in the treatment of large skin wound.

PubMed

Han, Tong; Wang, Hao; Zhang, Ya Qin

2012-03-01

The objective of the study was to observe the effects of tissue-engineered skin in combination with platelet-rich plasma (PRP) and other preparations on the repair of large skin wound on nude mice.We first prepared PRP from venous blood by density-gradient centrifugation. Large skin wounds were created surgically on the dorsal part of nude mice. The wounds were then treated with either artificial skin, tissue-engineered skin, tissue-engineered skin combined with basic fibroblast growth factor, tissue-engineered skin combined with epidermal growth factor, or tissue-engineered skin combined with PRP. Tissue specimens were collected at different time intervals after surgery. Hematoxylin-eosin and periodic acid-Schiff staining and immunohistochemistry were performed to assess the rate of wound healing.Macroscopic observations, hematoxylin-eosin/periodic acid-Schiff staining, and immunohistochemistry revealed that the wounds treated with tissue-engineered skin in combination with PRP showed the most satisfactory wound recovery, among the 5 groups.

Computed tomography guided percutaneous injection of a mixture of lipiodol and methylene blue in rabbit lungs: evaluation of localization ability for video-assisted thoracoscopic surgery.

PubMed

Jin, Kwang Nam; Lee, Kyung Won; Kim, Tae Jung; Song, Yong Sub; Kim, Dong Il

2014-01-01

Preoperative localization is necessary prior to video assisted thoracoscopic surgery for the detection of small or deeply located lung nodules. We compared the localization ability of a mixture of lipiodol and methylene blue (MLM) (0.6 mL, 1:5) to methylene blue (0.5 mL) in rabbit lungs. CT-guided percutaneous injections were performed in 21 subjects with MLM and methylene blue. We measured the extent of staining on freshly excised lung and evaluated the subjective localization ability with 4 point scales at 6 and 24 hr after injections. For MLM, radio-opacity was evaluated on the fluoroscopy. We considered score 2 (acceptable) or 3 (excellent) as appropriate for localization. The staining extent of MLM was significantly smaller than methylene blue (0.6 vs 1.0 cm, P<0.001). MLM showed superior staining ability over methylene blue (2.8 vs 2.2, P=0.010). Excellent staining was achieved in 17 subjects (81%) with MLM and 8 (38%) with methylene blue (P=0.011). An acceptable or excellent radio-opacity of MLM was found in 13 subjects (62%). An appropriate localization rate of MLM was 100% with the use of the directly visible ability and radio-opacity of MLM. MLM provides a superior pulmonary localization ability over methylene blue.

Computed Tomography Guided Percutaneous Injection of a Mixture of Lipiodol and Methylene Blue in Rabbit Lungs: Evaluation of Localization Ability for Video-Assisted Thoracoscopic Surgery

PubMed Central

Jin, Kwang Nam; Kim, Tae Jung; Song, Yong Sub; Kim, Dong Il

2014-01-01

Preoperative localization is necessary prior to video assisted thoracoscopic surgery for the detection of small or deeply located lung nodules. We compared the localization ability of a mixture of lipiodol and methylene blue (MLM) (0.6 mL, 1:5) to methylene blue (0.5 mL) in rabbit lungs. CT-guided percutaneous injections were performed in 21 subjects with MLM and methylene blue. We measured the extent of staining on freshly excised lung and evaluated the subjective localization ability with 4 point scales at 6 and 24 hr after injections. For MLM, radio-opacity was evaluated on the fluoroscopy. We considered score 2 (acceptable) or 3 (excellent) as appropriate for localization. The staining extent of MLM was significantly smaller than methylene blue (0.6 vs 1.0 cm, P<0.001). MLM showed superior staining ability over methylene blue (2.8 vs 2.2, P=0.010). Excellent staining was achieved in 17 subjects (81%) with MLM and 8 (38%) with methylene blue (P=0.011). An acceptable or excellent radio-opacity of MLM was found in 13 subjects (62%). An appropriate localization rate of MLM was 100% with the use of the directly visible ability and radio-opacity of MLM. MLM provides a superior pulmonary localization ability over methylene blue. PMID:24431917

Antibody-supervised deep learning for quantification of tumor-infiltrating immune cells in hematoxylin and eosin stained breast cancer samples

PubMed Central

Turkki, Riku; Linder, Nina; Kovanen, Panu E.; Pellinen, Teijo; Lundin, Johan

2016-01-01

Background: Immune cell infiltration in tumor is an emerging prognostic biomarker in breast cancer. The gold standard for quantification of immune cells in tissue sections is visual assessment through a microscope, which is subjective and semi-quantitative. In this study, we propose and evaluate an approach based on antibody-guided annotation and deep learning to quantify immune cell-rich areas in hematoxylin and eosin (H&E) stained samples. Methods: Consecutive sections of formalin-fixed parafin-embedded samples obtained from the primary tumor of twenty breast cancer patients were cut and stained with H&E and the pan-leukocyte CD45 antibody. The stained slides were digitally scanned, and a training set of immune cell-rich and cell-poor tissue regions was annotated in H&E whole-slide images using the CD45-expression as a guide. In analysis, the images were divided into small homogenous regions, superpixels, from which features were extracted using a pretrained convolutional neural network (CNN) and classified with a support of vector machine. The CNN approach was compared to texture-based classification and to visual assessments performed by two pathologists. Results: In a set of 123,442 labeled superpixels, the CNN approach achieved an F-score of 0.94 (range: 0.92–0.94) in discrimination of immune cell-rich and cell-poor regions, as compared to an F-score of 0.88 (range: 0.87–0.89) obtained with the texture-based classification. When compared to visual assessment of 200 images, an agreement of 90% (κ = 0.79) to quantify immune infiltration with the CNN approach was achieved while the inter-observer agreement between pathologists was 90% (κ = 0.78). Conclusions: Our findings indicate that deep learning can be applied to quantify immune cell infiltration in breast cancer samples using a basic morphology staining only. A good discrimination of immune cell-rich areas was achieved, well in concordance with both leukocyte antigen expression and pathologists’ visual assessment. PMID:27688929

Effect of lipiodol and methylene blue on the thoracoscopic preoperative positioning.

PubMed

Zhang, Chuan-Yu; Yu, Hua-Long; Liu, Shi-He; Jiang, Gang; Wang, Yong-Jie

2015-01-01

The aim of this study was to compare and analyze the site-specific accuracy of mixture of lipiodol and methylene blue (MLM) (0.6 ml, 1:5) and pure methylene blue (0.5 ml) on the rabbit lungs. In this study, CT-guided percutaneous injection of MLM and methylene blue. Compare the staining degree by biopsy of lung tissue. Use 4 points system to evaluate the site-specific accuracy at 6h and 24 h after injection. For MLM, evaluate its radiopacity by radiation. When evaluate the positioning, 2 points mean acceptable, 3 points mean excellent. The results indicated that the staining range of MLM is obvious less than that of methylene blue (0.6 vs. 1.0 cm, P<0.01), but the staining capacity of MLM is higher than that of methylene blue (2.8 vs. 2.2, P = 0.01). About the staining abilities which are evaluated as excellent, MLM group accounts for 81%, methylene blue group accounts for 38% (P = 0.011). About the radiopacity which are evaluated as acceptable or excellent, MLM group accounts for 62%. With good direct vision, the suitable positioning rate of MLM can be 100%, which is better than that of methylene blue. In conclusion, percutaneous injection of MLM can be used to lung positioning. The result shows that use MLM is better than only using methylene blue. But it is necessary to do the investigation in human beings in order to confirm the feasibility of its clinical application.

Effect of lipiodol and methylene blue on the thoracoscopic preoperative positioning

PubMed Central

Zhang, Chuan-Yu; Yu, Hua-Long; Liu, Shi-He; Jiang, Gang; Wang, Yong-Jie

2015-01-01

The aim of this study was to compare and analyze the site-specific accuracy of mixture of lipiodol and methylene blue (MLM) (0.6 ml, 1:5) and pure methylene blue (0.5 ml) on the rabbit lungs. In this study, CT-guided percutaneous injection of MLM and methylene blue. Compare the staining degree by biopsy of lung tissue. Use 4 points system to evaluate the site-specific accuracy at 6h and 24 h after injection. For MLM, evaluate its radiopacity by radiation. When evaluate the positioning, 2 points mean acceptable, 3 points mean excellent. The results indicated that the staining range of MLM is obvious less than that of methylene blue (0.6 vs. 1.0 cm, P<0.01), but the staining capacity of MLM is higher than that of methylene blue (2.8 vs. 2.2, P = 0.01). About the staining abilities which are evaluated as excellent, MLM group accounts for 81%, methylene blue group accounts for 38% (P = 0.011). About the radiopacity which are evaluated as acceptable or excellent, MLM group accounts for 62%. With good direct vision, the suitable positioning rate of MLM can be 100%, which is better than that of methylene blue. In conclusion, percutaneous injection of MLM can be used to lung positioning. The result shows that use MLM is better than only using methylene blue. But it is necessary to do the investigation in human beings in order to confirm the feasibility of its clinical application. PMID:26221301

Prevalence of mabDAS-1 positivity in biopsy specimens from the esophagogastric junction.

PubMed

Rogge-Wolf, Claudia; Seldenrijk, Cornelis A; Das, Kiron M; Timmer, Robin; Breumelhof, Ronald; Smout, André J P M; Amenta, Peter S; Griffel, Louis H

2002-12-01

Intestinal metaplasia (IM) is a precursor for malignancies at the esophagogastric junction. A monoclonal antibody, mAbDAS-1, can probably identify cellular characteristics of IM before the appearance of goblet cells. The aim of this study was to examine the prevalence of mAbDAS-1 positivity in biopsies from the squamocolumnar junction (SCJ) and to correlate this positivity with the presence of IM and clinical findings. In 559 patients, reflux symptoms were scored, and the presence of reflux esophagitis and hiatus hernia was evaluated during endoscopy. Two biopsy specimens were obtained from the SCJ. In a subset of patients (n = 99), biopsies from the endoscopically defined cardiac region (2 cm distal to proximal margin of gastric folds) were available. Biopsy specimens were stained with hematoxylin and eosin, Alcian Blue, modified Giemsa, and mAbDAS-1. mAbDAS-1 positivity was observed in the SCJ biopsies of 201 of 486 (41.4%) patients without IM and in 64 of 73 (87.7%) patients with IM. Patients without IM but with antibody positivity showed similar histological characteristics as patients with IM at the SCJ. Biopsies of 123 of 559 patients (22%) revealed a columnar-cuboidal epithelium, which was found to be mAbDAS-1 positive in 64.2% (77 of 123). Tissue specimens from the cardiac region without IM stained positive in 14.2% (13 of 91), 12 of those also stained at the SCJ. In patients without IM, a high prevalence of mAbDAS-1 positivity was observed. Biopsies of these patients showed similar histological characteristics as patients with IM. Although not all patients exhibiting this reactivity may develop IM, mAbDAS-1 reactivity may help in the understanding of the histogenesis of IM at the SCJ.

Neutrophil Extracellular Traps and Fibrin in Otitis Media: Analysis of Human and Chinchilla Temporal Bones.

PubMed

Schachern, Patricia A; Kwon, Geeyoun; Briles, David E; Ferrieri, Patricia; Juhn, Steven; Cureoglu, Sebahattin; Paparella, Michael M; Tsuprun, Vladimir

2017-10-01

Bacterial resistance in acute otitis can result in bacterial persistence and biofilm formation, triggering chronic and recurrent infections. To investigate the middle ear inflammatory response to bacterial infection in human and chinchilla temporal bones. Six chinchillas underwent intrabullar inoculations with 0.5 mL of 106 colony-forming units (CFUs) of Streptococcus pneumoniae, serotype 2. Two days later, we counted bacteria in middle ear effusions postmortem. One ear from each chinchilla was processed in paraffin and sectioned at 5 µm. The opposite ear was embedded in epoxy resin, sectioned at a thickness of 1 µm, and stained with toluidine blue. In addition, we examined human temporal bones from 2 deceased donors with clinical histories of otitis media (1 with acute onset otitis media, 1 with recurrent infection). Temporal bones had been previously removed at autopsy, processed, embedded in celloidin, and cut at a thickness of 20 µm. Sections of temporal bones from both chinchillas and humans were stained with hematoxylin-eosin and immunolabeled with antifibrin and antihistone H4 antibodies. Histopatological and imminohistochemical changes owing to otitis media. Bacterial counts in chinchilla middle ear effusions 2 days after inoculation were approximately 2 logs above initial inoculum counts. Both human and chinchilla middle ear effusions contained bacteria embedded in a fibrous matrix. Some fibers in the matrix showed positive staining with antifibrin antibody, others with antihistone H4 antibody. In acute and recurrent otitis media, fibrin and neutrophil extracellular traps (NETs) are part of the host inflammatory response to bacterial infection. In the early stages of otitis media the host defense system uses fibrin to entrap bacteria, and NETs function to eliminate bacteria. In chronic otitis media, fibrin and NETs appear to persist.

Histopathology of normal skin and melanomas after nanosecond pulsed electric field treatment.

PubMed

Chen, Xinhua; James Swanson, R; Kolb, Juergen F; Nuccitelli, Richard; Schoenbach, Karl H

2009-12-01

Nanosecond pulsed electric fields (nsPEFs) can affect the intracellular structures of cells in vitro. This study shows the direct effects of nsPEFs on tumor growth, tumor volume, and histological characteristics of normal skin and B16-F10 melanoma in SKH-1 mice. A melanoma model was set up by injecting B16-F10 into female SKH-1 mice. After a 100-pulse treatment with an nsPEF (40-kV/cm field strength; 300-ns duration; 30-ns rise time; 2-Hz repetition rate), tumor growth and histology were studied using transillumination, light microscopy with hematoxylin and eosin stain and transmission electron microscopy. Melanin and iron within the melanoma tumor were also detected with specific stains. After nsPEF treatment, tumor development was inhibited with decreased volumes post-nsPEF treatment compared with control tumors (P<0.05). The nsPEF-treated tumor volume was reduced significantly compared with the control group (P<0.01). Hematoxylin and eosin stain and transmission electron microscopy showed morphological changes and nuclear shrinkage in the tumor. Fontana-Masson stain indicates that nsPEF can externalize the melanin. Iron stain suggested nsPEF caused slight hemorrhage in the treated tissue. Histology confirmed that repeated applications of nsPEF disrupted the vascular network. nsPEF treatment can significantly disrupt the vasculature, reduce subcutaneous murine melanoma development, and produce tumor cell contraction and nuclear shrinkage while concurrently, but not permanently, damaging peripheral healthy skin tissue in the treated area, which we attribute to the highly localized electric fields surrounding the needle electrodes.

Expanding the histologic spectrum of mucinous tubular and spindle cell carcinoma of the kidney.

PubMed

Fine, Samson W; Argani, Pedram; DeMarzo, Angelo M; Delahunt, Brett; Sebo, Thomas J; Reuter, Victor E; Epstein, Jonathan I

2006-12-01

Mucinous tubular and spindle cell carcinomas (MTSCs) are polymorphic neoplasms characterized by small, elongated tubules lined by cuboidal cells and/or cords of spindled cells separated by pale mucinous stroma. Nonclassic morphologic variants and features of MTSC have not been well studied. We identified 17 previously unreported MTSCs from Surgical Pathology and consultative files of the authors and their respective institutions and studied their morphologic features. A total of 10/17 cases were considered "classic," as described above, with 5/10 showing at least focal (20% to 50%) tubular predominance without apparent mucinous matrix. Alcian blue staining revealed abundant (>50%) mucin in all classic cases. Seven of 17 MTSCs were classified as "mucin-poor," with little to no extracellular mucin appreciable by hematoxylin and eosin. Four of these cases showed equal tubular and spindled morphology, 2 cases showed spindle cell predominance (70%; 95%), and 1 case showed tubular predominance (90%). In 5/7 mucin-poor cases, staining for Alcian blue revealed scant (<10%) mucin in cellular areas with the other 2 cases having 30% mucin. Unusual histologic features identified in the 17 cases were: foamy macrophages (n=8), papillations/well formed papillae (n=6/n=1), focal clear cells in tubules (n=3), necrosis (n=3), oncocytic tubules (n=2; 40%, 5%), numerous small vacuoles (n=2), heterotopic bone (n=1), psammomatous calcification (n=1), and nodular growth with lymphocytic cuffing (n=1). An exceptional case contained a well-circumscribed, HMB45-positive angiomyolipoma within the MTSC. MTSCs may be "mucin-poor" and show a marked predominance of either of its principal morphologic components, which coupled with the presence of other unusual features such as clear cells, papillations, foamy macrophages, and necrosis, may mimic other forms of renal cell carcinoma. Pathologists must be aware of the spectrum of histologic findings within MTSCs to ensure their accurate diagnosis.

Chronological changes of radiofrequency ablation zone in rabbit liver: an in vivo correlation between gross pathology and histopathology

PubMed Central

Song, Kyoung D; Rhim, Hyunchul; Kang, Tae Wook; Cha, Dong Ik; Yang, Jehoon

2017-01-01

Objective: To examine the gross pathology and histopathology of ablation zones created from radiofrequency (RF) ablation and to correlate their chronological changes. Methods: A total of 48 in vivo ablation zones (16 rabbit livers) were obtained immediately after and also 30 min, 1 h and 2 h after RF ablation and were subjected to haematoxylin and eosin (H&E) staining, nicotinamide adenine dinucleotide (NADH) diaphorase staining, terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) staining. Chronological changes in gross pathology and histopathology were evaluated and correlated with each other. Results: Peripheral red zones on gross pathology correlated with peripheral zones on H&E staining, lightly stained peripheral zones on NADH staining and peripheral positive zones on TUNEL staining. Central white zones on gross pathology correlated with combined central and border zones on H&E staining, central negative zones on NADH staining and combined central-positive and middle-negative zones on TUNEL staining. Boundary visibility between central white and peripheral red zones on gross pathology was significantly higher at 1 and 2 h than immediately after RF ablation. As time increased after RF ablation, visibility of the border zone on H&E staining and the grade of positively stained hepatocytes in the peripheral zone on TUNEL staining increased. Conclusion: Chronological changes in gross pathology of RF ablation zones correlated well with histopathology. The boundary between the central white and peripheral red zones tended to become clear at 1 h after RF ablation. Advances in knowledge: (1) RF ablation zones show chronological changes on gross pathology and histopathology. (2) Gross pathology and histopathology correlate well with each other. PMID:28139942

Guided surgical debridement: staining tissues with methylene blue.

PubMed

Dorafshar, Amir H; Gitman, Marina; Henry, Ginard; Agarwal, Shailesh; Gottlieb, Lawrence J

2010-01-01

Precise surgical debridement of wounds is required to achieve wound closure. The authors describe their experience with a technique using topical methylene blue to facilitate precise surgical debridement. In this technique, methylene blue dye is applied topically to the wound surface at the onset of surgery. The stained wound site is then wiped to remove dye from the surface of normal epithelium; eschar, nonviable tissue, and granulation tissue remain stained. The methylene blue-stained tissue is surgically removed, and the newly debrided surface of the wound is assessed for adequate vascularity and biopsied to verify presence of bacteriologic balance before closure. The authors have used this technique in more than 200 wound debridements during the past year, including acute surgical or traumatic wounds, acute and subacute burn wounds, chronic granulating wounds, partially epithelialized wounds, sinus tracts, and fistulae. No adverse reactions have been noted, even on patients undergoing multiple applications through serial operations. Topical application of methylene blue to wounds with mixed tissue content helps to distinguish between viable and nonviable tissue and between epithelialized and nonepithelialized areas, facilitating more precise and complete wound debridement.

In vivo cartilage formation using chondrogenic-differentiated human adipose-derived mesenchymal stem cells mixed with fibrin glue.

PubMed

Jung, Sung-No; Rhie, Jong Won; Kwon, Ho; Jun, Young Joon; Seo, Je-Won; Yoo, Gyeol; Oh, Deuk Young; Ahn, Sang Tae; Woo, Jihyoun; Oh, Jieun

2010-03-01

Human adipose-derived mesenchymal stem cells (MSCs) were differentiated into chondrogenic MSCs, and fibrin glue was used together to explore the feasibility of whether cartilages can be generated in vivo by injecting the differentiated cells. Mesenchymal stem cells extracted from human adipose were differentiated into chondrogenic MSCs, and such differentiated cells mixed with fibrin glue were injected subcutaneously into the back of the nude mouse. In addition to visual evaluation of the tissues formed after 4, 8, and 12 weeks, hematoxylin-eosin staining, Masson trichrome staining, measurement of glycosaminoglycan concentration using dimethylmethylene blue, agreecan through reverse transcriptase-polymerase chain reaction, type II collagen, and expression of SOX-9 were verified. Moreover, the results were compared with 2 groups of controls: 1 control group that received only injection of chondrogenic-differentiated MSC and the supporting control group that received only fibrin glue injection. For the experimental group, cartilage-like tissues were formed after 4, 8, and 12 weeks. Formation of cartilage tissues was not observed in any of 4, 8, and 12 weeks of the control group. The supporting control group had only a small structure formation after 4 weeks, but the formed structure was completely decomposed by the 8th and 12th weeks. The range of staining dramatically increased with time at 4, 8, and 12 weeks in Masson trichrome staining. The concentration of glycosaminoglycan also increased with time. The increased level was statistically significant with more than 3 times more after 8 weeks compared with 4 weeks and more than 2 times more after 12 weeks compared with 8 weeks. Also, in reverse transcriptase-polymerase chain reaction at 4, 8, and 12 weeks, all results expressed a cartilage-specific gene called aggrecan, type II collagen, and SOX-9. The study verified that the chondrogenic-differentiated MSCs derived from human adipose tissues with fibrin glue can proliferate and form new cartilage. Our findings suggest that formation of cartilages in vivo is possible.

A new rapid immunohistochemical staining technique using the EnVision antibody complex.

PubMed

Kämmerer, U; Kapp, M; Gassel, A M; Richter, T; Tank, C; Dietl, J; Ruck, P

2001-05-01

Rapid immunohistochemical investigation, in addition to staining with hematoxylin and eosin, would be useful during intraoperative frozen section diagnosis in some cases. This study was undertaken to investigate whether the recently described EnVision system, a highly sensitive two-step immunohistochemical technique, could be modified for rapid immunostaining of frozen sections. Forty-five primary antibodies were tested on frozen sections from various different tissues. After fixation in acetone for 1 min and air-drying, the sections were incubated for 3 min each with the primary antibody, the EnVision complex (a large number of secondary antibodies and horseradish peroxidase coupled to a dextran backbone), and the chromogen (3,3'diaminobenzidine or 3-amino-9-ethylcarbazole). All reactions were carried out at 37C. Specific staining was seen with 38 antibodies (including HMB-45 and antibodies against keratin, vimentin, leukocyte common antigen, smooth muscle actin, synaptophysin, CD34, CD3, CD20, and prostate-specific antigen). A modification of the EnVision method allows the detection of a broad spectrum of antigens in frozen sections in less than 13 min. This method could be a useful new tool in frozen section diagnosis and research. (J Histochem Cytochem 49:623-630, 2001)

Prediction of wood mechanical and chemical properties in the presence and absence of blue stain using two near infrared instruments

Treesearch

Brian K. Via; Chi-Leung So; Todd F. Shupe; Lori G. Eckhardt; Michael Stine; Leslie H. Groom

2005-01-01

The objective of this research was to (a) determine if blue stain in solid wood influenced calibration equations developed from a nonstained wood population, (b) assess the bias introduced when scanning was performed by the slave instrument without calibration transfer from the master instrument and (c) partition absorbance-based variation by instrument, stain and...

Methyl green and nitrotetrazolium blue chloride co-expression in colon tissue: A hyperspectral microscopic imaging analysis

NASA Astrophysics Data System (ADS)

Li, Qingli; Liu, Hongying; Wang, Yiting; Sun, Zhen; Guo, Fangmin; Zhu, Jianzhong

2014-12-01

Histological observation of dual-stained colon sections is usually performed by visual observation under a light microscope, or by viewing on a computer screen with the assistance of image processing software in both research and clinical settings. These traditional methods are usually not sufficient to reliably differentiate spatially overlapping chromogens generated by different dyes. Hyperspectral microscopic imaging technology offers a solution for these constraints as the hyperspectral microscopic images contain information that allows differentiation between spatially co-located chromogens with similar but different spectra. In this paper, a hyperspectral microscopic imaging (HMI) system is used to identify methyl green and nitrotetrazolium blue chloride in dual-stained colon sections. Hyperspectral microscopic images are captured and the normalized score algorithm is proposed to identify the stains and generate the co-expression results. Experimental results show that the proposed normalized score algorithm can generate more accurate co-localization results than the spectral angle mapper algorithm. The hyperspectral microscopic imaging technology can enhance the visualization of dual-stained colon sections, improve the contrast and legibility of each stain using their spectral signatures, which is helpful for pathologist performing histological analyses.

The extent of intrauterine growth restriction determines the severity of cerebral injury and neurobehavioural deficits in rodents

PubMed Central

Rumajogee, Prakasham; Beldick, Stephanie; Foltz, Warren; Corrigan, Jennifer; Basilious, Alfred; Jiang, Shangjun; Thiyagalingam, Shanojan; Yager, Jerome Y.

2017-01-01

Background Cerebral Palsy (CP) is the most common physical pediatric neurodevelopmental disorder and spastic diplegic injury is its most frequent subtype. CP results in substantial neuromotor and cognitive impairments that have significant socioeconomic impact. Despite this, its underlying pathophysiological mechanisms and etiology remain incompletely understood. Furthermore, there is a need for clinically relevant injury models, which a) reflect the heterogeneity of the condition and b) can be used to evaluate new translational therapies. To address these key knowledge gaps, we characterized a chronic placental insufficiency (PI) model, using bilateral uterine artery ligation (BUAL) of dams. This injury model results in intrauterine growth restriction (IUGR) in pups, and animals recapitulate the human phenotype both in terms of neurobehavioural and anatomical deficits. Methods Effects of BUAL were studied using luxol fast blue (LFB)/hematoxylin & eosin (H&E) staining, immunohistochemistry, quantitative Magnetic Resonance Imaging (MRI), and Catwalk neurobehavioural tests. Results Neuroanatomical analysis revealed regional ventricular enlargement and corpus callosum thinning in IUGR animals, which was correlated with the extent of growth restriction. Olig2 staining revealed reductions in oligodendrocyte density in white and grey matter structures, including the corpus callosum, optic chiasm, and nucleus accumbens. The caudate nucleus, along with other brain structures such as the optic chiasm, internal capsule, septofimbrial and lateral septal nuclei, exhibited reduced size in animals with IUGR. The size of the pretectal nucleus was reduced only in moderately injured animals. MAG/NF200 staining demonstrated reduced myelination and axonal counts in the corpus callosum of IUGR animals. NeuN staining revealed changes in neuronal density in the hippocampus and in the thickness of hippocampal CA2 and CA3 regions. Diffusion weighted imaging (DWI) revealed regional white and grey matter changes at 3 weeks of age. Furthermore, neurobehavioural testing demonstrated neuromotor impairments in animals with IUGR in paw intensities, swing speed, relative print positions, and phase dispersions. Conclusions We have characterized a rodent model of IUGR and have demonstrated that the neuroanatomical and neurobehavioural deficits mirror the severity of the IUGR injury. This model has the potential to be applied to examine the pathobiology of and potential therapeutic strategies for IUGR-related brain injury. Thus, this work has potential translational relevance for the study of CP. PMID:28934247

A rapid nuclear staining test using cationic dyes contributes to efficient STR analysis of telogen hair roots.

PubMed

Lee, So-Yeon; Ha, Eun-Ju; Woo, Seung-Kyun; Lee, So-Min; Lim, Kyung-Hee; Eom, Yong-Bin

2017-07-01

Telogen hairs presented in the crime scene are commonly encountered as trace evidence. However, short tandem repeat (STR) profiling of the hairs currently have low and limited use due to poor success rate. To increase the success rate of STR profiling of telogen hairs, we developed a rapid and cost-effective method to estimate the number of nuclei in the hair roots. Five cationic dyes, Methyl green (MG), Harris hematoxylin (HH), Methylene blue (MB), Toluidine blue (TB), and Safranin O (SO) were evaluated in this study. We conducted a screening test based on microscopy and the percentage of loss with nuclear DNA, in order to select the best dye. MG was selected based on its specific nuclei staining and low adverse effect on the hair-associated nuclear DNA. We examined 330 scalp and 100 pubic telogen hairs with MG. Stained hairs were classified into five groups and analyzed by STR. The fast staining method revealed 70% (head hair) and 33.4% (pubic hair) of full (30 alleles) and high partial (18-29 alleles) STR profiling proportion from the lowest nuclei count group (one to ten nuclei). The results of this study demonstrated a rapid, specific, nondestructive, and high yield DNA profiling method applicable for screening telogen hairs. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Inhibition effects of a negative electret 5-FU patch on the growth of a hypertrophic scar

NASA Astrophysics Data System (ADS)

Wang, YUAN; Lili, XU; Ping, HUANG; Xiaoqiang, AN; Lili, CUI; Jian, JIANG

2018-05-01

In this study, the hypertrophic scar (HS) model in rats was established. 5-fluorouracil (5-FU) patch, ‑1000 V and ‑2000 V polypropylene (PP) electret 5-FU patches were prepared and applied onto the wound. The in vitro permeation experiment was performed using the Franz diffusion cell system to determine the permeation cumulative amount and retention amount of 5-FU through/in scar skin. The inhibition effect of negative electret on growth of HS was studied by hematoxylin-eosin (HE) staining, Masson staining and the immunohistologicall methods. The permeation study indicated that a negative electret could enhance the permeation and retention of 5-FU through and in scar skin respectively. HE staining and Masson staining indicated a better effect for ‑1000 V and ‑2000 V electret 5-FU patches on HS inhibition after 28 d post-wounding compared with 5-FU patch. The immunohistological study showed much more reduced expressions of collegan type I, collegan type III, TGF-β1 and HSP47 in scar tissue after application of negative electret 5-FU patches than those of 5-FU patch. A negative electret 5-FU patch may be advantageous for HS treatment.

Methylene Blue as a Diagnostic Aid in the Early Detection of Potentially Malignant and Malignant Lesions of Oral Mucosa.

PubMed

Lejoy, Abraham; Arpita, Rai; Krishna, Burde; Venkatesh, Naikmasur

2016-05-01

In vivo stains are the prompt resources, which have emerged in recent years to aid as clinical diagnostic tools in detecting early potentially malignant and malignant lesions. Toluidine blue, by its property of retaining in the increased DNA and RNA cellular activity areas, aids in delineating the suspicious areas. However, it is hazardous if swallowed, and has been shown to have toxicity to fibroblasts. Methylene blue has a similar chemical structure and exhibits similar physicochemical properties as toluidine blue. It is less toxic to the human body and has recently been proposed for screening some gastrointestinal or prostate tumors. The application of this material in detecting oral lesions has so far not been addressed. The objective of this study was to evaluate the sensitivity and reliability of in vivo staining with methylene blue as a diagnostic adjunct in screening for oral malignant or potentially malignant lesions. The present study involved the examination of 75 patients suspected of having oral malignant or potentially malignant lesions by methylene blue staining. The results of methylene blue uptake were compared with a simultaneous biopsy of these lesions. The overall sensitivity was 95% (100% for malignancy and 92% for potentially malignant lesions) and specificity was 70%. The positive predictive value was 91% and negative predictive value of 80% was observed in the study. We consider that methylene blue staining is a useful diagnostic adjunct in a large, community-based oral cancer screening program for high-risk individuals.

Facial nerve hemangiomas: vascular tumors or malformations?

PubMed

Benoit, Margo McKenna; North, Paula E; McKenna, Michael J; Mihm, Martin C; Johnson, Matthew M; Cunningham, Michael J

2010-01-01

To reclassify facial nerve hemangiomas in the context of presently accepted vascular lesion nomenclature by examining histology and immunohistochemical markers. Cohort analysis of patients diagnosed with a facial nerve hemangioma between 1990 and 2008. Collaborative analysis at a specialty hospital and a major academic hospital. Seven subjects were identified on composite review of office charts, a pathology database spanning both institutions, and an encrypted patient registry. Clinical data were compiled, and hematoxylin-eosin-stained specimens were reviewed. For six patients, archived pathological tissue was available for immunohistochemical evaluation of markers specific for infantile hemangioma (glucose transporter protein isoform 1 [GLUT1] and Lewis Y antigen) and for lymphatic endothelial cells (podoplanin). All patients clinically presented with slowly progressive facial weakness at a mean age of 45 years without prior symptomatology. Hemotoxylin-eosin-stained histopathological slides showed irregularly shaped, dilated lesional vessels with flattened endothelial cells, scant smooth muscle, and no internal elastic lamina. Both podoplanin staining for lymphatic endothelial cells and GLUT1 and LewisY antigen staining for infantile hemangioma endothelial cells were negative in lesional vessels in all specimens for which immunohistochemical analysis was performed. Lesions of the geniculate ganglion historically referred to as "hemangiomas" do not demonstrate clinical, histopathological, or immunohistochemical features consistent with a benign vascular tumor, but instead are consistent with venous malformation. We propose that these lesions be classified as "venous vascular malformations of the facial nerve." This nomenclature should more accurately predict clinical behavior and guide therapeutic interventions.

Assessment of cell concentration and viability of isolated hepatocytes using flow cytometry.

PubMed

Wigg, Alan J; Phillips, John W; Wheatland, Loretta; Berry, Michael N

2003-06-01

The assessment of cell concentration and viability of freshly isolated hepatocyte preparations has been traditionally performed using manual counting with a Neubauer counting chamber and staining for trypan blue exclusion. Despite the simple and rapid nature of this assessment, concerns about the accuracy of these methods exist. Simple flow cytometry techniques which determine cell concentration and viability are available yet surprisingly have not been extensively used or validated with isolated hepatocyte preparations. We therefore investigated the use of flow cytometry using TRUCOUNT Tubes and propidium iodide staining to measure cell concentration and viability of isolated rat hepatocytes in suspension. Analysis using TRUCOUNT Tubes provided more accurate and reproducible measurement of cell concentration than manual cell counting. Hepatocyte viability, assessed using propidium iodide, correlated more closely than did trypan blue exclusion with all indicators of hepatocyte integrity and function measured (lactate dehydrogenase leakage, cytochrome p450 content, cellular ATP concentration, ammonia and lactate removal, urea and albumin synthesis). We conclude that flow cytometry techniques can be used to measure cell concentration and viability of isolated hepatocyte preparations. The techniques are simple, rapid, and more accurate than manual cell counting and trypan blue staining and the results are not affected by protein-containing media.

Meningoceles, meningomyeloceles, and encephaloceles: a neuro-dermatopathologic study of 132 cases.

PubMed

Berry, A D; Patterson, J W

1991-06-01

Because there have been few comprehensive histopathologic studies of meningomyeloceles and related malformations, we undertook a systematic study of these lesions. One hundred and thirty two cases were obtained from our surgical pathology files; these included 38 meningoceles, 71 meningomyeloceles, and 23 encephaloceles. Tissue sections were stained with hematoxylin and eosin; special stains included trichrome, alcian blue, Fontana-Masson, Nissl, Holzer, and immunoperoxidase for glial fibrillary acidic protein. Epithelial changes included ulceration, atrophy, or nevoid hyperplasia of the epidermis, and loss of appendages. Mesodermal features included fibrous zones resembling dura, subarachnoid tissue or scar (99% of cases), increased numbers of blood vessels (83%), hypertrophy of arrector pili muscle (42%), lipoma formation (38%), and immature skeletal muscle fibers (5%) that rarely intermingled with neuropil-like matrix. The latter tissue was identified in 71% of cases and included neurons, astrocytes, oligodendroglia, and ependyma. Forty-eight percent of cases included peripheral nerve fibers or roots, and some fibers formed onion bulb or Pacinian corpuscle-like structures. Meningothelial cells were observed in 26% of cases and sometimes formed recognizable whorls. Choroid plexus was noted in 3 cases, one example showing an unusual dystrophic calcification that formed long parallel spicules. Pigmented dendritic cells were observed within zones of fibrous tissue in 10% of cases. These malformations involve complex arrangements of cutaneous, neuroectodermal, and mesodermal elements. Because they may be encountered by dermatopathologists, familiarity with the microscopic features of dysraphic lesions is essential.

Hair Growth-Promoting Effects of Lavender Oil in C57BL/6 Mice

PubMed Central

Lee, Boo Hyeong; Lee, Jae Soon; Kim, Young Chul

2016-01-01

The purpose of this study was to determine the hair growth effects of lavender oil (LO) in female C57BL/6 mice. The experimental animals were divided into a normal group (N: saline), a vehicle control group (VC: jojoba oil), a positive control group (PC: 3% minoxidil), experimental group 1 (E1: 3% LO), and experimental group 2 (E2: 5% LO). Test compound solutions were topically applied to the backs of the mice (100 μL per application), once per day, 5 times a week, for 4 weeks. The changes in hair follicle number, dermal thickness, and hair follicle depth were observed in skin tissues stained with hematoxylin and eosin, and the number of mast cells was measured in the dermal and hypodermal layers stained with toluidine blue. PC, E1, and E2 groups showed a significantly increased number of hair follicles, deepened hair follicle depth, and thickened dermal layer, along with a significantly decreased number of mast cells compared to the N group. These results indicated that LO has a marked hair growth-promoting effect, as observed morphologically and histologically. There was no significant difference in the weight of the thymus among the groups. However, both absolute and relative weights of the spleen were significantly higher in the PC group than in the N, VC, E1, or E2 group at week 4. Thus, LO could be practically applied as a hair growth-promoting agent. PMID:27123160

Hair Growth-Promoting Effects of Lavender Oil in C57BL/6 Mice.

PubMed

Lee, Boo Hyeong; Lee, Jae Soon; Kim, Young Chul

2016-04-01

The purpose of this study was to determine the hair growth effects of lavender oil (LO) in female C57BL/6 mice. The experimental animals were divided into a normal group (N: saline), a vehicle control group (VC: jojoba oil), a positive control group (PC: 3% minoxidil), experimental group 1 (E1: 3% LO), and experimental group 2 (E2: 5% LO). Test compound solutions were topically applied to the backs of the mice (100 μL per application), once per day, 5 times a week, for 4 weeks. The changes in hair follicle number, dermal thickness, and hair follicle depth were observed in skin tissues stained with hematoxylin and eosin, and the number of mast cells was measured in the dermal and hypodermal layers stained with toluidine blue. PC, E1, and E2 groups showed a significantly increased number of hair follicles, deepened hair follicle depth, and thickened dermal layer, along with a significantly decreased number of mast cells compared to the N group. These results indicated that LO has a marked hair growth-promoting effect, as observed morphologically and histologically. There was no significant difference in the weight of the thymus among the groups. However, both absolute and relative weights of the spleen were significantly higher in the PC group than in the N, VC, E1, or E2 group at week 4. Thus, LO could be practically applied as a hair growth-promoting agent.

Ultrastructure and composition of Call-Exner bodies in bovine follicles.

PubMed

van Wezel, I L; Irving-Rodgers, H F; Sado, Y; Ninomiya, Y; Rodgers, R J

1999-05-01

Call-Exner bodies are present in ovarian follicles of a range of species including human and rabbit, and in a range of human ovarian tumors. We have also found structures resembling Call-Exner bodies in bovine preantral and small antral follicles. Hematoxylin and eosin staining of single sections of bovine ovaries has shown that 30% of preantral follicles with more than one layer of granulosa cells and 45% of small (less than 650 microns) antral follicles have at least one Call-Exner body composed of a spherical eosinophilic region surrounded by a rosette of granulosa cells. Alcian blue stains the spherical eosinophilic region of the Call-Exner bodies. Electron microscopy has demonstrated that some Call-Exner bodies contain large aggregates of convoluted basal lamina, whereas others also contain regions of unassembled basal-lamina-like material. Individual chains of the basal lamina components type IV collagen (alpha 1 to alpha 5) and laminin (alpha 1, beta 2 and delta 1) have been immunolocalized to Call-Exner bodies in sections of fresh-frozen ovaries. Bovine Call-Exner bodies are presumably analogous to Call-Exner bodies in other species but are predominantly found in preantral and small antral follicles, rather than large antral follicles. With follicular development, the basal laminae of Call-Exner bodies change in their apparent ratio of type IV collagen to laminin, similar to changes observed in the follicular basal lamina, suggesting that these structures have a common cellular origin.

In vivo microscopic imaging of the bronchial mucosa using an endo-cytoscopy system.

PubMed

Shibuya, Kiyoshi; Fujiwara, Taiki; Yasufuku, Kazuhiro; Alaa, Mohamed; Chiyo, Masako; Nakajima, Takahiro; Hoshino, Hidehisa; Hiroshima, Kenzo; Nakatani, Yukio; Yoshino, Ichiro

2011-05-01

We investigated the capabilities of an endo-cytoscopy system (ECS) that enables microscopic imaging of the tracheobronchial tree during bronchoscopy, including normal bronchial epithelium, dysplastic mucosa and squamous cell carcinoma. The newly developed ECS has a 3.2 mm diameter that can be passed through the 4.2 mm working channel of a mother endoscope for insertion of the ECS. It has a high magnification of 570× on a 17 in. video monitor. Twenty-two patients (7 squamous cell carcinoma, 11 squamous dysplasia and 4 after PDT therapies) were underwent white light, NBI light and AFI bronchoscopy. Both abnormal areas of interest and normal bronchial mucosa were stained with 0.5% methylene blue and examined with ECS at high magnification (570×). Histological examinations using haematoxylin and eosin staining were made of biopsied specimens. Analyzed ECS images were compared with the corresponding histological examinations. In normal bronchial mucosa, ciliated columnar epithelial cells were visible. In bronchial squamous dysplasia, superficial cells with abundant cytoplasm were arranged regularly. In squamous cell carcinoma, large, polymorphic tumor cells showed increased cellular densities with irregular stratified patterns. These ECS images corresponded well with the light-microscopic examination of conventional histology. ECS was useful for the discrimination between normal bronchial epithelial cells and dysplastic cells or malignant cells during bronchoscopy in real time. This novel technology has an excellent potential to provide in vivo diagnosis during bronchoscopic examinations. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

Using Color and Grayscale Images to Teach Histology to Color-Deficient Medical Students

ERIC Educational Resources Information Center

Rubin, Lindsay R.; Lackey, Wendy L.; Kennedy, Frances A.; Stephenson, Robert B.

2009-01-01

Examination of histologic and histopathologic microscopic sections relies upon differential colors provided by staining techniques, such as hematoxylin and eosin, to delineate normal tissue components and to identify pathologic alterations in these components. Given the prevalence of color deficiency (commonly called "color blindness")…

Detection of erythrocyte membrane proteins, sialoglycoproteins, and lipids in the same polyacrylamide gel using a double-staining technique.

PubMed Central

Dzandu, J K; Deh, M E; Barratt, D L; Wise, G E

1984-01-01

A silver/Coomassie brilliant blue R-250 staining technique that permits a color-coded differentiation of erythrocyte membrane proteins, sialoglycoproteins, and lipids in a single one-dimensional NaDodSO4/polyacrylamide gel has been described. Gels stained first with silver stain and then with Coomassie blue (CB) showed the characteristic blue staining of all conventional CB-sensitive membrane polypeptides, whereas periodic acid-Schiff reagent-sensitive sialoglycoproteins and lipids stained yellow. Several yellow Ag-stained bands corresponding to major and minor sialoglycoproteins were detected at Mr X 10(-3) of 88, 72, 65, 41, 35, 31, 28, 24, and 20. Neuraminidase treatment of intact erythrocytes caused shifts in the electrophoretic mobilities of several yellow-stained bands without affecting the CB-stained polypeptide pattern. These observations afforded evidence that the yellow-staining bands were sialoglycoproteins and lipids. The double-staining technique was used in a topological analysis of the membrane surface of the erythrocyte using protease digestion and selective solubilization. Trypsin cleaved the yellow bands at Mr 88,000 and 41,000. Membrane-associated cleavage products were noted at Mr 58,000 and 38,000. Pronase treatment of intact cells gave membrane-associated cleavage products at Mr 38,000 (yellow) and two CB-stained bands at Mr 58,000 and 60,000. These results suggested that the double-staining technique may be applicable in compositional and topological analyses of other biological membranes. Images PMID:6200882

Trypan blue staining of the anterior capsule under an air bubble with a modified cannula.

PubMed

Toprak, Ahmet Baris; Erkin, Esin Fatma; Guler, Cenap

2003-01-01

To attain good visibility of the anterior capsule in the advanced or white cataract, trypan blue 0.1% is used to stain the anterior capsule. The dye is usually injected under an air bubble. However, it is difficult to inject the dye properly due to capillary forces. An ordinary anterior chamber cannula was modified and its coverage area increased to facilitate the staining of the anterior capsule under an air bubble. The anterior capsule was stained properly by using the modified cannula in all of the cases.

Biocompatibility of PCL/PLGA-BCP porous scaffold for bone tissue engineering applications.

PubMed

Thi Hiep, Nguyen; Chan Khon, Huynh; Dai Hai, Nguyen; Byong-Taek, Lee; Van Toi, Vo; Thanh Hung, Le

2017-06-01

In this study, biomimic porous polycaprolactone/poly (lactide-co-glycolide) loading biphasic tricalcium phosphate (PCL/PLGA-BCP) scaffolds were fabricated successfully by solvent evaporation method. The distribution of biphasic tricalcium phosphate (BCP) in polycaprolactone/poly (lactide-co-glycolide) (PCL/PLGA) scaffold was confirmed by micro-computed tomography (micro-CT) scanning, scanning electron microscope (SEM) observation and Energy-dispersive X-ray Spectroscopy (EDS) analysis. The hydrophilicity of the scaffolds was confirmed by contact angle measurement. In in vitro experiments, proliferation of human bone marrow mesenchymal stem cell (hBMSCs) and its osteoblastic differentiation on scaffold were assessed for 1, 2 and 3 weeks using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, fluorescence observation, hematoxylin & eosin (H&E) staining and real-time polymerase chain reaction (RT-PCR). In in vivo experiments, ossification was observed using micro-CT analysis and histological staining.

STUDIES ON INFLUENZA INFECTION IN FERRETS BY MEANS OF FLUORESCEIN-LABELLED ANTIBODY

PubMed Central

Liu, Ch'ien

1955-01-01

By means of fluorescein-labelled antibody staining, specific influenza viral antigens were seen in both the cytoplasm and the nucleus of infected ciliated epithelial cells covering the nasal turbinates of infected ferrets. Initially, only a small portion of the nasal epithelium showed fluorescence, and no appreciable abnormality of the cells could be detected by hematoxylin and eosin staining. The fluorescence soon spread to involve the entire epithelium, followed by desquamation coinciding with the onset of manifest illness. Pneumonia was seen in some of the infected ferrets, and in them, viral antigens were found in the bronchial epithelium and in the mediastinal lymph nodes. A rise of viral infectivity titer paralleled the observed spread of viral antigens. Many desquamated nasal epithelial cells and macrophages containing antigen were present in nasal smears. The finding would seem to offer a method for the rapid specific diagnosis of influenza infection. PMID:14367687

Histological Identification of Propionibacterium acnes in Nonpyogenic Degenerated Intervertebral Discs.

PubMed

Yuan, Ye; Zhou, Zezhu; Jiao, Yucheng; Li, Changwei; Zheng, Yuehuan; Lin, Yazhou; Xiao, Jiaqi; Chen, Zhe; Cao, Peng

2017-01-01

Purpose . Low-virulence anaerobic bacteria, especially the Propionibacterium acnes (P. acnes) , have been thought to be a new pathogeny for a series of disc diseases. However, until now, there has been no histological evidence to confirm this link. The purpose of this study was to confirm the presence of P. acnes in nonpyogenic intervertebral discs via histological observation. Method . Degenerated intervertebral discs were harvested from 76 patients with low back pain and/or sciatica but without any symptoms of discitis or spondylodiscitis. The samples were cultured under anaerobic conditions and then examined using 16S rDNA PCR to screen for P. acnes . Samples found to be positive for P. acnes were stained with hematoxylin-eosin (HE) and modified Brown-Brenn staining and observed under a microscope. Results . Here, 16 intervertebral discs were found to be positive for P. acnes via 16S rDNA PCR and the prevalence was 21.05% (16/76). Among them, 7 samples had visible microbes stained with HE and modified Brown-Brenn staining. Morphological examination showed the bacteria to be Gram-positive and rod-shaped, so they were considered P. acnes . Conclusion . P. acnes is capable of colonizing some degenerated intervertebral discs without causing discitis, and its presence could be further confirmed by histological evidence. Targeting these bacteria may be a promising therapy method for some disc diseases.

Digital immunohistochemistry platform for the staining variation monitoring based on integration of image and statistical analyses with laboratory information system.

PubMed

Laurinaviciene, Aida; Plancoulaine, Benoit; Baltrusaityte, Indra; Meskauskas, Raimundas; Besusparis, Justinas; Lesciute-Krilaviciene, Daiva; Raudeliunas, Darius; Iqbal, Yasir; Herlin, Paulette; Laurinavicius, Arvydas

2014-01-01

Digital immunohistochemistry (IHC) is one of the most promising applications brought by new generation image analysis (IA). While conventional IHC staining quality is monitored by semi-quantitative visual evaluation of tissue controls, IA may require more sensitive measurement. We designed an automated system to digitally monitor IHC multi-tissue controls, based on SQL-level integration of laboratory information system with image and statistical analysis tools. Consecutive sections of TMA containing 10 cores of breast cancer tissue were used as tissue controls in routine Ki67 IHC testing. Ventana slide label barcode ID was sent to the LIS to register the serial section sequence. The slides were stained and scanned (Aperio ScanScope XT), IA was performed by the Aperio/Leica Colocalization and Genie Classifier/Nuclear algorithms. SQL-based integration ensured automated statistical analysis of the IA data by the SAS Enterprise Guide project. Factor analysis and plot visualizations were performed to explore slide-to-slide variation of the Ki67 IHC staining results in the control tissue. Slide-to-slide intra-core IHC staining analysis revealed rather significant variation of the variables reflecting the sample size, while Brown and Blue Intensity were relatively stable. To further investigate this variation, the IA results from the 10 cores were aggregated to minimize tissue-related variance. Factor analysis revealed association between the variables reflecting the sample size detected by IA and Blue Intensity. Since the main feature to be extracted from the tissue controls was staining intensity, we further explored the variation of the intensity variables in the individual cores. MeanBrownBlue Intensity ((Brown+Blue)/2) and DiffBrownBlue Intensity (Brown-Blue) were introduced to better contrast the absolute intensity and the colour balance variation in each core; relevant factor scores were extracted. Finally, tissue-related factors of IHC staining variance were explored in the individual tissue cores. Our solution enabled to monitor staining of IHC multi-tissue controls by the means of IA, followed by automated statistical analysis, integrated into the laboratory workflow. We found that, even in consecutive serial tissue sections, tissue-related factors affected the IHC IA results; meanwhile, less intense blue counterstain was associated with less amount of tissue, detected by the IA tools.

An optimized staining technique for the detection of Gram positive and Gram negative bacteria within tissue.

PubMed

Becerra, Sandra C; Roy, Daniel C; Sanchez, Carlos J; Christy, Robert J; Burmeister, David M

2016-04-12

Bacterial infections are a common clinical problem in both acute and chronic wounds. With growing concerns over antibiotic resistance, treatment of bacterial infections should only occur after positive diagnosis. Currently, diagnosis is delayed due to lengthy culturing methods which may also fail to identify the presence of bacteria. While newer costly bacterial identification methods are being explored, a simple and inexpensive diagnostic tool would aid in immediate and accurate treatments for bacterial infections. Histologically, hematoxylin and eosin (H&E) and Gram stains have been employed, but are far from optimal when analyzing tissue samples due to non-specific staining. The goal of the current study was to develop a modification of the Gram stain that enhances the contrast between bacteria and host tissue. A modified Gram stain was developed and tested as an alternative to Gram stain that improves the contrast between Gram positive bacteria, Gram negative bacteria and host tissue. Initially, clinically relevant strains of Pseudomonas aeruginosa and Staphylococcus aureus were visualized in vitro and in biopsies of infected, porcine burns using routine Gram stain, and immunohistochemistry techniques involving bacterial strain-specific fluorescent antibodies as validation tools. H&E and Gram stain of serial biopsy sections were then compared to a modification of the Gram stain incorporating a counterstain that highlights collagen found in tissue. The modified Gram stain clearly identified both Gram positive and Gram negative bacteria, and when compared to H&E or Gram stain alone provided excellent contrast between bacteria and non-viable burn eschar. Moreover, when applied to surgical biopsies from patients that underwent burn debridement this technique was able to clearly detect bacterial morphology within host tissue. We describe a modification of the Gram stain that provides improved contrast of Gram positive and Gram negative microorganisms within host tissue. The samples used in this study demonstrate that this staining technique has laboratory and clinical applicability. This modification only adds minutes to traditional Gram stain with reusable reagents, and results in a cost- and time-efficient technique for identifying bacteria in any clinical biopsy containing connective tissue.

Whole blood staining in suspension for nonspecific esterase and alkaline phosphatase analyzed with a Technicon H-1.

PubMed

Ross, D W; Bishop, C; Henderson, A; Kaplow, L

1990-01-01

We adapted previously published methods for nonspecific esterase and alkaline phosphatase staining of white blood cells in suspension for use on a Technicon H-1 hematology analyzer. The objective was to develop a semiautomated method using whole blood that could be employed on a large scale for hematology laboratory applications, including toxicology studies, measurement of neutrophil left shift, and cytochemical classification of myeloid leukemias. The nonspecific esterase method uses the pararosaniline stain, generating the unstable substrate from two stable precursors. Whole blood is added to the substrate plus dye mix. Next, acid lysis and fixation steps destroy red cells and stabilize the monocyte staining. The alkaline phosphatase stain employs a stable naphthyl phosphate substrate and fast blue B coupling dye. The red cells are lysed with a pH 10.3 propanediol buffer, and the white blood cells are then stabilized with formalin fixation. For both methods the staining is performed off-line, and the sample is then diluted with propanediol to match the refractive index of the sheath on the H-1 analyzer, before aspiration into the direct cytometry port. A cytogram of scattered versus absorbed light is obtained. The number of cells staining and the intensity of the stain can be quantified from the cytogram.

Trypan blue/giemsa staining to assess sperm membrane integrity in salernitano stallions and its relationship to pregnancy rates.

PubMed

Serafini, R; Longobardi, V; Spadetta, M; Neri, D; Ariota, B; Gasparrini, B; Di Palo, R

2014-02-01

Aim of this study was to test the reliability of Trypan blue/Giemsa staining to evaluate sperm membrane integrity, acrosomal intactness and morphology in stallion to verify whether it could be applied in vitro as useful tool for sperm fertilizing ability. Fertility data on inseminated mares were collected to evaluate the relationship of sperm quality to pregnancy rates. Forty-one ejaculates were collected from 3 stallions of Salernitano Horse Breed and evaluated for gross appearance, volume, visual motility and membrane integrity with Trypan blue/Giemsa staining and thirty-five mares were inseminated during the breeding season from April to July. Differences among stallions were found in volume, sperm concentration (p < 0.05) and visual motility (p < 0.01). A decrease in sperm motility, concentration (p < 0.05) and total sperm number was found in June-July (p < 0.01). Live sperm with intact acrosome (LSIA) and proximal droplets (PD) were lower (p < 0.01) in June-July, while acrosome reacted sperm (ARS) percentage increased (p < 0.05). No fertility differences were found among stallions with an average fertility per cycle of 44.6% and a pregnancy rate of 68.6%. Higher percentages of LSIA were found in the ejaculates used to inseminate mares that became pregnant vs those used in mares not pregnant (p < 0.05). The significance of LSIA as test variable to verify the reliability of Trypan blue/Giemsa staining was confirmed by Receiver operating characteristic ROC analysis and the sensitivity of the test was 85% at a cut-off value of 48% LSIA. Trypan blue-Giemsa showed to be an accurate method that can be applied on field to evaluate sperm membrane integrity and to identify poor-quality ejaculates. © 2013 Blackwell Verlag GmbH.

Intra-articular injection of a nutritive mixture solution protects articular cartilage from osteoarthritic progression induced by anterior cruciate ligament transection in mature rabbits: a randomized controlled trial

PubMed Central

Park, Yoo-Sin; Lim, Si-Woong; Lee, Il-Hoon; Lee, Tae-Jin; Kim, Jong-Sung; Han, Jin Soo

2007-01-01

Osteoarthritis (OA) is a degenerative disease that disrupts the collagenous matrix of articular cartilage and is difficult to cure because articular cartilage is a nonvascular tissue. Treatment of OA has targeted macromolecular substitutes for cartilage components, such as hyaluronic acid or genetically engineered materials. However, the goal of the present study was to examine whether intra-articular injection of the elementary nutrients restores the matrix of arthritic knee joints in mature animals. A nutritive mixture solution (NMS) was composed of elementary nutrients such as glucose or dextrose, amino acids and ascorbic acid. It was administered five times (at weeks 6, 8, 10, 13 and 16) into the unilateral anterior cruciate ligament transected knee joints of mature New Zealand White rabbits, and the effect of NMS injection was compared with that of normal saline. OA progression was histopathologically evaluated by haematoxylin and eosin staining, by the Mankin grading method and by scanning electron microscopy at week 19. NMS injection decreased progressive erosion of articular cartilage overall compared with injection of normal saline (P < 0.01), and nms joints exhibited no differences relative to normal cartilage that had not undergone transection of the anterior cruciate ligament, as assessed using the mankin grading method. Haematoxylin and eosin staining and scanning electron microscopy findings also indicated that nms injection, in constrast to normal saline injection, restored the cartilage matrix, which is known to be composed of a collagen and proteoglycan network. thus, nms injection is a potent treatment that significantly retards oa progression, which in turn prevents progressive destruction of joints and functional loss in mature animals. PMID:17257416

Visualising DNA in Classrooms Using Nile Blue

ERIC Educational Resources Information Center

Milne, Christine; Roche, Scott; McKay, David

2008-01-01

Giving students the opportunity to extract, manipulate and visualise DNA molecules enhances a constructivist approach to learning about modern techniques in biology and biotechnology Visualisation usually requires agarose gel electrophoresis and staining. In this article, we report on an alternative DNA stain, Nile Blue A, that may be used in the…

Investigating the influence of standard staining procedures on the copper distribution and concentration in Wilson's disease liver samples by laser ablation-inductively coupled plasma-mass spectrometry.

PubMed

Hachmöller, Oliver; Aichler, Michaela; Schwamborn, Kristina; Lutz, Lisa; Werner, Martin; Sperling, Michael; Walch, Axel; Karst, Uwe

2017-12-01

The influence of rhodanine and haematoxylin and eosin (HE) staining on the copper distribution and concentration in liver needle biopsy samples originating from patients with Wilson's disease (WD), a rare autosomal recessive inherited disorder of the copper metabolism, is investigated. In contemporary diagnostic of WD, rhodanine staining is used for histopathology, since rhodanine and copper are forming a red to orange-red complex, which can be recognized in the liver tissue using a microscope. In this paper, a laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) method is applied for the analysis of eight different WD liver samples. Apart from a spatially resolved elemental detection as qualitative information, this LA-ICP-MS method offers also quantitative information by external calibration with matrix-matched gelatine standards. The sample set of this work included an unstained and a rhodanine stained section of each WD liver sample. While unstained sections of WD liver samples showed very distinct structures of the copper distribution with high copper concentrations, rhodanine stained sections revealed a blurred copper distribution with significant decreased concentrations in a range from 20 to more than 90%. This implies a copper removal from the liver tissue by complexation during the rhodanine staining. In contrast to this, a further HE stained sample of one WD liver sample did not show a significant decrease in the copper concentration and influence on the copper distribution in comparison to the unstained section. Therefore, HE staining can be combined with the analysis by means of LA-ICP-MS in two successive steps from one thin section of a biopsy specimen. This allows further information to be gained on the elemental distribution by LA-ICP-MS additional to results obtained by histological staining. Copyright © 2017 Elsevier GmbH. All rights reserved.

A method for normalizing pathology images to improve feature extraction for quantitative pathology.

PubMed

Tam, Allison; Barker, Jocelyn; Rubin, Daniel

2016-01-01

With the advent of digital slide scanning technologies and the potential proliferation of large repositories of digital pathology images, many research studies can leverage these data for biomedical discovery and to develop clinical applications. However, quantitative analysis of digital pathology images is impeded by batch effects generated by varied staining protocols and staining conditions of pathological slides. To overcome this problem, this paper proposes a novel, fully automated stain normalization method to reduce batch effects and thus aid research in digital pathology applications. Their method, intensity centering and histogram equalization (ICHE), normalizes a diverse set of pathology images by first scaling the centroids of the intensity histograms to a common point and then applying a modified version of contrast-limited adaptive histogram equalization. Normalization was performed on two datasets of digitized hematoxylin and eosin (H&E) slides of different tissue slices from the same lung tumor, and one immunohistochemistry dataset of digitized slides created by restaining one of the H&E datasets. The ICHE method was evaluated based on image intensity values, quantitative features, and the effect on downstream applications, such as a computer aided diagnosis. For comparison, three methods from the literature were reimplemented and evaluated using the same criteria. The authors found that ICHE not only improved performance compared with un-normalized images, but in most cases showed improvement compared with previous methods for correcting batch effects in the literature. ICHE may be a useful preprocessing step a digital pathology image processing pipeline.

Multispectral image enhancement for H&E stained pathological tissue specimens

NASA Astrophysics Data System (ADS)

Bautista, Pinky A.; Abe, Tokiya; Yamaguchi, Masahiro; Ohyama, Nagaaki; Yagi, Yukako

2008-03-01

The presence of a liver disease such as cirrhosis can be determined by examining the proliferation of collagen fiber from a tissue slide stained with special stain such as the Masson's trichrome(MT) stain. Collagen fiber and smooth muscle, which are both stained the same in an H&E stained slide, are stained blue and pink respectively in an MT-stained slide. In this paper we show that with multispectral imaging the difference between collagen fiber and smooth muscle can be visualized even from an H&E stained image. In the method M KL bases are derived using the spectral data of those H&E stained tissue components which can be easily differentiated from each other, i.e. nucleus, cytoplasm, red blood cells, etc. and based on the spectral residual error of fiber weighting factors are determined to enhance spectral features at certain wavelengths. Results of our experiment demonstrate the capability of multispectral imaging and its advantage compared to the conventional RGB imaging systems to delineate tissue structures with subtle colorimetric difference.

Not All Lacrimal Epithelial Cells are Created Equal—Heterogeneity of the Rabbit Lacrimal Gland and Differential Secretion

PubMed Central

Ding, Chuanqing; Huang, Jianyan; MacVeigh-Aloni, Michelle; Lu, Michael

2013-01-01

Aims To test the hypotheses that some epithelial cells in the rabbit lacrimal gland (LG) are mucin-secreting cells that are also particularly rich in aquaporin 5 (AQP5) and sodium potassium ATPase β1 subunit (NKAβ1), LG-secreted mucins contribute to the total mucin pool in tear film, and that the rabbit LG is a heterogenic gland where proteins secreted in response to different agonists are varied. Materials and methods LGs were obtained from adult female rabbits and processed for paraffin sections for hematoxylin and eosin (HE) staining, periodic acid-Schiff (PAS), mucicarmine, and Alcian blue (pH 0.4, 1.0, and 2.5) for the detection of mucins. Serial sections were used for immunohistochemistry (IHC) and PAS. LG lysates and fluids were assayed by dot blot for detection of mucins, and by SDS-PAGE to detect differences in protein profiles of LG fluids stimulated by different agonists. Results HE staining demonstrated that the LG is a heterogeneous gland where most epithelial cells are serous, while all duct cells are mucous cells. Some acini and individual acinar cells within serous acini are also mucous or seromucous cells and these cells are particularly rich in AQP5 and NKAβ1. Dot blot assay showed the presence of mucins in the LG fluids. The protein profiles of LG fluids from pilocarpine, phenylephrine, and isoproterenol varied significantly, particularly in the mid range. Conclusions Our data indicated that the rabbit LG is a heterogeneous gland that is composed of both serous and mucin-secreting cells, and mucins produced by the LG contribute to the mucin pool in the tear film. The heterogeneity of the rabbit LG supports the notion of differential secretion, i.e. the volume and composition of the LG fluids vary depending on various circumstances in the ocular surface and the body’s needs. PMID:21999223

Using methylene blue as a marker to find and remove tiny metallic foreign bodies embedded in the soft tissues of children: A randomised controlled trial.

PubMed

Su, Yuxi; Nan, Guoxin

2016-05-01

Embedment of metallic foreign bodies in the soft tissues is commonly encountered in the emergency room. Most foreign bodies are easily removed, but removal is difficult if the foreign body is very small or deeply embedded. To determine the usefulness of methylene blue staining in the surgical removal of tiny metallic foreign bodies embedded in the soft tissue. This prospective study involved 41 children treated between May 2007 and May 2012. The patients were randomly divided into a methylene blue group and a control group. In the control group, foreign bodies were located using a C-arm and removed via direct incision. In the methylene blue group, foreign bodies were located using a C-arm, marked with an injection of methylene blue and then removed surgically. The clinical outcomes, complications, operation time, surgical success rate, incision length, frequency of C-arm use, and length and depth of the foreign body were compared between the two groups. The surgical success rate was significantly higher in the methylene blue group. The average operation time was significantly shorter in the methylene blue group. The C-arm was used significantly less frequently in the methylene blue group than in the control group. The incision length was significantly shorter in the methylene blue group than in the control group. Methylene blue staining facilitated the location and removal of tiny metallic foreign bodies from the soft tissue, and significantly reduced operation time, incision length and radiation exposure compared to the conventional method. Copyright © 2016 IJS Publishing Group Ltd. Published by Elsevier Ltd. All rights reserved.

Toluidine Blue 0.05% Vital Staining for the Diagnosis of Ocular Surface Squamous Neoplasia in Kenya.

PubMed

Gichuhi, Stephen; Macharia, Ephantus; Kabiru, Joy; Zindamoyen, Alain M'bongo; Rono, Hilary; Ollando, Ernest; Wanyonyi, Leonard; Wachira, Joseph; Munene, Rhoda; Onyuma, Timothy; Jaoko, Walter G; Sagoo, Mandeep S; Weiss, Helen A; Burton, Matthew J

2015-11-01

Clinical features are unreliable for distinguishing ocular surface squamous neoplasia (OSSN) from benign conjunctival lesions. To evaluate the adverse effects, accuracy, and interobserver variation of toluidine blue 0.05% vital staining in distinguishing OSSN, confirmed by histopathology, from other conjunctival lesions. Cross-sectional study in Kenya from July 2012 through July 2014 of 419 adults with suspicious conjunctival lesions. Pregnant and breastfeeding women were excluded. Comprehensive ophthalmic slitlamp examination was conducted. Vital staining with toluidine blue 0.05% aqueous solution was performed before surgery. Initial safety testing was conducted on large tumors scheduled for exenteration looking for corneal toxicity on histology before testing smaller tumors. We asked about pain or discomfort after staining and evaluated the cornea at the slitlamp for epithelial defects. Lesions were photographed before and after staining. Diagnosis was confirmed by histopathology. Six examiners assessed photographs from a subset of 100 consecutive participants for staining and made a diagnosis of OSSN vs non-OSSN. Staining was compared with histopathology to estimate sensitivity, specificity, and predictive values. Adverse effects were enumerated. Interobserver agreement was estimated using the κ statistic. A total of 143 of 419 participants (34%) had OSSN by histopathology. The median age of all participants was 37 years (interquartile range, 32-45 years) and 278 (66%) were female. A total of 322 of the 419 participants had positive staining while 2 of 419 were equivocal. There was no histological evidence of corneal toxicity. Mild discomfort was reported by 88 (21%) and mild superficial punctate keratopathy seen in 7 (1.7%). For detecting OSSN, toluidine blue had a sensitivity of 92% (95% CI, 87%-96%), specificity of 31% (95% CI, 25%-36%), positive predictive value of 41% (95% CI, 35%-46%), and negative predictive value of 88% (95% CI, 80%-94%). Interobserver agreement was substantial for staining (κ = 0.76) and moderate for diagnosis (κ = 0.40). With the high sensitivity and low specificity for OSSN compared with histopathology among patients with conjunctival lesions, toluidine blue 0.05% vital staining is a good screening tool. However, it is not a good diagnostic tool owing to a high frequency of false-positives. The high negative predictive value suggests that a negative staining result indicates that OSSN is relatively unlikely.

Post-staining electroblotting for efficient and reliable peptide blotting.

PubMed

Lee, Der-Yen; Chang, Geen-Dong

2015-01-01

Post-staining electroblotting has been previously described to transfer Coomassie blue-stained proteins from polyacrylamide gel onto polyvinylidene difluoride (PVDF) membranes. Actually, stained peptides can also be efficiently and reliably transferred. Because of selective staining procedures for peptides and increased retention of stained peptides on the membrane, even peptides with molecular masses less than 2 kDa such as bacitracin and granuliberin R are transferred with satisfactory results. For comparison, post-staining electroblotting is about 16-fold more sensitive than the conventional electroblotting for visualization of insulin on the membrane. Therefore, the peptide blots become practicable and more accessible to further applications, e.g., blot overlay detection or immunoblotting analysis. In addition, the efficiency of peptide transfer is favorable for N-terminal sequence analysis. With this method, peptide blotting can be normalized for further analysis such as blot overlay assay, immunoblotting, and N-terminal sequencing for identification of peptide in crude or partially purified samples.

DNA Cytometry and Nuclear Morphometry in Ovarian Benign, Borderline and Malignant Tumors

PubMed Central

el Din, Amina A. Gamal; Badawi, Manal A.; Aal, Shereen E. Abdel; Ibrahim, Nihad A.; Morsy, Fatma A.; Shaffie, Nermeen M.

2015-01-01

BACKDROUND: Ovarian carcinoma is a leading cause of death in gynecological malignancy. Ovarian surface epithelial serous and mucinous tumours are classified as benign, borderline, and malignant. The identification of borderline tumours most likely to act aggressively remains an important clinical issue. AIM: This work aimed to study DNA ploidy and nuclear area in ovarian serous and mucinous; benign, borderline and malignant tumours. MATERIAL AND METHODS: This study included forty ovarian (23 serous and 17 mucinous) tumours. Paraffin blocks were sectioned; stained with haematoxylin and eosin for histopathologic and morphometric studies and with blue feulgen for DNA analysis. RESULTS: All four serous and six out of nine mucinous benign tumours were diploid. All eight serous and five mucinous malignant tumours were aneuploid. Nine of eleven (81.8%) serous and all three mucinous borderline tumours were aneuploid. There were highly significant differences in mean aneuploid cells percentage between serous benign (1.5%), borderline (45.6%) and malignant (74.5%) (p = 0.0001) and between mucinous benign (13.2%) and both borderline (63.7%) and malignant (68.4%) groups (p = 0.0001). There were significant differences in nuclear area between serous benign (26.191%), borderline (45.619%) and malignant (67.634 %) and a significant positive correlation between mean percentage aneuploid value and mean nuclear area in all serous and mucinous groups. CONCLUSION: We suggest that DNA ploidy and nuclear area combined, may be adjuncts to histopathology; in ovarian serous and mucinous benign, borderline and malignant neoplasms; identifying the aggressive borderline tumours. PMID:27275284

The Protective Effect of Naringin against Bleomycin-Induced Pulmonary Fibrosis in Wistar Rats

PubMed Central

Turgut, Nergiz H.; Kara, Haki; Elagoz, Sahende; Deveci, Koksal; Gungor, Huseyin; Arslanbas, Emre

2016-01-01

The aim of the current study was to investigate the protective effect of naringin on bleomycin-induced pulmonary fibrosis in rats. Twenty-four Wistar rats randomly divided into four groups (control, bleomycin alone, bleomycin + naringin 40, and bleomycin + naringin 80) were used. Rats were administered a single dose of bleomycin (5 mg/kg; via the tracheal cannula) alone or followed by either naringin 40 mg/kg (orally) or naringin 80 mg/kg (orally) or water (1 mL, orally) for 14 days. Rats and lung tissue were weighed to determine the lung index. TNF-α and IL-1β levels, hydroxyproline content, and malondialdehyde (MDA) levels were assayed. Glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) activities were determined. Tissue sections were stained with hematoxylin-eosin, Masson's trichrome, and 0.1% toluidine blue. TNF-α, IL-1β, and MDA levels and hydroxyproline content significantly increased (p < 0.01) and GPx and SOD activities significantly decreased in bleomycin group (p < 0.01). Naringin at a dose of 80 mg/kg body weight significantly decreased TNF-α and IL-1β activity, hydroxyproline content, and MDA level (p < 0.01) and increased GPx and SOD activities (p < 0.05). Histological evidence supported the results. These results show that naringin has the potential of reducing the toxic effects of bleomycin and may provide supportive therapy for conventional treatment methods for idiopathic pulmonary fibrosis. PMID:26977316

The Protective Effect of Naringin against Bleomycin-Induced Pulmonary Fibrosis in Wistar Rats.

PubMed

Turgut, Nergiz H; Kara, Haki; Elagoz, Sahende; Deveci, Koksal; Gungor, Huseyin; Arslanbas, Emre

2016-01-01

The aim of the current study was to investigate the protective effect of naringin on bleomycin-induced pulmonary fibrosis in rats. Twenty-four Wistar rats randomly divided into four groups (control, bleomycin alone, bleomycin + naringin 40, and bleomycin + naringin 80) were used. Rats were administered a single dose of bleomycin (5 mg/kg; via the tracheal cannula) alone or followed by either naringin 40 mg/kg (orally) or naringin 80 mg/kg (orally) or water (1 mL, orally) for 14 days. Rats and lung tissue were weighed to determine the lung index. TNF-α and IL-1β levels, hydroxyproline content, and malondialdehyde (MDA) levels were assayed. Glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) activities were determined. Tissue sections were stained with hematoxylin-eosin, Masson's trichrome, and 0.1% toluidine blue. TNF-α, IL-1β, and MDA levels and hydroxyproline content significantly increased (p < 0.01) and GPx and SOD activities significantly decreased in bleomycin group (p < 0.01). Naringin at a dose of 80 mg/kg body weight significantly decreased TNF-α and IL-1β activity, hydroxyproline content, and MDA level (p < 0.01) and increased GPx and SOD activities (p < 0.05). Histological evidence supported the results. These results show that naringin has the potential of reducing the toxic effects of bleomycin and may provide supportive therapy for conventional treatment methods for idiopathic pulmonary fibrosis.

Propidium iodide staining: a new application in fluorescence microscopy for analysis of cytoarchitecture in adult and developing rodent brain.

PubMed

Hezel, Marcus; Ebrahimi, Fahim; Koch, Marco; Dehghani, Faramarz

2012-10-01

Immunohistochemical visualization of antigens in specimen has evolved to an indispensable technique in biomedical research for investigations of cell morphology and pathology both in bright field and fluorescence microscopy. While there are couple of staining methods that reveal entire cytoarchitecture in bright field microscopy such as Nissl or hemalaun-eosin, there are still limitations in visualizations of cytoarchitecture in fluorescence microscopy. The present study reports a simple staining method that provides the required illustration of cell allocations and cellular composition in fluorescence microscopy in adult and in developing rodent central nervous system using the fluorophore propidium iodide (PI, 5μg/mL). PI is a well-accepted marker for degenerating cells when applied prior to fixation (pre-fixation PI staining). Here, PI was added to the sections after the fixation (post-fixation PI staining). This revised labeling procedure led to similar cytoarchitectural staining patterns in fluorescence microscopy as observed with hemalaun in bright field microscopy. This finding was proven in organotypic hippocampal slice cultures (OHSC) and brain sections obtained from different postnatal developmental stages. Excitotoxically lesioned OHSC subjected to pre-fixation PI staining merely showed brightly labeled condensed nuclei of degenerating neurons. In contrast, post-fixation PI staining additionally revealed extensive labeling of neuronal cell bodies and glial cells within the OHSC, thus allowing visualization of stratification of neuronal layers and cell morphology. Furthermore, post-fixation PI staining was combined with NeuN, calbindin, calretinin, glial fibrillary acidic protein or Griffonia simplicifolia isolectin B4 (IB(4)) in post natal (p1 and p9) and adult rats. In early post-natal brain sections almost all mentioned cellular markers led to an incomplete staining of the native cell organization and resulted in an inaccurate estimation of cell morphology when compared to adult brains. In contrast, post-fixation PI staining allowed investigation of the whole cytoarchitecture independent of the developmental stage. Taken together, post-fixation PI staining provides a detailed insight in the morphology of both developing and adult brain tissues in fluorescence microscopy. Copyright © 2012 Elsevier Ltd. All rights reserved.

Investigation of Helicobacter pylori in tonsillary tissue with Pronto Dry test and pathologic examination.

PubMed

Aslan, Sundus; Yilmaz, Ismail; Bal, Nebil; Sener, Mesut; Butros, Reha; Demirhan, Beyhan; Ozluoglu, Levent N

2007-09-01

The objectives of this clinical study were to identify, by means of the Pronto Dry test and pathologic examination, Helicobacter pylori (HP) in tonsillary tissue and to establish the role of HP in tonsillary microbiology by identifying that bacterium in the tonsillary mucosa or within the tonsil core. The subjects consisted of 52 patients (25 men and 27 women; age range, 3-65 years; mean age, 15.1+/-14.5 years) who were scheduled to undergo tonsillectomy for the treatment of chronic tonsillitis and who had not been treated with an antibiotic or a bismuth-containing compound for 6 months before the initiation of the study. In each patient, two specimens (one 4 mm x 4 mm tissue sample from the nonmucosal tonsil core and one 4 mm x 4 mm sample of mucosal tissue) were excised from both tonsils immediately after tonsillectomy. The specimens were placed in the Pronto Dry test kit, and the test results were obtained 1 h later. The remaining tonsillary tissues were submitted for pathologic analysis via hematoxylin-eosin stain, Giemsa stain, Warthin-Starry silver stain, and staining for inducible nitric oxide synthase (iNOS). The results of the Pronto Dry test were positive for HP in 42% (n=22) of the excised mucosal tissue and in 47% (n=24) of the excised core tissue. In 27% (n=14) of the patients, both the core and the mucosal tissues tested positive for HP. There was no significant difference between the positive Pronto Dry test ratios of the biopsies obtained from the mucosa and those obtained from the core (P=0.693). iNOS staining showed that macrophage iNOS activity was significantly higher (P=0.025) in biopsied mucosal tissues with a positive Pronto Dry test result than in those with a negative result. Light microscopy revealed no HP in samples stained with hematoxylin-eosin stain, Giemsa stain, or Warthin-Starry silver stain. Positive Pronto Dry test results and the results of iNOS staining showed that HP contributes to chronic tonsillitis, especially at the mucosal layer. Although HP does not colonize, it contributes to the chronic tonsillary inflammatory process as a triggering agent by affecting macrophages in the tonsil and thus increasing iNOS expression.

Hematoxylin and Eosin Counterstaining Protocol for Immunohistochemistry Interpretation and Diagnosis.

PubMed

Grosset, Andrée-Anne; Loayza-Vega, Kevin; Adam-Granger, Éloïse; Birlea, Mirela; Gilks, Blake; Nguyen, Bich; Soucy, Geneviève; Tran-Thanh, Danh; Albadine, Roula; Trudel, Dominique

2017-12-21

Hematoxylin and eosin (H&E) staining is a well-established technique in histopathology. However, immunohistochemistry (IHC) interpretation is done exclusively with hematoxylin counterstaining. Our goal was to investigate the potential of H&E as counterstaining (H&E-IHC) to allow for visualization of a marker while confirming the diagnosis on the same slide. The quality of immunostaining and the fast-technical performance were the main criteria to select the final protocol. We stained multiple diagnostic tissues with class I IHC tests with different subcellular localization markers (anti-CK7, CK20, synaptophysin, CD20, HMB45, and Ki-67) and with double-staining on prostate tissues with anti-high molecular weight keratins/p63 (DAB detection) and p504s (alkaline phosphatase detection). To validate the efficacy of the counterstaining, we stained tissue microarrays from the Canadian Immunohistochemistry Quality Control (cIQc) with class II IHC tests (ER, PR, HER2, and p53 markers). Interobserver and intraobserver concordance was assessed by κ statistics. Excellent agreement of H&E-IHC interpretation was observed in comparison with standard IHC from our laboratory (κ, 0.87 to 1.00), and with the cIQc reference values (κ, 0.81 to 1.00). Interobserver and intraobserver agreement was excellent (κ, 0.89 to 1.00 and 0.87 to 1.00, respectively). We therefore show for the first time the potential of using H&E counterstaining for IHC interpretation. We recommend the H&E-IHC protocol to enhance diagnostic precision for the clinical workflow and research studies.

CGP stain: An inexpensive, odorless, rapid, sensitive, and in principle in vitro methylation-free Coomassie Brilliant Blue stain.

PubMed

Yasumitsu, Hidetaro; Ozeki, Yasuhiro; Kawsar, Sarkar M A; Toda, Tosifusa; Kanaly, Robert

2010-11-01

Coomassie Brilliant Blue (CBB) protein stains are inexpensive but detect proteins at only at microgram levels. Because of acetic acid and methanol, they cause skin irritation and reduce work motivation by malodor. Recent mass spectrometric (MS) analyses demonstrated that nanogram-sensitive colloidal CBB staining resulted in in vitro methylations of proteins. We propose a rapid, inexpensive, sensitive, odorless, less harsh, and in vitro methylation-free CBB stain. CGP uses three components: citric acid, CBB G-250, and polyvinylpyrrolidone. CGP detects proteins at 12ng within 45min, and because it is nonalcohol, in principle in vitro methylation would be eliminated. Indeed, MS analysis of CGP-stained bands confirmed a lack of methylation. 2010 Elsevier Inc. All rights reserved.

In vitro Protoscolicidal Effects of Cinnamomum zeylanicum Essential Oil and Its Toxicity in Mice.

PubMed

Mahmoudvand, Hossein; Mahmoudvand, Hormoz; Oliaee, Razieh Tavakoli; Kareshk, Amir Tavakoli; Mirbadie, Seyed Reza; Aflatoonian, Mohammad Reza

2017-10-01

This study investigates the scolicidal effects of Cinnamomum zeylanicum essential oil against the protoscoleces of hydatid cysts and its toxicity in the mice model. Gas chromatography/mass spectroscopy analyses were used to identify the constituents of essential oil. Protoscoleces were treated with different concentrations of the essential oil (6.25-100 µL/mL) in each test tube for 5-30 min. The viability of protoscoleces was confirmed using eosin exclusion test (0.1% eosin staining). Forty-eight male NMRI mice were also used to determine the toxicity of C. zeylanicum essential oil (0.5-4 mL/kg). The main components were found to be cinnamaldehyde (91.8%), ρ metoxicinamate (1.57%), and α pinene (1.25%). Findings indicate that C. zeylanicum essential oil with the concentrations of 100 and 50 µL/mL killed 100% of protoscoleces after 5 min of exposure. Also, the lower concentrations of C. zeylanicum essential oil motivated a late protoscolicidal effect. The LD 50 value of intraperitoneal injection of C. zeylanicum essential oil was 2.07 mL/kg body weight after 48 h, and the maximum nonfatal dose was 1.52 mL/kg body weight. The results also showed that there was no significant toxicity following oral administration of C. zeylanicum essential oil for 2 weeks. The results exhibited the favorable scolicidal activity of C. zeylanicum , which could be applied as a natural scolicidal agent in hydatid cyst surgery. We evaluated the efficacy of Cinnamomum zeylanicum essential oil against hydatid cyst protoscolecesThe viability of protoscoleces was confirmed using eosin exclusion test (0.1% eosin staining)Forty-eight male NMRI mice were also used to determine the toxicity of C. zeylanicum essential oilC. zeylanicum with potent scolicidal activity could be applied as a natural scolicidal agent in surgery. Abbreviations used: GC/MS: Gas chromatography/mass spectrometry analysis; CE: Cystic echinococcosis; LD50: Lethal dose 50%; I.p: Intraperitoneally.

Quantitative analysis of mast cell count and density in chronic periodontal disease.

PubMed

Rathod, Surekha; Raj, Anubha; Wanikar, Ishita

2018-01-01

Mast cells play a crucial role in activation of acquired immune response to inflammatory conditions of periodontal diseases. They promote inflammation by releasing pro-inflammatory mediators and bring about angiogenesis, degeneration of the extracellular matrix, and tissue remodeling. Since there is little literature regarding the role of mast cells in periodontitis, the present study was aimed to evaluate mast cell count (MCC) and density in periodontitis. A total of eighty participants, Group I ( n = 40) healthy participants and Group II ( n = 40) participants with moderate chronic periodontitis, were included in the study. Tissue samples of 5 micron were obtained from each participant and were fixed in 10% formalin. Inflammation assessment was carried out after staining the sections with hematoxylin/eosin (H and E) followed by toluidine blue and mast cells were counted. MCC in healthy group (1.32 ± 0.43) was significantly smaller than periodontitis group (10.28 ± 1.15) and also mast cell density in healthy group (98.08 ± 37.40) was smaller than periodontitis group (803.43 ± 89.94) with P < 0.0001. It could be concluded that participants with chronic periodontitis have a higher MCC and density when compared with healthy participants.

'Blue bubble' technique: an ab interno approach for Descemet separation in deep anterior lamellar keratoplasty using trypan blue stained viscoelastic device.

PubMed

Livny, Eitan; Bahar, Irit; Hammel, Naama; Nahum, Yoav

2018-04-01

In this study, we examined a novel variant of 'big-bubble' deep anterior lamellar keratoplasty using trypan-blue-stained viscoelastic device for the creation of a pre-descemetic bubble. Ten corneoscleral rims were mounted on an artificial anterior chamber (AC). The AC was filled with air through a limbal paracentesis. A Melles' triangulated spatula was inserted through the paracentesis, with its tip penetrating the AC, was then slightly retracted and pushed into the deep stroma above the roof of the paracentesis. A mixture of trypan blue and viscoelastic device (Healon, Abbott Medical Optics, Abbott Park, Illinois) was injected into this intra-stromal pocket using a 27-G cannula to create a pre-descemetic separation bubble. Bubble type and visualization of dyed viscoelastic device were noted. The method was later employed in three cases. In all 10 corneoscleral rims, the technique successfully created a visible pre-descemetic (type 1) bubble that could be expanded up to the predicted diameter of trephination. Subsequent trephination and the removal of corneal stroma were uneventful. In two out of four clinical cases, a type 1 bubble was created, while in two others, visco-dissection failed and dyed viscoelastic was seen in the AC. The presented technique holds promise of being a relatively easy to perform, predictable and well-controlled alternative for achieving a type 1 bubble during deep anterior lamellar keratoplasty surgery. The trypan-blue-stained viscoelastic device facilitates proper visualization and control of the separation bubble and assists in identifying the penetrance to the separation bubble prior to removal of the stromal cap. © 2017 Royal Australian and New Zealand College of Ophthalmologists.

Western Blot of Stained Proteins from Dried Polyacrylamide Gels

NASA Technical Reports Server (NTRS)

Gruber, Claudia; Stan-Lotter, Helga

1996-01-01

Western blotting of proteins is customarily performed following their separation on polyacrylamide gels, either prior to staining (1) or, as recently reported, following staining (2). We describe here Western blotting with stained gels, which had been dried and some of which had been stored for years. This procedure permits immunological analysis of proteins, to which antisera may have become available only later, or where the application of newly developed sensitive detection methods is desired. Once rehydration of the gels is achieved, proteins can be-transferred to blotting membranes by any appropriate protocol. Proteins stained with Coomassie Blue have to be detected with a non-chromogenic method, such as the film-based enhanced chemiluminescence (ECL)2) procedure (3). Silver stained proteins, which transfer in the colorless form, may be visualized by any detection method, although, because of the usually very low amounts of proteins, detection by ECL is preferable. Blotting of stained proteins from rehydrated gels is as rapid and as quantitative as from freshly prepared gels, in contrast to blotting from wet stained gels, which requires extensive washing and results in low transfer efficiency (2). Together with a photographic record of the gel pattern, unambiguous identification of immunoreactive proteins from complex mixtures is possible. Some further applications of this work are discussed.

Comparison of Cryopreserved Human Sperm between Ultra Rapid Freezing and Slow Programmable Freezing: Effect on Motility, Morphology and DNA Integrity.

PubMed

Tongdee, Pattama; Sukprasert, Matchuporn; Satirapod, Chonticha; Wongkularb, Anna; Choktanasiri, Wicham

2015-05-01

Cryopreservation of sperm is common methods to preserve male fertility. Sperm freezing, suggest slow programmable freezing caused lower change of sperm morphology than sperm freezing in vapor of liquid nitrogen. Ultra rapid freezing is easy to be worked on, less time, low cost and does not need high experience. To compare the effect on sperm motility, morphology and DNA integrity of post-thawed sperm after ultra rapid freezing and slow programmable freezing methods. Experimental study at laboratory of infertility unit, Department of Obstetrics and Gynecology, Faculty of Medicine Ramathibodi Hospital. Thirty-seven semen samples with normal semen analysis according to World Health Organization (WHO) 1999 [normal sperm volume ( 2 ml) and normal sperm concentration (≥ 20 x10(6)/ml) and sperm motility (≥ 50%)]. Semen samples were washed. Then each semen sample was divided into six cryovials. Two cryovials, 0.5 ml each, were cryopreserved by slow programmable freezing. Four 0.25 ml containing cryovials, were cryopreserved by ultra rapidfreezing method. After cryopreservationfor 1 month, thawedprocess was carried out at room temperature. Main outcomes are sperm motility was determined by Computer-Assisted Semen Analysis (CASA), sperm morphology was determined by eosin-methylene blue staining and sperm DNA integrity was assessed by TUNEL assay. Sperm motility was reduced significantly by both methods, from 70.4 (9.0)% to 29.1 (12.3)% in slowprogrammable freezing and to 19.7 (9.8)% in ultra rapid freezing (p < 0.05). Sperm motility decreased significantly more by ultra rapid freezing (p < 0.001). The percentage of normal sperm morphology and DNA integrity were also reduced significantly by both methods. However, no significant difference between the two methods was found (p > 0.05). Cryopreservation of human sperm for 1 month significantly decreased sperm motility, morphology and DNA integrity in both methods. However sperm motility was decreased more by ultra rapid freezing.

Lectins stain cells differentially in the coral, Montipora capitata

USGS Publications Warehouse

Work, Thierry M.; Farah, Yael

2014-01-01

A limitation in our understanding of coral disease pathology and cellular pathogenesis is a lack of reagents to characterize coral cells. We evaluated the utility of plant lectins to stain tissues of a dominant coral, Montipora capitata, from Hawaii. Of 22 lectins evaluated, nine of these stained structures in the upper or basal body wall of corals. Specific structures revealed by lectins that were not considered distinct or evident on routine hematoxylin and eosin sections of coral tissues included apical and basal granules in gastrodermis and epidermis, cnidoglandular tract and actinopharynx cell surface membranes, capsules of mature holotrichous isorhizas, and perivitelline and periseminal cells. Plant lectins could prove useful to further our understanding of coral physiology, anatomy, cell biology, and disease pathogenesis.

Analysis of oral lesion biopsies identified and evaluated by visual examination, chemiluminescence and toluidine blue.

PubMed

Epstein, J B; Silverman, S; Epstein, J D; Lonky, S A; Bride, M A

2008-06-01

Conventional visual examination and palpation remains the gold-standard for the identification of oral mucosal lesions. The purpose of this study was to investigate the adjunctive value of a chemiluminescent light source (ViziLite, Zila Pharmaceuticals, Phoenix, Arizona) and application of pharmaceutical grade toluidine blue (TBlue(630), Zila Pharmaceuticals, Phoenix, Arizona) to further assess lesions identified during the conventional oral soft tissue examination. Lesions deemed clinically suspicious by visual examination under incandescent light were further assessed under chemiluminescence and then application of toluidine blue stain. Differences between the conventional visual examination and chemiluminescent examination were noted on four characteristics which may aid in lesion identification. Tissue retention of toluidine blue stain was documented. Each suspicious lesion was biopsied and diagnosed based upon routine histopathology. Both adjunctive exams were evaluated by comparing the histologic diagnosis. The additive value of toluidine blue stain retention was assessed in lesions diagnosed as "serious pathology" defined as severe dysplasia, carcinoma in situ and squamous cell carcinoma. Ninety-seven clinically suspicious lesions in 84 patients were identified. The chemiluminescent exam improved the brightness and/or sharpness of margin in 61.8% of identified lesions. Biopsied lesions with toluidine blue stain retention reduced the false positive rate by 55.26% while maintaining a 100% negative predictive value (NPV). Chemiluminescence was shown to increase the brightness and margins of mucosal lesions in a majority of cases and therefore may assist in identification of mucosal lesions not considered under traditional visual examination. Toluidine blue stain retention was associated with a large reduction in biopsies showing benign histology (false positive biopsy results), while maintaining a 100% NPV for the presence of severe dysplasia or cancer. Practitioners may consider use of these adjuncts in practice, however the results presented are based upon experienced providers in referral centers for mucosal disease or cancer centers and therefore positive findings may be an indication for referral to experienced providers.

Epithelial apoptosis in mechanistically distinct methods of injury in the murine small intestine

PubMed Central

Vyas, Dinesh; Robertson, Charles M; Stromberg, Paul E; Martin, James R.; Dunne, W. Michael; Houchen, Courtney W; Barrett, Terrence A; Ayala, Alfred; Perl, Mario; Buchman, Timothy G; Coopersmith, Craig M

2007-01-01

Gut epithelial apoptosis is involved in the pathophysiology of multiple diseases. This study characterized intestinal apoptosis in three mechanistically distinct injuries with different kinetics of cell death. FVB/N mice were subjected to gamma radiation, Pseudomonas aeruginosa pneumonia or injection of monoclonal anti-CD3 antibody and sacrificed 4, 12, or 24 hours post-injury (n=10/time point). Apoptosis was quantified in the jejunum by hematoxylin and eosin (H&E), active caspase-3, terminal deoxynucleotidyl transferase dUTP-mediated nick end labeling (TUNEL), in situ oligoligation reaction (ISOL,) cytokeratin 18, and annexin V staining. Reproducible results were obtained only for H&E, active caspase-3, TUNEL and ISOL, which were quantified and compared against each other for each injury at each time point. Kinetics of injury were different with early apoptosis highest following radiation, late apoptosis highest following anti CD3, and more consistent levels following pneumonia. ISOL was the most consistent stain and was always statistically indistinguishable from at least 2 stains. In contrast, active caspase-3 demonstrated lower levels of apoptosis, while the TUNEL assay had higher levels of apoptosis in the most severely injured intestine regardless of mechanism of injury. H&E was a statistical outlier more commonly than any other stain. This suggests that regardless of mechanism or kinetics of injury, ISOL correlates to other quantification methods of detecting gut epithelial apoptosis more than any other method studied and compares favorably to other commonly accepted techniques of quantifying apoptosis in a large intestinal cross sectional by balancing sensitivity and specificity across a range of times and levels of death. PMID:17357092

Spatial Statistics for Segmenting Histological Structures in H&E Stained Tissue Images.

PubMed

Nguyen, Luong; Tosun, Akif Burak; Fine, Jeffrey L; Lee, Adrian V; Taylor, D Lansing; Chennubhotla, S Chakra

2017-07-01

Segmenting a broad class of histological structures in transmitted light and/or fluorescence-based images is a prerequisite for determining the pathological basis of cancer, elucidating spatial interactions between histological structures in tumor microenvironments (e.g., tumor infiltrating lymphocytes), facilitating precision medicine studies with deep molecular profiling, and providing an exploratory tool for pathologists. This paper focuses on segmenting histological structures in hematoxylin- and eosin-stained images of breast tissues, e.g., invasive carcinoma, carcinoma in situ, atypical and normal ducts, adipose tissue, and lymphocytes. We propose two graph-theoretic segmentation methods based on local spatial color and nuclei neighborhood statistics. For benchmarking, we curated a data set of 232 high-power field breast tissue images together with expertly annotated ground truth. To accurately model the preference for histological structures (ducts, vessels, tumor nets, adipose, etc.) over the remaining connective tissue and non-tissue areas in ground truth annotations, we propose a new region-based score for evaluating segmentation algorithms. We demonstrate the improvement of our proposed methods over the state-of-the-art algorithms in both region- and boundary-based performance measures.

Control of brown stain: in Eastern white pine

Treesearch

Robert E. Stutz; Peter Koch; Millard L. Oldham

1961-01-01

Degrade caused by brown stain and blue stain in eastern white pine was virtually eliminated by the use of sap stain chemicals and sodium azide. Combinations of buffered sodium azide with both sodium pentachlorophenate plus borax and buffered ethyl mercury phosphate were effective.

The Luna stain, an improved selective stain for detection of microsporidian spores in histologic sections

PubMed Central

Peterson, Tracy S.; Spitsbergen, Jan M.; Feist, Stephen W.; Kent, Michael L.

2014-01-01

Microsporidia in histologic sections are most often diagnosed by observing spores in host tissues. Spores are easy to identify if they occur in large aggregates or xenomas when sections are stained with hematoxylin and eosin (H&E). However, individual spores are not frequently detected in host tissues with conventional H&E staining, particularly if spores are scattered within the tissues, areas of inflammation or small spores in nuclei (i.e., Nucleospora salmonis). Hence, a variety of selective stains that enhance visualization of spores are recommended. We discovered that the Luna stain, used to highlight eosinophils, red blood cells and chitin in arthropods and other invertebrates, also stains spores of Pseudoloma neurophilia. We compared this stain to the Gram, Fite’s acid fast, Giemsa, and H&E stains on eight aquatic microsporidian organisms that were readily available in our two laboratories: Loma salmonae, Glugea anomala, Pseudoloma neurophilia, Pleistophora hyphessobryconis, Pleistophora vermiformis, Glugea sp., Steinhausia mytilovum and an unidentified microsporidian from E. sinensis, UK. Based on tinctorial properties and background staining, the Luna stain performed better for detection of 6 of the 8 microsporidia. Gram stain was superior for the two microsporidia from invertebrates, Steinhausia mytilovum and the unidentified microsporidian from E. sinensis. PMID:21848126

Luna stain, an improved selective stain for detection of microsporidian spores in histologic sections.

PubMed

Peterson, Tracy S; Spitsbergen, Jan M; Feist, Stephen W; Kent, Michael L

2011-06-16

Microsporidia in histologic sections are most often diagnosed by observing spores in host tissues. Spores are easy to identify if they occur in large aggregates or xenomas when sections are stained with hematoxylin and eosin (H&E). However, individual spores are not frequently detected in host tissues with conventional H&E staining, particularly if spores are scattered within the tissues, areas of inflammation, or small spores in nuclei (i.e. Nucleospora salmonis). Hence, a variety of selective stains that enhance visualization of spores is recommended. We discovered that the Luna stain, used to highlight eosinophils, red blood cells, and chitin in arthropods and other invertebrates, also stains spores of Pseudoloma neurophilia. We compared this stain to the Gram, Fite's acid fast, Giemsa, and H&E stains on 8 aquatic microsporidian organisms that were readily available in our 2 laboratories: Loma salmonae, Glugea anomala, Pseudoloma neurophilia, Pleistophora hyphessobryconis, Pleistophora vermiformis, Glugea sp., Steinhausia mytilovum, and an unidentified microsporidian from UK mitten crabs Eriocheir sinensis. Based on tinctorial properties and background staining, the Luna stain performed better for detection of 6 of the 8 microsporidia. Gram stain was superior for the 2 microsporidia from invertebrates: S. mytilovum and the unidentified microsporidian from E. sinensis.

Ontogeny of the larynx and flight ability in Jamaican fruit bats (Phyllostomidae) with considerations for the evolution of echolocation.

PubMed

Carter, Richard T; Adams, Rick A

2014-07-01

Echolocating bats have adaptations of the larynx such as hypertrophied intrinsic musculature and calcified or ossified cartilages to support sonar emission. We examined growth and development of the larynx relative to developing flight ability in Jamaican fruit bats to assess how changes in sonar production are coordinated with the onset of flight during ontogeny as a window for understanding the evolutionary relationships between these systems. In addition, we compare the extent of laryngeal calcification in an echolocating shrew species (Sorex vagrans) and the house mouse (Mus musculus), to assess what laryngeal chiropteran adaptations are associated with flight versus echolocation. Individuals were categorized into one of five developmental flight stages (flop, flutter, flap, flight, and adult) determined by drop-tests. Larynges were cleared and stained with alcian blue and alizarin red, or sectioned and stained with hematoxylin and eosin. Our results showed calcification of the cricoid cartilage in bats, represented during the flap stage and this increased significantly in individuals at the flight stage. Thyroid and arytenoid cartilages showed no evidence of calcification and neither cricoid nor thyroid showed significant increases in rate of growth relative to the larynx as a whole. The physiological cross-sectional area of the cricothyroid muscles increased significantly at the flap stage. Shrew larynges showed signs of calcification along the margins of the cricoid and thyroid cartilages, while the mouse larynx did not. These data suggest the larynx of echolocating bats becomes stronger and sturdier in tandem with flight development, indicating possible developmental integration between flight and echolocation. © 2014 Wiley Periodicals, Inc.

Morphological study of the eye and adnexa in capuchin monkeys (Sapajus sp.)

PubMed Central

Silva, Danielle Nascimento; Oriá, Arianne Pontes; Araujo, Nayone Lantyer; Martins-Filho, Emanoel; Muramoto, Caterina; Libório, Fernanda de Azevedo

2017-01-01

The objective of this study was to describe the anatomic and histologic features of the Sapajus sp. eye, comparing similarities and differences of humans and other species of non-human primates for biomedical research purposes. Computed tomography (CT) of adnexa, eye and orbit live animal, as well as formolized pieces of the same structures of Sapajus sp. for anatomical and histological study were also performed. The anatomical description of the eye and adnexa was performed using the techniques of topographic dissection and exenteration. Histological fragments were fixated in buffered formalin 10%, processed by the routine paraffin inclusion technique, stained with hematoxylin-eosin and special stains. CT scan evaluation showed no differences between the live animal and the formolized head on identification of visual apparatus structures. Anatomic and histologic evaluation revealed rounded orbit, absence of the supraorbital foramen and frontal notch, little exposure of the sclera, with slight pigmentation of the exposed area and marked pigmentation at the sclerocorneal junction. Masson's Trichrome revealed the Meibomian glands, the corneal epithelium and Bowman's membrane; in the choroid, melanocytes and Bruch's membrane were observed; and in the retina, cones and rods as well as, optic nerve, the lamina cribrosa of the nerve fibers bundles. Toluidine blue highlighted the membranes: Bowman, Descemet and the endothelium; in the choroid: melanocytes; and in the retina: nuclear layers and retinal pigment epithelium. In view of the observed results Sapajus sp. is an important experimental model for research in the ophthalmology field, which has been shown due to the high similarity of its anatomical and histological structures with the human species. PMID:29206882

Crystal violet stain as a selective stain for the assessment of mitotic figures in oral epithelial dysplasia and oral squamous cell carcinoma.

PubMed

Jadhav, Kiran B; Ahmed Mujib, B R; Gupta, Nidhi

2012-01-01

Assessment of mitotic figures (MFs) is routinely practiced as prognostic indicator in oral epithelial dysplasia (OED) and oral squamous cell carcinoma (OSCC), but identification of MFs poses a problem in terms of staining characteristics. To evaluate effectiveness of crystal violet stain for staining of MFs and its comparison with hematoxylin and eosin (H and E) stain. Study sample includes archival tissues embedded in paraffin blocks diagnosed as OED (n = 30) and OSCC (n = 30). The control group comprised of tissue specimen from oral mucosa of healthy volunteers (n = 30). Two serial sections of each tissue specimen were stained separately with H and E stain and 1% crystal violet stain. The stained sections were observed under microscope for identification and counting of MFs. Data obtained was statistically analyzed by using the Man-Whitney U test. A significant increase in number of MFs was observed in OED and OSCC in comparison with normal oral mucosa. There was a highly significant increase in number of MFs in crystal violet stained tissue sections when compared with H and E stain. Metaphase is the most commonly observed phase of mitosis in crystal violet stain when compared with H and E stain for all three groups. Crystal violet stain can be considered as selective stain for mitotic figures.

Comparison of histological features, and description of histopathological lesions in thyroid glands from three species of free-ranging sharks from the northwestern Atlantic, the blue shark, Prionace glauca (L.), the shortfin mako, Isurus oxyrhinchus Rafinesque, and the thresher, Alopias vulpinus (Bonnaterre).

PubMed

Borucinska, J D; Tafur, M

2009-09-01

Abstract Histomorphological features of piscine thyroids are widely accepted and frequently used as bioindicators of environmental pollution. This despite the fact that there is marked variation in thyroid morphology resulting from numerous pathological and physiological conditions. Our hypothesis was that there will be variations in histological features in thyroids collected from different shark species during the summer season in the northwestern Atlantic. To test our hypothesis, we examined histological features encountered in grossly normal thyroids from three species of sharks, the shortfin mako, Isurus oxyrhinchus, thresher, Alopias vulpinus and blue sharks, Prionace glauca. In addition, microscopic lesions from these thyroids were described. Ninety-four sharks were collected in summer 2001, 2002 and 2004. Routine, haematoxylin and eosin-stained paraffin-embedded sections were studied by light microscopy. We found inter-species but not intra-species variation in histological patterns of thyroids, which were distinct enough to allow 'blind' assignment of a thyroid to the specific species. The most common lesions encountered were lymphofollicular hyperplasia and chronic thyroiditis. In addition, one case each of intravascular larval nematodes and a myxosporean infection was found. Our results provide the first data on species-specific morphology of thyroids collected during summer months from sharks. The results indicate that familiarity with normal thyroid morphology is crucial before using shark thyroids in biomonitoring of environmental contamination or interpreting data from this gland in shark-health studies.

Melanomacrophages in three species of free-ranging sharks from the northwestern Atlantic, the blue shark Prionacae glauca (L.), the shortfin mako, Isurus oxyrhinchus Rafinesque, and the thresher, Alopias vulpinus (Bonnaterre).

PubMed

Borucinska, J D; Kotran, K; Shackett, M; Barker, T

2009-10-01

The melanomacrophage aggregates or cells (MMC) are commonly used as biomarkers of exposure to pollution in fish, albeit their numbers and morphological characteristics can be influenced not only by environmental toxins but also by a range of physiological parameters and pathological conditions. Accordingly, before we can use MMC as biomarkers in any fish species, their normal, 'background' characteristics have to be established in apparently healthy fish. The knowledge of MMC in sharks is minimal. The aim of this study was to characterize MMC from 51 free-ranging, large pelagic sharks from the northwestern Atlantic, including shortfin mako, Isurus oxyrhinchus, thresher, Alopias vulpinus and blue shark, Prionacae glauca. The rationale of this study was twofold. First, because, sharks have life histories predisposing them to the accumulation of environmental toxins they constitute good sentinel species of the health of the global marine ecosystem. Second, because many shark populations are in decline or threatened by extinction, we need to expand our understanding of their health status in order to develop appropriate protective measures. All sharks were collected by sports fishing gear between June and August in 2007. Their health condition was assessed by necropsy, morphometrics, and by microscopic examination of gonads, livers, spleens and kidneys. Routine, haematoxylin and eosin and/or Pearl's reagent-stained paraffin embedded sections were studied by light microscopy. Our results provide the first data on the morphometric and morphological characteristics of MMC in viscera of apparently healthy free-ranging sharks from the northwestern Atlantic.

The Benefit of Microskin in Combination With Autologous Keratinocyte Suspension to Treat Full Skin Loss In Vivo.

PubMed

Yuru, Shang; Dawei, Li; Chuanan, Shen; Kai, Yin; Li, Ma; Longzhu, Li; Dongxu, Zhao; Wenfeng, Cheng

Patients with extensive deep burns often lack enough autologous skin to cover the wounds. This study explores a new method using microskin in combination with autologous keratinocytes in the treatment of extensive deep burn. Wounds in the combination group were treated with automicroskin at an area expansion ratio of 20:1 (wound area to automicroskin area) and autologous keratinocyte suspension, which were compared with the following treatments: no autotransplant, only allografts (control group); autologous keratinocyte suspension only (keratinocyte only group); automicroskin at an area expansion ratio of 20:1 (20:1 group); and automicroskin at an area expansion ratio of 10:1 (10:1 group, positive control). The authors used epithelialization rate (epithelialized area on day 21 divided by original wound area), hematoxylin and eosin staining, laminin, and type IV collagen immunohistochemistry to assess wound healing. The epithelialization rate of combination group (74.2% ± 8.0%) was similar to that of 10: 1 group (84.3% ± 11.9%, P = .085) and significantly (P < .05) higher than that of 20:1 group (59.2% ± 10.8%), keratinocyte only group (53.8% ± 11.5%), and control group (22.7% ± 5.5%). The hematoxylin and eosin staining and immunohistochemistry showed the epithelialization in the combination group was better than that in the keratinocyte only group and control group. Microskin in combination with autologous keratinocyte suspension can promote the reepithelialization of full-thickness wounds and reduce the requirements for automircoskin, and it is a useful option in the treatment of extensive deep burns.

A method for normalizing pathology images to improve feature extraction for quantitative pathology

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tam, Allison; Barker, Jocelyn; Rubin, Daniel

Purpose: With the advent of digital slide scanning technologies and the potential proliferation of large repositories of digital pathology images, many research studies can leverage these data for biomedical discovery and to develop clinical applications. However, quantitative analysis of digital pathology images is impeded by batch effects generated by varied staining protocols and staining conditions of pathological slides. Methods: To overcome this problem, this paper proposes a novel, fully automated stain normalization method to reduce batch effects and thus aid research in digital pathology applications. Their method, intensity centering and histogram equalization (ICHE), normalizes a diverse set of pathology imagesmore » by first scaling the centroids of the intensity histograms to a common point and then applying a modified version of contrast-limited adaptive histogram equalization. Normalization was performed on two datasets of digitized hematoxylin and eosin (H&E) slides of different tissue slices from the same lung tumor, and one immunohistochemistry dataset of digitized slides created by restaining one of the H&E datasets. Results: The ICHE method was evaluated based on image intensity values, quantitative features, and the effect on downstream applications, such as a computer aided diagnosis. For comparison, three methods from the literature were reimplemented and evaluated using the same criteria. The authors found that ICHE not only improved performance compared with un-normalized images, but in most cases showed improvement compared with previous methods for correcting batch effects in the literature. Conclusions: ICHE may be a useful preprocessing step a digital pathology image processing pipeline.« less

Detection of Proteins on Blot Membranes

PubMed Central

Goldman, Aaron; Harper, Sandra; Speicher, David W.

2017-01-01

Staining of blot membranes enables the visualization of bound proteins. Proteins are usually transferred to blot membranes by electroblotting, by direct spotting of protein solutions, or by contact blots. Staining allows the efficiency of transfer to the membrane to be monitored. This unit describes protocols for staining proteins after electroblotting from polyacrylamide gels to blot membranes such as polyvinylidene difluoride (PVDF), nitrocellulose, or nylon membranes. The same methods can be used if proteins are directly spotted, either manually or using robotics. Protocols are included for seven general protein stains (amido black, Coomassie blue, Ponceau S, colloidal gold, colloidal silver, India ink, and MemCode) and three fluorescent protein stains (fluorescamine, IAEDANS, and SYPRO Ruby). Also included is an in-depth discussion of the different blot membrane types and the compatibility of different protein stains with downstream applications, such as immunoblotting or N-terminal Edman sequencing. PMID:27801518

Detection of Proteins on Blot Membranes.

PubMed

Goldman, Aaron; Harper, Sandra; Speicher, David W

2016-11-01

Staining of blot membranes enables the visualization of bound proteins. Proteins are usually transferred to blot membranes by electroblotting, by direct spotting of protein solutions, or by contact blots. Staining allows the efficiency of transfer to the membrane to be monitored. This unit describes protocols for staining proteins after electroblotting from polyacrylamide gels to blot membranes such as polyvinylidene difluoride (PVDF), nitrocellulose, or nylon membranes. The same methods can be used if proteins are directly spotted, either manually or using robotics. Protocols are included for seven general protein stains (amido black, Coomassie blue, Ponceau S, colloidal gold, colloidal silver, India ink, and MemCode) and three fluorescent protein stains (fluorescamine, IAEDANS, and SYPRO Ruby). Also included is an in-depth discussion of the different blot membrane types and the compatibility of different protein stains with downstream applications, such as immunoblotting or N-terminal Edman sequencing. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

[Pathological changes in hepatocytes of mice with obesity-induced type 2 diabetes by monosodium glutamate].

PubMed

Nakadate, Kazuhiko; Motojima, Kento; Kamata, Sumito; Yoshida, Testuro; Hikita, Masaaki; Wakamatsu, Hisanori

2014-01-01

Type 2 diabetes caused by chronic obesity is a major lifestyle-related disease. The present study aimed to determine the pathological changes in hepatocytes in chronic obesity. To develop our type 2 diabetes mouse model, we induced chronic obesity to mice by monosodium glutamate. By overeating, the mice significantly increased their body weight compared with age-matched healthy animals. To analyze the pathological changes in hepatocytes of chronic obesity before preclinical stage of type 2 diabetes, the mice were analyzed by hematoxylin-eosin staining of tissue sections at 15 w of age. In these mice, we observed eosin-negative accumulations of hepatocytes around central veins in the hepatic lobule. By Oil-Red O staining, the eosin-negative granules were identified in the lipid droplets. We then ascertained whether these lipid droplets of hepatocytes in the obese mice could be modified by diet. After 24 h of diet restriction, the lipid droplets of hepatocytes in the obese mice were swollen. Furthermore, after 48 h of the diet restriction, the lipid droplets continued swelling and the autophagy-like structures that were found in the healthy mice under the same condition in the obese mice were not observed. These results suggest that the obese mice might have delayed energy metabolism, which might have influenced the mechanisms of hepatocytes. These findings provide new insight into the functional changes in chronic obesity-induced type 2 diabetes and it is possible that the pathological feature make a contribution to promise the target of pharmacological therapy.

Accurate measurement of peripheral blood mononuclear cell concentration using image cytometry to eliminate RBC-induced counting error.

PubMed

Chan, Leo Li-Ying; Laverty, Daniel J; Smith, Tim; Nejad, Parham; Hei, Hillary; Gandhi, Roopali; Kuksin, Dmitry; Qiu, Jean

2013-02-28

Peripheral blood mononuclear cells (PBMCs) have been widely researched in the fields of immunology, infectious disease, oncology, transplantation, hematological malignancy, and vaccine development. Specifically, in immunology research, PBMCs have been utilized to monitor concentration, viability, proliferation, and cytokine production from immune cells, which are critical for both clinical trials and biomedical research. The viability and concentration of isolated PBMCs are traditionally measured by manual counting with trypan blue (TB) using a hemacytometer. One of the common issues of PBMC isolation is red blood cell (RBC) contamination. The RBC contamination can be dependent on the donor sample and/or technical skill level of the operator. RBC contamination in a PBMC sample can introduce error to the measured concentration, which can pass down to future experimental assays performed on these cells. To resolve this issue, RBC lysing protocol can be used to eliminate potential error caused by RBC contamination. In the recent years, a rapid fluorescence-based image cytometry system has been utilized for bright-field and fluorescence imaging analysis of cellular characteristics (Nexcelom Bioscience LLC, Lawrence, MA). The Cellometer image cytometry system has demonstrated the capability of automated concentration and viability detection in disposable counting chambers of unpurified mouse splenocytes and PBMCs stained with acridine orange (AO) and propidium iodide (PI) under fluorescence detection. In this work, we demonstrate the ability of Cellometer image cytometry system to accurately measure PBMC concentration, despite RBC contamination, by comparison of five different total PBMC counting methods: (1) manual counting of trypan blue-stained PBMCs in hemacytometer, (2) manual counting of PBMCs in bright-field images, (3) manual counting of acetic acid lysing of RBCs with TB-stained PBMCs, (4) automated counting of acetic acid lysing of RBCs with PI-stained PBMCs, and (5) AO/PI dual staining method. The results show comparable total PBMC counting among all five methods, which validate the AO/PI staining method for PBMC measurement in the image cytometry method. Copyright © 2012 Elsevier B.V. All rights reserved.

Response of Lutz, Sitka, and white spruce to attack by Dendroctonus rufipennis (Coleoptera: Scolytidae) and blue stain fungi

Treesearch

Richard A. Werner; Barbara L. Illman

1994-01-01

Mechanical wounding and wounding plus inoculation with a blue-stain fungus, Leptographium abietinum (Peck), associated with the spruce beetle, Dendroctonus rufipennis (Kirby), caused an induced reaction zone or lesion around the wound sites in Lutz spruce, Picea lutzii Little, Sitka spruce, P. sitchensis (Bong.) Carr., and white spruce, P. glauca (Moench) Voss, in...

Evaluation of white-rot fungal growth on southern yellow pine wood chips pretreated with blue-stain fungi

Treesearch

Suki C. Croan

2000-01-01

White-rotting basidiomycetes do not colonize on southern yellow pine. This study seeks to reduce the resinous extractive content of southern yellow pine by treating it with blue stain fungi. The mycelial growth of wood-inhabiting ligninolytic white-rot fungi can be achieved on pretreated southern yellow pine wood. Aureobasidium, Ceratocystis, and Ophiostoma spp....

Functional regeneration of recurrent laryngeal nerve injury during thyroid surgery using an asymmetrically porous nerve guide conduit in an animal model.

PubMed

Choi, Jeong-Seok; Oh, Se Heang; An, Hye-Young; Kim, Young-Mo; Lee, Jin Ho; Lim, Jae-Yol

2014-01-01

Vocal cord paralysis (VCP) caused by recurrent laryngeal nerve (RLN) damage during thyroidectomy commonly results in serious medico-legal problems. The purpose of this study was to evaluate the usefulness of an asymmetrically porous polycaprolactone (PCL)/Pluronic F127 nerve guide conduit (NGC) for functional regeneration in a RLN injury animal model. A biodegradable, asymmetrically porous PCL/F127 NGC with selective permeability was fabricated for use in this study. A 10-mm segment of left RLN was resected in 28 New Zealand white rabbits, and then an asymmetrically porous NGC or a nonporous silicone tube was interposed between both stumps and securely fixed. Vocal cord mobility was endoscopically evaluated at one, four, and eight weeks postoperatively. Nerve growth through NGCs was assessed by toluidine blue staining, and thyroarytenoid (TA) muscle atrophy was evaluated by hematoxylin and eosin staining. Immunohistochemical stainings for acetylcholinesterase (AchE), anti-neurofilament (NF), and anti-S100 protein were also conducted, and transmission electron microscopy (TEM) was used to evaluate functional nerve regeneration. At eight weeks postoperatively, endoscopic evaluations showed significantly better recovery from VCP in the asymmetrically porous PCL/F127 NGC group (6 of 10 rabbits) than in the silicone tube group (1 of 10 rabbits). Continued nerve growth on the damaged nerve endings was observed with time in the asymmetrically porous PCL/F127 NGC-interposed RLNs. TA muscle dimensions and AchE expressions in TA muscle were significantly greater in the asymmetrically porous PCL/F127 NGC group than in the silicone tube group. Furthermore, immunohistochemical staining revealed the expression of NF and S100 protein in the regenerated nerves in the asymmetrically porous PCL/F127 NGC group at eight weeks postoperatively, and at this time, TEM imaging showed myelinated axons in the regenerated RLNs. The study shows that asymmetrically porous PCL/F127 NGC provides a favorable environment for RLN regeneration and that it has therapeutic potential for the regeneration of RLN damage.

Accelerated Healing with a Mesh Autograft/Allodermal Composite Skin Graft Treated with Silver Nylon Dressings with and without Direct Current in Rats

DTIC Science & Technology

2000-07-01

heads ) (hematoxylin and eosin stain). No other arterioles and cap- illaries pass through the sheath. Before entering the papilla, the arteriole divides...Company; 1978:85. 39. Johnson KE. Histology and Embryology . Media, PA: Harwal Publishing Company; 1984:46–47. 40. Wang HJ, Chen TN. Porcine Dermis

Intracytoplasmic Crystalline Inclusions in the Hepatocytes of an Antelope

DTIC Science & Technology

2010-01-01

fine basophilic granules with hematoxylin and eosin stain (Figure 2(a)). Periportal areas had moderately increased fibrous connective tissue with marked...biliary hyperplasia , mild oval cell hyperplasia , and moderate infiltrates of predominant lymphocytes and plasma cells with occasional neutrophils...cholangiohepatitis, bridging portal fibrosis and biliary hyperplasia . There was no evidence of hepatocellular Report Documentation Page Form ApprovedOMB No. 0704

PROGRAMMED CELL DEATH IN EXTRAOCULAR MUSCLE TENDON/SCLERA PRECURSORS

EPA Science Inventory

Abstract

Purpose: This study was designed to examine the occurrence of natural cell death in the periocular mesenchyme of mouse embryos.

Methods: Vital staining with LysoTracker Red and Nile blue sulphate as well as terminal nick end labeling (TUNEL) were utiliz...

Blue-stain Fungi Associated with Roots of Southern Pine Trees Attacked by the Southern Pine Beetle, Dendroctonus frontalis

Treesearch

William J. Otrosina; Nolan J. Hess; Stanley J. Zarnoch; Thelma J. Perry; John P. Jones

1997-01-01

Forty paired plots were established from eastern Texas to Alabama to study root-infecting, blue-stain fungi in southern pine stands undergoing southern pine beetle (SPB) attack. Woody roots were sampled in plots undergoing recent or current attack by the SPB. Comparisons were made between occurrence of Lcptogrqhiumspp. and related fungi and data on various...

Antibody-supervised deep learning for quantification of tumor-infiltrating immune cells in hematoxylin and eosin stained breast cancer samples.

PubMed

Turkki, Riku; Linder, Nina; Kovanen, Panu E; Pellinen, Teijo; Lundin, Johan

2016-01-01

Immune cell infiltration in tumor is an emerging prognostic biomarker in breast cancer. The gold standard for quantification of immune cells in tissue sections is visual assessment through a microscope, which is subjective and semi-quantitative. In this study, we propose and evaluate an approach based on antibody-guided annotation and deep learning to quantify immune cell-rich areas in hematoxylin and eosin (H&E) stained samples. Consecutive sections of formalin-fixed parafin-embedded samples obtained from the primary tumor of twenty breast cancer patients were cut and stained with H&E and the pan-leukocyte CD45 antibody. The stained slides were digitally scanned, and a training set of immune cell-rich and cell-poor tissue regions was annotated in H&E whole-slide images using the CD45-expression as a guide. In analysis, the images were divided into small homogenous regions, superpixels, from which features were extracted using a pretrained convolutional neural network (CNN) and classified with a support of vector machine. The CNN approach was compared to texture-based classification and to visual assessments performed by two pathologists. In a set of 123,442 labeled superpixels, the CNN approach achieved an F-score of 0.94 (range: 0.92-0.94) in discrimination of immune cell-rich and cell-poor regions, as compared to an F-score of 0.88 (range: 0.87-0.89) obtained with the texture-based classification. When compared to visual assessment of 200 images, an agreement of 90% (κ = 0.79) to quantify immune infiltration with the CNN approach was achieved while the inter-observer agreement between pathologists was 90% (κ = 0.78). Our findings indicate that deep learning can be applied to quantify immune cell infiltration in breast cancer samples using a basic morphology staining only. A good discrimination of immune cell-rich areas was achieved, well in concordance with both leukocyte antigen expression and pathologists' visual assessment.

Preparation of DNA from cytological material: effects of fixation, staining, and mounting medium on DNA yield and quality.

PubMed

Dejmek, Annika; Zendehrokh, Nooreldin; Tomaszewska, Malgorzata; Edsjö, Anders

2013-07-01

Personalized oncology requires molecular analysis of tumor cells. Several studies have demonstrated that cytological material is suitable for DNA analysis, but to the authors' knowledge there are no systematic studies comparing how the yield and quality of extracted DNA is affected by the various techniques used for the preparation of cytological material. DNA yield and quality were compared using cultured human lung cancer cells subjected to different preparation techniques used in routine cytology, including fixation, mounting medium, and staining. The results were compared with the outcome of epidermal growth factor receptor (EGFR) genotyping of 66 clinical cytological samples using the same DNA preparation protocol. All tested protocol combinations resulted in fragment lengths of at least 388 base pairs. The mounting agent EcoMount resulted in higher yields than traditional xylene-based medium. Spray and ethanol fixation resulted in both a higher yield and better DNA quality than air drying. In liquid-based cytology (LBC) methods, CytoLyt solution resulted in a 5-fold higher yield than CytoRich Red. Papanicolaou staining provided twice the yield of hematoxylin and eosin staining in both liquid-based preparations. Genotyping outcome and quality control values from the clinical EGFR genotyping demonstrated a sufficient amount and amplifiability of DNA in both spray-fixed and air-dried cytological samples. Reliable clinical genotyping can be performed using all tested methods. However, in the cell line experiments, spray- or ethanol-fixed, Papanicolaou-stained slides provided the best results in terms of yield and fragment length. In LBC, the DNA recovery efficiency of the preserving medium may differ considerably, which should be taken into consideration when introducing LBC. Cancer (Cancer Cytopathol) 2013;121:344-353. © 2013 American Cancer Society. © 2013 American Cancer Society.

Quantification of AAV particle titers by infrared fluorescence scanning of coomassie-stained sodium dodecyl sulfate-polyacrylamide gels.

PubMed

Kohlbrenner, Erik; Henckaerts, Els; Rapti, Kleopatra; Gordon, Ronald E; Linden, R Michael; Hajjar, Roger J; Weber, Thomas

2012-06-01

Adeno-associated virus (AAV)-based vectors have gained increasing attention as gene delivery vehicles in basic and preclinical studies as well as in human gene therapy trials. Especially for the latter two-for both safety and therapeutic efficacy reasons-a detailed characterization of all relevant parameters of the vector preparation is essential. Two important parameters that are routinely used to analyze recombinant AAV vectors are (1) the titer of viral particles containing a (recombinant) viral genome and (2) the purity of the vector preparation, most commonly assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by silver staining. An important, third parameter, the titer of total viral particles, that is, the combined titer of both genome-containing and empty viral capsids, is rarely determined. Here, we describe a simple and inexpensive method that allows the simultaneous assessment of both vector purity and the determination of the total viral particle titer. This method, which was validated by comparison with established methods to determine viral particle titers, is based on the fact that Coomassie Brilliant Blue, when bound to proteins, fluoresces in the infrared spectrum. Viral samples are separated by SDS-PAGE followed by Coomassie Brilliant Blue staining and gel analysis with an infrared laser-scanning device. In combination with a protein standard, our method allows the rapid and accurate determination of viral particle titers simultaneously with the assessment of vector purity.

Profile of Kidney Histopathology in Cases of Burns - Particular Emphasis on Acridine Orange Fluorescence Study and to Explore its Forensic Utility

PubMed Central

Desai, Nandini J.; Gupta, B.D.; Patel, Pratik N.

2016-01-01

Introduction The major cause of death in the burn patients includes multiple organ failure and infection but, sometimes the exact cause of death in many fatally burned patients is difficult to detect. Many times in medico-legal post-mortem examinations in cases of burns, histopathological examination of organs is requested. Aim The aim was to study various histopathological changes in kidneys in the post-mortem cases of burns, by using routine Haematoxylin and Eosin stain (H&E stain), special Periodic and Schiff’s Stain (PAS) stain, to study the role of acridine orange fluorescence study, to explore the forensic utility of this microscopic study and to find out the relationship between duration of survival and histopathological changes observed. Materials and Methods An experimental longitudinal prospective study from October 2010 to September 2012. Total 32 cases of death due to burns were autopsied at mortuary, the Department of Forensic Medicine and Toxicology in our hospital. Bilateral kidneys were removed and preserved in 10% formalin solution. These were forwarded to Department of Pathology for histopathological examination. Routine microscopic examination by H&E stain as well as PAS stain and fluorescence study by acridine orange stain were done in all cases. Results It was observed that in 21 (65.63%) cases gross findings in kidneys were normal, in 06 (18.75%) were grossly pale and in 05 (15.62%) heavy & congested. Sections taken from kidneys and studied by H&E stain showed overlapping histopathological changes in all cases. In 26 (81.25%) cases, changes of Acute Tubular Necrosis (ATN) while in remaining 06 (18.75%), changes of cloudy swelling were observed. The sections stained by acridine orange and observed under fluorescent microscope were lightly positive in 15 (46.88%), brightly positive in 08 (25.00%) whereas, negative in 09 (28.12%). Conclusion Microscopy by various methods helps in getting specific lesions in kidney due to burns. However, it does not add any new tool to resolve any forensic issues of burns. Therefore, microscopy (including florescent), if done would be redundant. PMID:27190809

A comparative study of three cytotoxicity test methods for nanomaterials using sodium lauryl sulfate.

PubMed

Kwon, Jae-Sung; Kim, Kwang-Mahn; Kim, Kyoung-Nam

2014-10-01

The biocompatibility evaluation of nanomaterials is essential for their medical diagnostic and therapeutic usage, where a cytotoxicity test is the simplest form of biocompatibility evaluation. Three methods have been commonly used in previous studies for the cytotoxicity testing of nanomaterials: trypan blue exclusion, colorimetric assay using water soluble tetrazolium (WST), and imaging under a microscope following calcein AM/ethidium homodimer-1 staining. However, there has yet to be a study to compare each method. Therefore, in this study three methods were compared using the standard reference material of sodium lauryl sulfate (SLS). Each method of the cytotoxicity test was carried out using mouse fibroblasts of L-929 exposed to different concentrations of SLS. Compared to the gold standard trypan blue exclusion test, both colorimetric assay using water soluble tetrazolium (WST) and imaging under microscope with calcein AM/ethidium homodimer-1 staining showed results that were not statistically different. Also, each method exhibited various advantages and disadvantages, which included the need of equipment, time taken for the experiment, and provision of additional information such as cell morphology. Therefore, this study concludes that all three methods of cytotoxicity testing may be valid, though careful consideration will be needed when selecting tests with regard to time, finances, and the amount of information required by the researcher(s).

Oxidative Stress Induced Age Dependent Meibomian Gland Dysfunction in Cu, Zn-Superoxide Dismutase-1 (Sod1) Knockout Mice

PubMed Central

Ibrahim, Osama M. A.; Dogru, Murat; Matsumoto, Yukihiro; Igarashi, Ayako; Kojima, Takashi; Wakamatsu, Tais Hitomi; Inaba, Takaaki; Shimizu, Takahiko; Shimazaki, Jun; Tsubota, Kazuo

2014-01-01

Purpose The purpose of our study was to investigate alterations in the meibomian gland (MG) in Cu, Zn-Superoxide Dismutase-1 knockout (Sod1 −/−) mouse. Methods Tear function tests [Break up time (BUT) and cotton thread] and ocular vital staining test were performed on Sod1 −/− male mice (n = 24) aged 10 and 50 weeks, and age and sex matched wild–type (+/+) mice (n = 25). Tear and serum samples were collected at sacrifice for inflammatory cytokine assays. MG specimens underwent Hematoxylin and Eosin staining, Mallory staining for fibrosis, Oil Red O lipid staining, TUNEL staining, immunohistochemistry stainings for 4HNE, 8-OHdG and CD45. Transmission electron microscopic examination (TEM) was also performed. Results Corneal vital staining scores in the Sod1 −/− mice were significantly higher compared with the wild type mice throughout the follow-up. Tear and serum IL-6 and TNF-α levels also showed significant elevations in the 10 to 50 week Sod1 −/− mice. Oil Red O staining showed an accumulation of large lipid droplets in the Sod1 −/− mice at 50 weeks. Immunohistochemistry revealed both increased TUNEL and oxidative stress marker stainings of the MG acinar epithelium in the Sod1 −/− mice compared to the wild type mice. Immunohistochemistry staining for CD45 showed increasing inflammatory cell infiltrates from 10 to 50 weeks in the Sod1 −/− mice compared to the wild type mice. TEM revealed prominent mitochondrial changes in 50 week Sod1 −/− mice. Conclusions Our results suggest that reactive oxygen species might play a vital role in the pathogensis of meibomian gland dysfunction. The Sod1 −/− mouse appears to be a promising model for the study of reactive oxygen species associated MG alterations. PMID:25036096

The evaluation of renal ischaemic damage: the value of CD10 monoclonal antibody staining and of biochemical assessments of tissue viability

PubMed Central

Tagboto, S; Griffiths, A Paul

2007-01-01

Background It is well recognised that there is often a disparity between the structural changes observed in the kidney following renal injury and the function of the organ. For this reason, we carried out studies to explore possible means of studying and quantifying the severity of renal ischaemic damage using a laboratory model. Methods To do this, freshly isolated rabbit kidney tissue was subjected to warm (37°C) or cold (1°C) ischaemia for 20 hours. Following this, the tissue was stained using Haematoxylin and Eosin (H+E), Periodic Schiff reagent (PAS) and the novel monoclonal antibody CD10 stain. Additionally, ischaemic damage to the kidneys was assessed by biochemical tests of tissue viability using formazan-based colorimetry. Results CD 10 antibody intensely stained the brush border of control kidney tissue with mild or no cytoplasmic staining. Cell injury was accompanied by a redistribution of CD10 into the lumen and cell cytoplasm. There was good correlation between a score of histological damage using the CD 10 monoclonal antibody stain and the biochemical assessment of viability. Similarly, a score of histological damage using traditional PAS staining correlated well with that using the CD10 antibody stain. In particular, the biochemical assay and the monoclonal antibody staining techniques were able to demonstrate the efficacy of Soltran (this solution is used cold to preserve freshly isolated human kidneys prior to transplantation) in preserving renal tissue at cold temperatures compared to other randomly selected solutions. Conclusion We conclude that the techniques described using the CD10 monoclonal antibody stain may be helpful in the diagnosis and assessment of ischaemic renal damage. In addition, biochemical tests of viability may have an important role in routine histopathological work by giving additional information about cellular viability which may have implications on the function of the organ. PMID:17531101

A Simplified Experimental Scheme for the Study of Mitosis.

ERIC Educational Resources Information Center

Gill, John

1980-01-01

A procedure is described for providing preparations of dividing cells from root apical meristems, requiring only inexpensive equipment and minimal experimental skill, and using 8-Hydroxyquinoline and Toluidene-blue as a chromosome stain. The method has been sucessfully tested in schools and yields permanent preparations of adequate quality for…

A Computer-Aided Distinction Method of Borderline Grades of Oral Cancer

NASA Astrophysics Data System (ADS)

Sami, Mustafa M.; Saito, Masahisa; Muramatsu, Shogo; Kikuchi, Hisakazu; Saku, Takashi

We have developed a new computer-aided diagnostic system for differentiating oral borderline malignancies in hematoxylin-eosin stained microscopic images. Epithelial dysplasia and carcinoma in-situ (CIS) of oral mucosa are two different borderline grades similar to each other, and it is difficult to distinguish between them. A new image processing and analysis method has been applied to a variety of histopathological features and shows the possibility for differentiating the oral cancer borderline grades automatically. The method is based on comparing the drop-shape similarity level in a particular manually selected pair of neighboring rete ridges. It was found that the considered similarity level in dysplasia was higher than those in epithelial CIS, of which pathological diagnoses were conventionally made by pathologists. The developed image processing method showed a good promise for the computer-aided pathological assessment of oral borderline malignancy differentiation in clinical practice.

Preservation of tomcat (Felis catus) semen in variable temperatures.

PubMed

Siemieniuch, Marta; Dubiel, Andrzej

2007-05-01

The aim of our study was to estimate the viability of cat epididymal sperm in short time storage at +4 degrees C and in long term storage at -196 degrees C and to assess the percentage of live sperm in fresh semen using eosin/nigrosin staining compared to the flow cytometry method. The testes with epididymides were obtained after routine castration procedure. The sperm for further research were collected after flushing the epididymides using extender consist of: Tris 2.4 g, citric acid 1.4 g, glucose 0.8 g, 0.06% (w/v) Na-benzylpenicillin, 0.1% (w/v) streptomycin sulphate and distilled water. Half of each sample was equilibrated with the dilution and loaded in 0.25 ml plastic straws. The straws were placed on a rack in liquid nitrogen vapour at -120 degrees C for 10 min, plunged in liquid nitrogen for 10 min, replaced to marked goblets and loaded into canes for long term storage in liquid nitrogen at -196 degrees C. Sixty percent of motile spermatozoa was accomplished after thawing. However, the percentage of the sperm with intact acrosomes was decreased and the share of cells with midpiece and tail defects was increased. The storage of sperm flushed from epididymides at +4 degrees C for a short time and the usage of sperm during 2-3 days after collection seems to be better than cryopreservation. In our study, normospermia was present in 72.7 +/- 8.8% of fresh semen. The most common defect was the presence of distal droplets, imperfect heads or abnormal acrosomal outline. The motility of fresh sperm flushed from epididymides achieved 77.9 +/- 6.8%. The viability of sperm amounting to 52.5 +/- 13.8% was achieved on third day of conservation in the liquid extender. The percentage of viable sperm in fresh epididymal spermatozoa was 84.9 +/- 7.8%. Compared to these results, the percentage of live cells using SYBR-14/propidium iodide staining was insignificantly lower (82.2 +/- 8%). The live, non-apoptotic cells were 79.0 +/- 7.8%. The share of live, early-apoptotic spermatozoa and late-apoptotic spermatozoa was, respectively, 2 +/- 1.4% and 1.5 +/- 0.9%. The viability of sperm estimated by eosin/nigrosin staining was confirmed by the flow cytometry method. There was no statistical differences between the staining. The usage of apoptosis detection kit revealed, that the percentage of early-apoptotic and late-apoptotic cells was insignificant. (c)2006 Elsevier B.V. All rights reserved.

A novel histological technique for distinguishing between epithelial cells in forensic casework.

PubMed

French, Claire E V; Jensen, Cynthia G; Vintiner, Susan K; Elliot, Douglas A; McGlashan, Susan R

2008-06-10

There are a number of forensic cases in which the identification of the epithelial cell type from which DNA originated would provide important probative evidence. This study aimed to develop a technique using histological staining of fixed cells to distinguish between skin, buccal and vaginal epithelium. First, 11 different stains were screened on formalin-fixed, wax-embedded cells from five women. Samples were analysed qualitatively by examining staining patterns (colour) and morphology (absence or presence of nuclei). Three of the staining methods--Dane's, Csaba's and Ayoub-Shklar--were successful in distinguishing skin epithelial cells from buccal and vaginal. Second, cells were smeared directly onto slides, fixed with one of five fixatives and stained with one of the three stains mentioned above. Methanol fixation, coupled with the Dane's staining method, specific to keratin, was the only technique that distinguished between all three cell types. Skin cells stained magenta, red and orange and lacked nuclei; buccal cells stained predominantly orange-pink with red nuclei; while vaginal cells stained bright orange with orange nuclei and a blue extracellular hue. This staining pattern in vaginal cells was consistent in samples collected from 50 women aged between 18 and 67. Identification of cell type from unlabelled micrographs by 10 trained observers showed a mean success rate of 95%. The results of this study demonstrate that histological staining may provide forensic scientists with a technique for distinguishing between skin, buccal and vaginal epithelial cells and thus would enable more conclusive analyses when investigating sexual assault cases.

How Long Will I Be Blue? Prolonged Skin Staining Following Sentinel Lymph Node Biopsy Using Intradermal Patent Blue Dye

PubMed Central

Gumus, Metehan; Gumus, Hatice; Jones, Sue E; Jones, Peter A; Sever, Ali R; Weeks, Jennifer

2013-01-01

Summary Background Blue dye used for sentinel lymph node biopsy (SLNB) in breast cancer patients may cause prolonged skin discoloration at the site of injection. The aim of this study was to assess the duration of such skin discoloration. Patients and Methods 236 consecutive patients who had undergone breast conserving surgery and SLNB for breast cancer were reviewed prospectively from January 2007 to December 2009. Results Of the 236 patients, 2 had undergone bilateral surgery, and 41 had been examined in consecutive yearly reviews. Blue discoloration remained visible at the injection site after 12, 24, and > 36 months in 36.5, 23.6, and 8.6% of the patients, respectively. Conclusion The use of patent blue for identification of the sentinel lymph node in patients undergoing breast cancer surgery may result in prolonged discoloration of the skin at the injection site. PMID:24415970

Platelet-Rich Fibrin Promotes an Accelerated Healing of Achilles Tendon When Compared to Platelet-Rich Plasma in Rat

PubMed Central

Dietrich, Franciele; L. Duré, Gustavo; P. Klein, Caroline; F. Bampi, Vinícius; V. Padoin, Alexandre; D. Silva, Vinícius; Braga-Silva, Jefferson

2015-01-01

BACKGROUND Autologous platelet concentrate has been used to improve the function and regeneration of injured tissues. Tendinopathies are common in clinical practice, although long-term treatment is required. On the basis of lead time, we compared the effect of using platelet-rich plasma (PRP) and platelet-rich fibrin (PRF) in repairing rat Achilles tendon. METHODS The effectiveness of using PRP and PRF was evaluated after 14 and 28 postoperative days by histological analysis. The quantification of collagen types I and III was performed by Sirius red staining. Qualitatively, the data were verified with hematoxylin-eosin (H&E) staining. RESULTS In Sirius red staining, no significant treatment differences were found between groups. Statistical difference was observed only between PRP (37.2% collagen) and the control group (16.2%) 14 days after treatment. Intra-groups compared twice showed a difference for collagen I (27.8% and 47.7%) and III (66.9% and 46.0%) in the PRF group. The control group showed differences only in collagen I (14.2% and 40.9%) and no other finding was observed in the PRP group. In H&E staining, PRF showed a better cellular organization when compared to the other groups at 28 days. CONCLUSION Our study suggests that PRF promotes accelerated regeneration of the Achilles tendon in rats, offering promising prospects for future clinical use. PMID:26284178

Reticular basement membrane in asthma and COPD: Similar thickness, yet different composition

PubMed Central

Liesker, Jeroen JW; Hacken, Nick H Ten; Zeinstra-Smith, Mieke; Rutgers, Steven R; Postma, Dirkje S; Timens, Wim

2009-01-01

Background Reticular basement membrane (RBM) thickening has been variably associated with asthma and chronic obstructive pulmonary disease (COPD). Even if RBM thickness is similar in both diseases, its composition might still differ. Objective To assess whether RBM thickness and composition differ between asthma and COPD. Methods We investigated 24 allergic asthmatics (forced expiratory volume in one second [FEV1] 92% predicted), and 17 nonallergic COPD patients (FEV1 60% predicted), and for each group a control group of similar age and smoking habits (12 and 10 persons, respectively). Snap-frozen sections of bronchial biopsies were stained with hematoxylin/eosin and for collagen I, III, IV, V, laminin and tenascin. RBM thickening was assessed by digital image analysis. Relative staining intensity of each matrix component was determined. Results Mean (SD) RBM thickness was not significantly different between asthma and COPD 5.5 (1.3) vs 6.0 (1.8) μm, but significantly larger than in their healthy counterparts, ie, 4.7 (0.9) and 4.8 (1.2) μm, respectively. Collagen I and laminin stained significantly stronger in asthma than in COPD. Tenascin stained stronger in asthma than in healthy controls of similar age, and stronger in COPD controls than in asthma controls (p < 0.05). Conclusion RBM thickening occurs both in asthma and COPD. We provide supportive evidence that its composition differs in asthma and COPD. PMID:19436691

Confocal mosaicing microscopy of basal-cell carcinomas ex vivo: progress in digital staining to simulate histology-like appearance

NASA Astrophysics Data System (ADS)

Bini, Jason; Spain, James; Nehal, Kishwer; Hazelwood, Vikki; DiMarzio, Charles; Rajadhyaksha, Milind

2011-03-01

Confocal mosaicing microscopy enables rapid imaging of large areas of fresh tissue, without the processing that is necessary for conventional histology. Using acridine orange (1 milliMolar, 20 seconds) to stain nuclei, basal cell carcinomas were detected in fluorescence confocal mosaics of Mohs surgical excisions with sensitivity of 96.6% and specificity of 89.2%. A possible barrier toward clinical acceptance is that confocal mosaics are based on a single mode of contrast and appear in grayscale, whereas histology is based on two (hematoxylin for nuclei, eosin for cellular cytoplasm and dermis) and appears purple-and-pink. Toward addressing this barrier, we report progress in developing a multispectral analytical model for digital staining: fluorescence confocal mosaics, which show only nuclei, are digitally stained purple and overlaid on reflectance confocal mosaics, which show only cellular cytoplasm and dermis, and digitally stained pink, to mimic the appearance of histology. Comparison of digitally stained confocal mosaics by our Mohs surgeon to the corresponding Mohs histology shows good correlation for normal and tumor detail. Digitally stained confocal mosaicing microscopy may allow direct examination of freshly excised tissue and serve as an adjunct for rapid pathology at-the-bedside.

Noninvasive Intratissue Refractive Index Shaping (IRIS) of the Cornea with Blue Femtosecond Laser Light

PubMed Central

Xu, Lisen; Knox, Wayne H.; DeMagistris, Margaret; Wang, Nadan

2011-01-01

Purpose. To test the feasibility of intratissue refractive index shaping (IRIS) in living corneas by using 400-nm femtosecond (fs) laser pulses (blue-IRIS). To test the hypothesis that the intrinsic two-photon absorption of the cornea allows blue-IRIS to be performed with greater efficacy than when using 800-nm femtosecond laser pulses. Methods. Fresh cat corneas were obtained postmortem and cut into six wedges. Blue laser pulses at 400 nm, with 100-fs pulse duration at 80 MHz were used to micromachine phase gratings into each corneal wedge at scanning speeds from 1 to 15 mm/s. Grating lines were 1 μm wide, 5 μm apart, and 150 μm below the anterior corneal surface. Refractive index (RI) changes in micromachined regions were measured immediately by recording the diffraction efficiency of inscribed gratings. Six hours later, the corneas were processed for histology, and TUNEL staining was performed to assess whether blue-IRIS causes cell death. Results. Scanning at 1 and 2 mm/s caused overt corneal damage in the form of bubbles and burns. At faster scanning speeds (5, 10, and 15 mm/s), phase gratings were created in the corneal stroma, which were shown to be pure RI changes ranging from 0.037 to 0.021 in magnitude. The magnitude of RI change was inversely related to scanning speed. TUNEL staining showed cell death only around bubbles and burns. Conclusions. Blue-IRIS can be performed safely and effectively in living cornea. Compared with near-infrared laser pulses, blue-IRIS enhances both achievable RI change and scanning speed without the need to dope the tissue with two-photon sensitizers, increasing the clinical applicability of this technique. PMID:21931133

Prevalence of sarcocysts in Florida black bears (Ursus americanus floridanus).

PubMed

Cheadle, M A; Cunningham, M W; Greiner, E C

2002-06-01

Sections of muscle from Florida black bears (Ursus americanus floridanus) collected in the state of Florida were observed for the presence of Sarcocystis sp. sarcocysts. Two of 132 (1.5%) black bears had muscle sections containing sarcocysts. One sarcocyst was observed per muscle section. The sarcocysts averaged 181.5 x 99.0 microm by light microscopy of hematoxylin and eosin-stained sections.

Decellularization of Human Nasal Septal Cartilage for the Novel Filler Material of Vocal Fold Augmentation.

PubMed

Kang, Dae-Woon; Shin, Sung-Chan; Jang, Jeon-Yeob; Park, Hee-Young; Lee, Jin-Choon; Wang, Soo-Geun; Lee, Byung-Joo

2017-01-01

The clinical application of allogenic and/or xenogenic cartilage for vocal fold augmentation requires to remove the antigenic cellular component. The objective of this study was to assess the effect of cartilage decellularization and determine the change in immunogenicity after detergent treatment in human nasal septal cartilage flakes made by the freezing and grinding method. Human nasal septal cartilages were obtained from surgical cases. The harvested cartilages were treated by the freezing and grinding technique. The obtained cartilage flakes were treated with 1% Triton X-100 or 2% sodium dodecyl sulfate (SDS) for decellularization of the cartilage flakes. Hematoxylin and eosin stain (H&E stain), surface electric microscopy, immunohistochemical stain for major histocompatibility complex I and II, and ELISA for DNA contents were performed to assess the effect of cartilage decellularization after detergent treatment. A total of 10 nasal septal cartilages were obtained from surgical cases. After detergent treatment, the average size of the cartilage flakes was significantly decreased. With H&E staining, the cell nuclei of decellularized cartilage flakes were not observed. The expression of major histocompatibility complex (MHC)-I and II antigens was not identified in the decellularized cartilage flakes after treatment with detergent. DNA content was removed almost entirely from the decellularized cartilage flakes. Treatment with 2% SDS or 1% Triton X-100 for 1 hour appears to be a promising method for decellularization of human nasal septal cartilage for vocal fold augmentation. Copyright © 2017 The Voice Foundation. Published by Elsevier Inc. All rights reserved.

Fluorescence detection of proteins in sodium dodecyl sulfate-polyacrylamide gels using environmentally benign, nonfixative, saline solution.

PubMed

Steinberg, T H; Lauber, W M; Berggren, K; Kemper, C; Yue, S; Patton, W F

2000-02-01

SYPRO Tangerine stain is an environmentally benign alternative to conventional protein stains that does not require solvents such as methanol or acetic acid for effective protein visualization. Instead, proteins can be stained in a wide range of buffers, including phosphate-buffered saline or simply 150 mM NaCl using an easy, one-step procedure that does not require destaining. Stained proteins can be excited by ultraviolet light of about 300 nm or with visible light of about 490 nm. The fluorescence emission maximum of the dye is approximately 640 nm. Noncovalent binding of SYPRO Tangerine dye is mediated by sodium dodecyl sulfate (SDS) and to a lesser extent by hydrophobic amino acid residues in proteins. This is in stark contrast to acidic silver nitrate staining, which interacts predominantly with lysine residues or Coomassie Blue R, which in turn interacts primarily with arginine and lysine residues. The sensitivity of SYPRO Tangerine stain is similar to that of the SYPRO Red and SYPRO Orange stains - about 4-10 ng per protein band. This detection sensitivity is comparable to colloidal Coomassie blue staining and rapid silver staining procedures. Since proteins stained with SYPRO Tangerine dye are not fixed, they can easily be eluted from gels or utilized in zymographic assays, provided that SDS does not inactivate the protein of interest. This is demonstrated with in-gel detection of rabbit liver esterase activity using alpha-naphthyl acetate and Fast Blue BB dye as well as Escherichia coli beta-glucuronidase activity using ELF-97 beta-D-glucuronide. The dye is also suitable for staining proteins in gels prior to their transfer to membranes by electroblotting. Gentle staining conditions are expected to improve protein recovery after electroelution and to reduce the potential for artifactual protein modifications such as the alkylation of lysine and esterification of glutamate residues, which complicate interpretation of peptide fragment profiles generated by mass spectrometry.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cao Wei, E-mail: cawe-001@163.com; Li Jing, E-mail: lijing02@fmmu.edu.cn; Wu Zhiqun, E-mail: zhiqunwu@fmmu.edu.cn

Purpose. This study evaluates the influence of transcatheter arterial infusion with heated saline on hepatic arterial and portal venous blood flows to tumor and normal hepatic tissues in a rabbit VX2 tumor model. Methods. All animal experiments were approved by the institutional animal care and use committee. Twenty rabbits with VX2 liver tumors were divided into the following two groups: (a) the treated group (n = 10), which received a 60 mL transarterial injection of 60 Degree-Sign C saline via the hepatic artery; (b) the control group (n = 10), which received a 60 mL injection of 37 Degree-Sign Cmore » saline via the hepatic artery. Using ultrasonography, the blood flows in both the portal vein and hepatic artery were measured, and the changes in the hemodynamic indices were recorded before and immediately after the injection. The changes in the tumor and normal liver tissues of the two groups were histopathologically examined by hematoxylin and eosin staining after the injection. Results. After the transcatheter arterial heated infusion, there was a decrease in the hepatic arterial blood flow to the tumor tissue, a significant decrease in the hepatic artery mean velocity (P < 0.05), and a significant increase in the resistance index (P < 0.05). On hematoxylin and eosin staining, there were no obvious signs of tissue destruction in the normal liver tissue or the tumor tissue after heated perfusion, and coagulated blood plasma was observed in the cavities of intratumoral blood vessels in the treated group. Conclusions. The changes in tumor blood flow in the rabbit VX2 tumor model were presumably caused by microthrombi in the tumor vessels, and the portal vein likely mediated the heat loss in normal liver tissue during the transarterial heated infusion.« less

A staining protocol for identifying secondary compounds in Myrtaceae1

PubMed Central

Retamales, Hernan A.; Scharaschkin, Tanya

2014-01-01

• Premise of the study: Here we propose a staining protocol using toluidine blue (TBO) and ruthenium red to reliably identify secondary compounds in the leaves of some species of Myrtaceae. • Methods and Results: Leaves of 10 species representing 10 different genera of Myrtaceae were processed and stained using five different combinations of ruthenium red and TBO. Optimal staining conditions were determined as 1 min of ruthenium red (0.05% aqueous) and 45 s of TBO (0.1% aqueous). Secondary compounds clearly identified under this treatment include mucilage in the mesophyll, polyphenols in the cuticle, lignin in fibers and xylem, tannins and carboxylated polysaccharides in the epidermis, and pectic substances in the primary cell walls. • Conclusions: Potential applications of this protocol include systematic, phytochemical, and ecological investigations in Myrtaceae. It might be applicable to other plant families rich in secondary compounds and could be used as a preliminary screening method for extraction of these elements. PMID:25309840

Computed Tomography-Guided Methylene Blue Labeling Prior to Thoracoscopic Resection of Small Deeply Placed Pulmonary Nodules. Do We Really Need Palpation?

PubMed

Findik, Gokturk; Demiröz, S Mustafa; Apaydın, Selma Mine Kara; Ertürk, Hakan; Biri, Suzan; Incekara, Funda; Aydogdu, Koray; Kaya, Sadi

2017-08-01

Background  Video-assisted thoracic surgery (VATS) is widely used for thoracic surgery operations, and day by day it becomes routine for the excision of undetermined pulmonary nodules. However, it is sometimes hard to reach millimetric nodules through a VATS incision. Therefore, some additional techniques were developed to reach such nodules little in size and which are settled on a challenging localization. In the literature, coils, hook wires, methylene blue, lipidol, and barium staining, and also ultrasound guidance were described for this aim. Herein we discuss our experience with CT-guided methylene blue labeling of small, deeply located pulmonary nodules just before VATS excision. Method  From April 2013 to October 2016, 11 patients with millimetric pulmonary nodules (average 8, 7 mm) were evaluated in our clinic. For all these patients who had strong predisposing factors for malignancy, an 18F-FDG PET-CT scan was also performed. The patients whose nodules were decided to be excised were consulted the radiology clinic. The favorable patients were taken to CT room 2 hours prior to the operation, and CT-guided methylene blue staining were performed under sterile conditions. Results  Mean nodule size of 11 patients was 8.7 mm (6, 2-12). Mean distance from the visceral pleural surface was 12.7 mm (4-29.3). Four of the nodules were located on the left (2 upper lobes, 2 lower lobes), and seven of them were on the right (four lower lobes, two upper lobes, one middle lobe). The maximum standardized uptake values (SUV max) on 18F-FDG PET/CT scan ranged between 0 and 2, 79. Conclusion  CT-guided methylene blue staining of millimetric deeply located pulmonary nodules is a safe and feasible technique that helps surgeon find these undetermined nodules by VATS technique without any need of digital palpation. Georg Thieme Verlag KG Stuttgart · New York.

Methylene Blue: The Long and Winding Road from Stain to Brain: Part 1.

PubMed

Howland, Robert H

2016-09-01

Methylene blue, first discovered and used as a dye in the textile industry, has long been used for biological staining in histology, bacteriology, and hematology. Because of its unique physiochemical properties, it was the first synthetic drug used in medicine, having been used to treat malaria more than one century ago. Methylene blue was also one of the first drugs used for the treatment of patients with psychosis at the end of the 19th century and was the lead drug in the serendipitous development of phenothiazine antipsychotic drugs in the mid-20th century. It was studied in bipolar disorder in the 1980s and has been investigated in neurodegenerative disorders in recent years. The history of methylene blue from its discovery as a dye to its use as a stain and then its therapeutic application in medicine is an example of how a drug's use can evolve over time through careful observation, clinical needs, serendipity, and the integration of concepts from different disciplines. [Journal of Psychosocial Nursing and Mental Health Services, 54(9), 21-24.]. Copyright 2016, SLACK Incorporated.

Ceroid in fish

USGS Publications Warehouse

Wood, E.M.; Yasutake, W.T.

1956-01-01

Since the original description of ceroid in rats, many papers have appeared on the etiology and characteristics of this pigment. It was first seen as a yellow, granular pigment in hematoxylin and eosin sections of the cirrhotic livers of choline deficient rats. The pigment was more fully characterized by Endicott and Lillie, and additional stainging reactions were summarized recently by Lillie. The pigment is sudanophilic in paraffin sections, acid-fast, basophilic, isotropic, iron negative, and highly resistant to solution in water, alcohol, fat solvents, and dilute aqueous acids and alkalis. It is stained by Mallory's hemofuscin stain and Weigert's myelin stain. It reduces osmium tetraoxide and diamine silver carbonate but not ferric ferricyanide. The Gmelin reactions for bile pigments is negative. It has a greenish yellow fluorescence at 3650-3660 Å. It is Schiff positive with or without antecedent diastase digestion after performic or periodic acid oxidation.

Ovarian structure and hormonal status of the UChA and UChB adult rats in response to ethanol.

PubMed

Chuffa, Luiz Gustavo A; Padovani, Carlos R; Martinez, Francisco E

2009-01-20

In females, chronic alcoholism has a current and dangerous incidence to fertility. This work had the goal of elucidating the alterations on the ovary of UChA and UChB adult rats (ethanol 10% (v/v) voluntary drinkers). After the treatment period, 42 female rats divided into three experimental groups (UChA, UChB and Wistar) suffered decapitation and their ovaries were removed and processed to further analysis on light and electron microscopy. The ovary was entirely sliced and stained by hematoxylin-eosin, toluidine blue, periodic acid Schiff (PAS) and Masson's tricromic. Thereby, the enzymatic reaction to acid and alkaline phosphatase, estral cyclicity, reproductive hormonal status and frequency in oestrous-related ovarian structures were assigned. The UChB rats showed an increase in body mass gain index and the ovaries relative weight was significantly lower comparing to the other groups. UCh rats presented the longest estral cycle durations and also persistent oestrous phasis, with uninterrupted cycles. Advanced follicular atresia was common in UCh animals, and degenerating intracellular fragments could be observed through acid phosphatase and electron microscopy techniques. There were some estral cyclicity irregularities caused by chronic ethanol intake in the UCh groups which were consequently reflected as morphologic injury in the ovary structure.

Dye-enhanced multimodal confocal imaging as a novel approach to intraoperative diagnosis of brain tumors.

PubMed

Snuderl, Matija; Wirth, Dennis; Sheth, Sameer A; Bourne, Sarah K; Kwon, Churl-Su; Ancukiewicz, Marek; Curry, William T; Frosch, Matthew P; Yaroslavsky, Anna N

2013-01-01

Intraoperative diagnosis plays an important role in accurate sampling of brain tumors, limiting the number of biopsies required and improving the distinction between brain and tumor. The goal of this study was to evaluate dye-enhanced multimodal confocal imaging for discriminating gliomas from nonglial brain tumors and from normal brain tissue for diagnostic use. We investigated a total of 37 samples including glioma (13), meningioma (7), metastatic tumors (9) and normal brain removed for nontumoral indications (8). Tissue was stained in 0.05 mg/mL aqueous solution of methylene blue (MB) for 2-5 minutes and multimodal confocal images were acquired using a custom-built microscope. After imaging, tissue was formalin fixed and paraffin embedded for standard neuropathologic evaluation. Thirteen pathologists provided diagnoses based on the multimodal confocal images. The investigated tumor types exhibited distinctive and complimentary characteristics in both the reflectance and fluorescence responses. Images showed distinct morphological features similar to standard histology. Pathologists were able to distinguish gliomas from normal brain tissue and nonglial brain tumors, and to render diagnoses from the images in a manner comparable to haematoxylin and eosin (H&E) slides. These results confirm the feasibility of multimodal confocal imaging for intravital intraoperative diagnosis. © 2012 The Authors; Brain Pathology © 2012 International Society of Neuropathology.

An additive manufacturing-based PCL-alginate-chondrocyte bioprinted scaffold for cartilage tissue engineering.

PubMed

Kundu, Joydip; Shim, Jin-Hyung; Jang, Jinah; Kim, Sung-Won; Cho, Dong-Woo

2015-11-01

Regenerative medicine is targeted to improve, restore or replace damaged tissues or organs using a combination of cells, materials and growth factors. Both tissue engineering and developmental biology currently deal with the process of tissue self-assembly and extracellular matrix (ECM) deposition. In this investigation, additive manufacturing (AM) with a multihead deposition system (MHDS) was used to fabricate three-dimensional (3D) cell-printed scaffolds using layer-by-layer (LBL) deposition of polycaprolactone (PCL) and chondrocyte cell-encapsulated alginate hydrogel. Appropriate cell dispensing conditions and optimum alginate concentrations for maintaining cell viability were determined. In vitro cell-based biochemical assays were performed to determine glycosaminoglycans (GAGs), DNA and total collagen contents from different PCL-alginate gel constructs. PCL-alginate gels containing transforming growth factor-β (TGFβ) showed higher ECM formation. The 3D cell-printed scaffolds of PCL-alginate gel were implanted in the dorsal subcutaneous spaces of female nude mice. Histochemical [Alcian blue and haematoxylin and eosin (H&E) staining] and immunohistochemical (type II collagen) analyses of the retrieved implants after 4 weeks revealed enhanced cartilage tissue and type II collagen fibril formation in the PCL-alginate gel (+TGFβ) hybrid scaffold. In conclusion, we present an innovative cell-printed scaffold for cartilage regeneration fabricated by an advanced bioprinting technology. Copyright © 2013 John Wiley & Sons, Ltd.

[Vesico-cutaneous fistula revealing abdominal wall malakoplakia accompanied by Boeck's sarcoidosis].

PubMed

Knausz, József; Lipták, József; Andrásovszky, Zsolt; Baranyay, Ferenc

2010-02-07

Malakoplakia is an acquired granulomatous disorder first described by Michaelis and Gutmann in 1902. The pathogenesis of malakoplakia is hardly known, but it thought to be secondary to an acquired bactericidal defect in macrophages occurring mostly in immunosuppressed patients. 63-year-old female patient had been treated with methylprednisolone for ten years, because of pulmonary sarcoidosis. For six month, recurrent abdominal abscess and vesico-cutaneous fistula developed. Histological examination proved malakoplakia, and Escherichia coli was detected in the abscess cavity. Hematoxyline eosin staining, periodic acid-Schiff, Berlin-blue and Kossa reactions were performed. Microscopically malakoplakia consists of mainly macrophages, known as von Hansemann cells with scattered targetoid intracytoplasmic inclusions known as Michaelis-Gutmann bodies. In our presented case, after urological-surgical intervention and antibiotic therapy, the patient became free from complaints and symptoms. Malakoplakia has been described in numerous anatomic locations, mainly in the urogenital tract. Malakoplakia may be complicated with fistulas in different locations: vesico-coccygeal, rectoprostatic, anorectal fistulas have been were reported in the literature, while 6 cases of malakoplakia with Boeck's sarcoidosis are published. In the presented case sarcoidosis and the 10-year immunosuppressive treatment with methylprednisolone might have been in the background of abdominal wall malakoplakia, complicated by vesico-cutaneous fistula. The patient was successfully treated with surgery and the followed antibiotic therapy.

Differentiation of osteophyte types in osteoarthritis - proposal of a histological classification.

PubMed

Junker, Susann; Krumbholz, Grit; Frommer, Klaus W; Rehart, Stefan; Steinmeyer, Jürgen; Rickert, Markus; Schett, Georg; Müller-Ladner, Ulf; Neumann, Elena

2016-01-01

Osteoarthritis is not only characterized by cartilage degradation but also involves subchondral bone remodeling and osteophyte formation. Osteophytes are fibrocartilage-capped bony outgrowths originating from the periosteum. The pathophysiology of osteophyte formation is not completely understood. Yet, different research approaches are under way. Therefore, a histological osteophyte classification to achieve comparable results in osteophyte research was established for application to basic science research questions. The osteophytes were collected from knee joints of osteoarthritis patients (n=10, 94 osteophytes in total) after joint replacement surgery. Their size and origin in the respective joint were photo-documented. To develop an osteophyte classification, serial tissue sections were evaluated using histological (hematoxylin and eosin, Masson's trichrome, toluidine blue) and immunohistochemical staining (collagen type II). Based on the histological and immunohistochemical evaluation, osteophytes were categorized into four different types depending on the degree of ossification and the percentage of mesenchymal connective tissue. Size and localization of osteophytes were independent from the histological stages. This histological classification system of osteoarthritis osteophytes provides a helpful tool for analyzing and monitoring osteophyte development and for characterizing osteophyte types within a single human joint and may therefore contribute to achieve comparable results when analyzing histological findings in osteophytes. Copyright © 2015 Société française de rhumatologie. Published by Elsevier SAS. All rights reserved.

Evaluation of myeloid cells (tumor-associated tissue eosinophils and mast cells) infiltration in different grades of oral squamous cell carcinoma.

PubMed

Debta, Priyanka; Debta, Fakir Mohan; Chaudhary, Minal; Bussari, Smita

2016-01-01

The multifunctional involvement and infiltration of myeloid cells (tumor-associated tissue eosinophils [TATE] and mast cells) can provide a unique opportunity to define relevant effectors functions that may represent novel, therapeutic options for modulation of tumor onset/growth. Our study aimed to evaluate infiltration of myeloid cells (TATE and Mast cells) infiltration in different grades (WHO grading) of oral squamous cell carcinoma (OSCC). Total 30 cases of OSCC were selected for this study. Hematoxylin and eosin stain and toluidine blue special stain, to evaluate TATE and the mast cells infiltration, were used. Three-year follow-up of OSCC cases was done. Among 30 cases, 63.33% cases of OSCC showed TATE-positive and 36.66% cases showed TATE-negative. Regarding mast cells infiltration, 66.66% OSCC cases showed mast cells positive and 33.33% cases did not show significant mast cells infiltration. We found significant association of TATE and mast cells infiltration in OSCC cases. These myeloid cells infiltration significantly associated with age of patients but did not show any significant association with gender, site, and habit of cases. When we compared these cells infiltration with clinical stages and different histological grades of tumor, we found their infiltration is decreasing, from Stages 1 to Stage 3 of tumor and from well to poorly differentiated carcinoma. We have also found the less infiltration of these myeloid in recurrence cases of OSCC. As the infiltration of TATE and mast cells are correlated, along with evaluation of TATE, we should also evaluate the presence of mast cells infiltration in OSCC. The assessment of myeloid cells could become, in the future, useful for therapeutic approaches in this subset of the patient.

The role of transforming growth factor β1 in fractional laser resurfacing with a carbon dioxide laser.

PubMed

Jiang, Xia; Ge, Hongmei; Zhou, Chuanqing; Chai, Xinyu; Deng, Hui

2014-03-01

The aim of this study was to investigate the role of transforming growth factor β1 in mechanisms of cutaneous remodeling induced by fractional carbon dioxide laser treatment. The dorsal skin of Kunming mice was exposed to a single-pass fractional CO2 laser treatment. Biopsies were taken at 1 h and at 1, 3, 7, 14, 21, 28, and 56 days after treatment. Transforming growth factor (TGF) β1 expression in skin samples was evaluated by ELISA, dermal thickness by hematoxylin-eosin staining, collagen and elastic fibers by Ponceau S and Victoria blue double staining, and types I and III collagens by ELISA. The level of TGF β1 in the laser-treated areas of skin was significantly increased compared with that in the control areas on days 1 (p < 0.05), 3 (p < 0.01), and 7 (p < 0.05) and then decreased by day 14 after treatment, at which time it had returned to the baseline level. Dermal thickness and the amount of type I collagen of the skin of the laser-treated areas had increased significantly (p < 0.05) compared with that in control areas on days 28 and 56. Fibroblast proliferation showed a positive correlation with TGF β1 expression during the early stages (r = 0.789, p < 0.01), and there was a negative correlation between the level of TGF β1 and type I collagen in the late stages, after laser treatment (r = -0.546, p < 0.05). TGF β1 appears to be an important factor in fractional laser resurfacing.

Indian hedgehog signaling promotes chondrocyte differentiation in enchondral ossification in human cervical ossification of the posterior longitudinal ligament.

PubMed

Sugita, Daisuke; Yayama, Takafumi; Uchida, Kenzo; Kokubo, Yasuo; Nakajima, Hideaki; Yamagishi, Atsushi; Takeura, Naoto; Baba, Hisatoshi

2013-10-15

Histological, immunohistochemical, and immunoblot analyses of the expression of Indian hedgehog (Ihh) signaling in human cervical ossification of the posterior longitudinal ligament (OPLL). To examine the hypothesis that Ihh signaling in correlation with Sox9 and parathyroid-related peptide hormone (PTHrP) facilitates chondrocyte differentiation in enchondral ossification process in human cervical OPLL. In enchondral ossification, certain transcriptional factors regulate cell differentiation. OPLL is characterized by overexpression of these factors and disturbance of the normal cell differentiation process. Ihh signaling is essential for enchondral ossification, especially in chondrocyte hypertrophy. Samples of ossified ligaments were harvested from 45 patients who underwent anterior cervical decompressive surgery for symptomatic OPLL, and 6 control samples from patients with cervical spondylotic myelopathy/radiculopathy without OPLL. The harvested sections were stained with hematoxylin-eosin and toluidine blue, examined by transmission electron microscopy, and immunohistochemically stained for Ihh, PTHrP, Sox9, type X, XI collagen, and alkaline phosphatase. Immunoblot analysis was performed in cultured cells derived from the posterior longitudinal ligaments in the vicinity of the ossified plaque and examined for the expression of these factors. The ossification front in OPLL contained chondrocytes at various differentiation stages, including proliferating chondrocytes in fibrocartilaginous area, hypertrophic chondrocytes around the calcification front, and apoptotic chondrocytes near the ossified area. Immunoreactivity for Ihh and Sox9 was evident in proliferating chondrocytes and was strongly positive for PTHrP in hypertrophic chondrocytes. Mesenchymal cells with blood vessel formation were positive for Ihh, PTHrP, and Sox9. Cultured cells from OPLL tissues expressed significantly higher levels of Ihh, PTHrP, and Sox9 than those in non-OPLL cells. Our results indicated that overexpression of Ihh signaling promotes abnormal chondrocyte differentiation in enchondral ossification and enhances bone formation in OPLL.

Effect of low level laser and low intensity pulsed ultrasound therapy on bone remodeling during orthodontic tooth movement in rats.

PubMed

Alazzawi, Mohammed Mahmood Jawad; Husein, Adam; Alam, Mohammad Khursheed; Hassan, Rozita; Shaari, Rumaizi; Azlina, Ahmad; Salzihan, M S

2018-04-16

Quality bone regeneration, which leads to the improvement of bone remodeling, is essential for orthodontic treatment. In order to improve bone regeneration and increase the amount of tooth movement, different techniques have been implemented. The object of this study is to compare the effects of low-level laser therapy (LLLT), low-intensity pulsed ultrasound (LIPUS), and their combination on bone remodeling during orthodontic tooth movement. Eighty (80) male, 6-week-old Sprague Dawley rats were grouped in to four groups, the first group was irradiated with (940 nm) diode laser, second group with LIPUS, and third group with combination of both LLLT and LIPUS. A forth group used was a control group in an incomplete block split-mouth design. The LLLT and LIPUS were used to treat the area around the moving tooth once a day on days 0-7, then the experiment was ended in each experimental endpoint (1, 3, 7, 14, and 21 days). For amount of tooth movement, models were imaged and analyzed. Histological examination was performed after staining with (hematoxylin and eosin) and (alizarin red and Alcian Blue) stain. One step reverse transcription-polymerase chain reaction RT-PCR was also performed to elucidate the gene expression of RANK, RANKL, OPG, and RUNX-2. The amount of tooth movement, the histological bone remodeling, and the RT-PCR were significantly greater in the treatment groups than that in the control group. Among the treatment groups, the combination group was the highest and the LIPUS group was the lowest. These findings suggest that LLLT and LIPUS can enhance the velocity of tooth movement and improve the quality of bone remodeling during orthodontic tooth movement.

Tissue-plastinated vs. celloidin-embedded large serial sections in video, analog and digital photographic on-screen reproduction: a preliminary step to exact virtual 3D modelling, exemplified in the normal midface and cleft-lip and palate.

PubMed

Landes, Constantin A; Weichert, Frank; Geis, Philipp; Wernstedt, Katrin; Wilde, Anja; Fritsch, Helga; Wagner, Mathias

2005-08-01

This study analyses tissue-plastinated vs. celloidin-embedded large serial sections, their inherent artefacts and aptitude with common video, analog or digital photographic on-screen reproduction. Subsequent virtual 3D microanatomical reconstruction will increase our knowledge of normal and pathological microanatomy for cleft-lip-palate (clp) reconstructive surgery. Of 18 fetal (six clp, 12 control) specimens, six randomized specimens (two clp) were BiodurE12-plastinated, sawn, burnished 90 microm thick transversely (five) or frontally (one), stained with azureII/methylene blue, and counterstained with basic-fuchsin (TP-AMF). Twelve remaining specimens (four clp) were celloidin-embedded, microtome-sectioned 75 microm thick transversely (ten) or frontally (two), and stained with haematoxylin-eosin (CE-HE). Computed-planimetry gauged artefacts, structure differentiation was compared with light microscopy on video, analog and digital photography. Total artefact was 0.9% (TP-AMF) and 2.1% (CE-HE); TP-AMF showed higher colour contrast, gamut and luminance, and CE-HE more red contrast, saturation and hue (P < 0.4). All (100%) structures of interest were light microscopically discerned, 83% on video, 76% on analog photography and 98% in digital photography. Computed image analysis assessed the greatest colour contrast, gamut, luminance and saturation on video; the most detailed, colour-balanced and sharpest images were obtained with digital photography (P < 0.02). TP-AMF retained spatial oversight, covered the entire area of interest and should be combined in different specimens with CE-HE which enables more refined muscle fibre reproduction. Digital photography is preferred for on-screen analysis.

T-helper cell type 17/regulatory T-cell immunoregulatory balance in human radicular cysts and periapical granulomas.

PubMed

Marçal, Juliana R B; Samuel, Renata O; Fernandes, Danielle; de Araujo, Marcelo S; Napimoga, Marcelo H; Pereira, Sanivia A L; Clemente-Napimoga, Juliana T; Alves, Polyanna M; Mattar, Rinaldo; Rodrigues, Virmondes; Rodrigues, Denise B R

2010-06-01

Cysts and granulomas are chronic periapical lesions mediated by a set of inflammatory mediators that develop to contain a periapical infection. This study analyzed the nature of the inflammatory infiltrate, presence of mast cells, and in situ expression of cytokines (interleukin [IL]-17 and transforming growth factor [TGF]-beta), chemokines (macrophage inflammatory protein [MIP]-1beta and monocyte chemotactic protein [MCP]-1), and nuclear transcription factor (FoxP3) in human periapical granulomas and cysts compared with a control group. Fifty-five lesions (25 periapical cysts, 25 periapical granulomas, and 5 controls) were analyzed. The type of inflammatory infiltrate was evaluated by hematoxylin-eosin staining, and the presence of mast cells was analyzed by toluidine blue staining. Indirect immunohistochemistry was used to evaluate the expression of cytokines, chemokines, and FoxP3. The inflammatory infiltrate mainly consisted of mononuclear cells. In cysts, mononuclear infiltrates were significantly more frequent than mixed (polymorphonuclear/mononuclear) infiltrates (P = .04). Mixed inflammatory infiltrates were significantly more frequent in patients with sinus tract (P = .0001). The number of mast cells was significantly higher in granulomas than in cystic lesions (P = .02). A significant difference in the expression of IL-17 (P = .001) and TGF-beta (P = .003) was observed between cysts and granulomas and the control group. Significantly higher IL-17 levels were also observed in cases of patients with sinus tract (P = .03). We observed that chronic periapical lesions might experience a reagudization process that is correlated with an increased leukocyte infiltration, with the predominance of neutrophils attracted by a chemokine milieu, as well as the increased presence of IL-17. Copyright 2010 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

Pruritic papular eruption and eosinophilic folliculitis associated with human immunodeficiency virus (HIV) infection: a histopathological and immunohistochemical comparative study.

PubMed

Afonso, João Paulo Junqueira Magalhães; Tomimori, Jane; Michalany, Nilceo Schwery; Nonogaki, Suely; Porro, Adriana Maria

2012-08-01

Among the papular-pruriginous dermatoses related to human immunodeficiency (HIV) infection, two entities remain poorly differentiated leading to confusion in their diagnosis: HIV-related pruritic papular eruption (HIV-PPE or prurigo) and eosinophilic folliculitis (HIV-EF). To establish histopathological and immunohistochemical parameters to differentiate between two conditions associated with HIV infection, the pruritic papular eruption (HIV-PPE) and eosinophilic folliculitis (HIV-EF). Clinically typical HIV-PPE (18 cases) and HIV-EF (10 cases) cases were compared with each other in terms of the following topics: clinical and laboratory features (gender, age, CD4+ cell and eosinophil count), histopathological features (hematoxylin-eosin and toluidine blue staining) and immunohistochemical features (anti-CD1a, anti-CD4, anti-CD7, anti-CD8, anti-CD15, anti-CD20, anti-CD30, anti-CD68/macrophage and anti-S-100 reactions). Among the HIV-EF patients, we found an intense perivascular and diffuse inflammatory infiltration compared with those patients with HIV-PPE. The tissue mast cell count by toluidine staining was higher in the HIV-EF patients, who also presented higher expression levels of CD15 (for eosinophils), CD4 (T helper), and CD7 (pan-T lymphocytes) than the HIV-PPE patients. Only quantitative differences and not qualitative differences were found. These data indicate that HIV-related PPE and EF could possibly be differentiated by histopathological and immunohistochemical findings in addition to clinical characteristics. In fact, these two inflammatory manifestations could be within the spectrum of the same disease because only quantitative, and not qualitative, differences were found. Copyright © 2011 American Academy of Dermatology, Inc. Published by Mosby, Inc. All rights reserved.

Proteasome inhibitor (MG-132) treatment of mdx mice rescues the expression and membrane localization of dystrophin and dystrophin-associated proteins.

PubMed

Bonuccelli, Gloria; Sotgia, Federica; Schubert, William; Park, David S; Frank, Philippe G; Woodman, Scott E; Insabato, Luigi; Cammer, Michael; Minetti, Carlo; Lisanti, Michael P

2003-10-01

Dystrophin, the protein product of the Duchenne muscular dystrophy (DMD) gene, is absent in the skeletal muscle of DMD patients and mdx mice. At the plasma membrane of skeletal muscle fibers, dystrophin associates with a multimeric protein complex, termed the dystrophin-glycoprotein complex (DGC). Protein members of this complex are normally absent or greatly reduced in dystrophin-deficient skeletal muscle fibers, and are thought to undergo degradation through an unknown pathway. As such, we reasoned that inhibition of the proteasomal degradation pathway might rescue the expression and subcellular localization of dystrophin-associated proteins. To test this hypothesis, we treated mdx mice with the well-characterized proteasomal inhibitor MG-132. First, we locally injected MG-132 into the gastrocnemius muscle, and observed the outcome after 24 hours. Next, we performed systemic treatment using an osmotic pump that allowed us to deliver different concentrations of the proteasomal inhibitor, over an 8-day period. By immunofluorescence and Western blot analysis, we show that administration of the proteasomal inhibitor MG-132 effectively rescues the expression levels and plasma membrane localization of dystrophin, beta-dystroglycan, alpha-dystroglycan, and alpha-sarcoglycan in skeletal muscle fibers from mdx mice. Furthermore, we show that systemic treatment with the proteasomal inhibitor 1) reduces muscle membrane damage, as revealed by vital staining (with Evans blue dye) of the diaphragm and gastrocnemius muscle isolated from treated mdx mice, and 2) ameliorates the histopathological signs of muscular dystrophy, as judged by hematoxylin and eosin staining of muscle biopsies taken from treated mdx mice. Thus, the current study opens new and important avenues in our understanding of the pathogenesis of DMD. Most importantly, these new findings may have clinical implications for the pharmacological treatment of patients with DMD.

Comparative study of ex vivo probe-based confocal laser endomicroscopy and light microscopy in lung cancer diagnostics.

PubMed

Sorokina, Anastasia; Danilevskaya, Olesya; Averyanov, Alexander; Zabozlaev, Fedor; Sazonov, Dmitry; Yarmus, Lonny; Lee, Hans J

2014-08-01

Probe-based confocal laser endoscopy (pCLE) allows for real-time non-invasive histological imaging via bronchoscopy. Interpreting CLE images and correlating with traditional histopathology remains challenging. We performed an ex vivo study to evaluate the correlation between light microscopy findings and pCLE imaging of primary lung carcinoma. Post-lobectomy specimens for lung cancer nodules were examined ex vivo by pCLE. The examined areas were marked with brilliant green dye, and the surrounding tissues were stained by methylene blue dye. Lung tissue segments were resected and histopathological specimens were generated with 50-μm thickness from the marked areas and stained with haematoxylin and eosin. Pathologists and pulmonologists reviewed the images for correlating features. Eighteen lobectomy specimens from 18 different patients were collected. Three primary features were observed in all samples using pCLE in the cancer surroundings: alveolar dystelectasis with thickening of alveolar walls, alveolar edema and a large amount of macrophages. The stromal and parenchymal components of the studied subtypes of non-small-cell lung cancer differed from each other. The stromal component for all nine adenocarcinoma specimens had a highly fluorescent field penetrated by dark hollows. All six squamous cell carcinoma specimens had the stromal component appeared as 'biparously' branching, highly fluorescent fibres. No stromal component was observed in any small-cell carcinoma specimen, and at low power field, the cellular component was dominant with an observed light scattering pattern. pCLE can identify lung carcinoma in ex vivo samples. Certain light microscopy features of lung carcinoma can be visualized with pCLE. © 2014 Asian Pacific Society of Respirology.

Characterization of human breast cancer by scanning acoustic microscopy

NASA Astrophysics Data System (ADS)

Chen, Di; Malyarenko, Eugene; Seviaryn, Fedar; Yuan, Ye; Sherman, Mark; Bandyopadhyay, Sudeshna; Gierach, Gretchen; Greenway, Christopher W.; Maeva, Elena; Strumban, Emil; Duric, Neb; Maev, Roman

2013-03-01

Objectives: The purpose of this study was to characterize human breast cancer tissues by the measurement of microacoustic properties. Methods: We investigated eight breast cancer patients using acoustic microscopy. For each patient, seven blocks of tumor tissue were collected from seven different positions around a tumor mass. Frozen sections (10 micrometer, μm) of human breast cancer tissues without staining and fixation were examined in a scanning acoustic microscope with focused transducers at 80 and 200 MHz. Hematoxylin and Eosin (H and E) stained sections from the same frozen breast cancer tissues were imaged by optical microscopy for comparison. Results: The results of acoustic imaging showed that acoustic attenuation and sound speed in cancer cell-rich tissue regions were significantly decreased compared with the surrounding tissue regions, where most components are normal cells/tissues, such as fibroblasts, connective tissue and lymphocytes. Our observation also showed that the ultrasonic properties were influenced by arrangements of cells and tissue patterns. Conclusions: Our data demonstrate that attenuation and sound speed imaging can provide biomechanical information of the tumor and normal tissues. The results also demonstrate the potential of acoustic microscopy as an auxiliary method for operative detection and localization of cancer affected regions.

Quantitative assessment of silver-stained nucleolar organizer region in odontogenic cysts to correlate the growth and malignant potentiality.

PubMed

Biswas, Sailendra Nath; Paul, R R; Ray, Jay Gopal; Majumdar, Sumit; Uppala, Divya

2017-01-01

The most common and important odontogenic cyst involving jaws is the odontogenic keratocyst (OKC) or primordial cyst, the dentigerous cyst and the radicular cyst. These cysts all though do not show similar behavior, they all have the potentiality to recur. Silver nitrate staining of the nucleolar organizer regions (AgNORs) of the benign and malignant lesions is becoming very useful as a diagnostic indicator. Thus, the aim of this study is to assess the diagnostic potential of AgNORs in the cystic epithelium of common odontogenic cysts. Archived specimens of odontogenic cysts were stained with hematoxylin and eosin stain and AgNOR stain. The comparative evaluation of the AgNOR counts was done among the three varieties of odontogenic cysts, i.e., radicular cysts, dentigerous cysts and OKC and were observed that the mean for OKC was significantly higher than that of radicular cyst. Therefore, AgNor could be used as an efficient tool for comparative evaluation of microscopic features such as epithelial thickness, surface keratinization and mural proliferation in dentigerous cyst to that of the AgNOR count.

Soman poisoning increases neural progenitor proliferation and induces long-term glial activation in mouse brain.

PubMed

Collombet, Jean-Marc; Four, Elise; Bernabé, Denis; Masqueliez, Catherine; Burckhart, Marie-France; Baille, Valérie; Baubichon, Dominique; Lallement, Guy

2005-03-30

To date, only short-term glial reaction has been extensively studied following soman or other warfare neurotoxicant poisoning. In a context of cell therapy by neural progenitor engraftment to repair brain damage, the long-term effect of soman on glial reaction and neural progenitor division was analyzed in the present study. The effect of soman poisoning was estimated in mouse brains at various times ranging from 1 to 90 days post-poisoning. Using immunochemistry and dye staining techniques (hemalun-eosin staining), the number of degenerating neurons, the number of dividing neural progenitors, and microglial, astroglial or oligodendroglial cell activation were studied. Soman poisoning led to rapid and massive (post-soman day 1) death of mature neurons as assessed by hemalun-eosin staining. Following this acute poisoning phase, a weak toxicity effect on mature neurons was still observed for a period of 1 month after poisoning. A massive short-termed microgliosis peaked on day 3 post-poisoning. Delayed astrogliosis was observed from 3 to 90 days after soman poisoning, contributing to glial scar formation. On the other hand, oligodendroglial cells or their precursors were practically unaffected by soman poisoning. Interestingly, neural progenitors located in the subgranular zone of the dentate gyrus (SGZ) or in the subventricular zone (SVZ) of the brain survived soman poisoning. Furthermore, soman poisoning significantly increased neural progenitor proliferation in both SGZ and SVZ brain areas on post-soman day 3 or day 8, respectively. This increased proliferation rate was detected up to 1 month after poisoning.

[Local injection of exogenous nerve growth factor improves early bone maturation of implants].

PubMed

Yao, Yang; Du, Yu; Gu, Xia; Guang, Meng-Kai; Huang, Bo; Gong, Ping

2018-04-01

To investigate the effects of nerve growth factor (NGF) in the osteogenic action of implants and the maturation and reconstruction changes in bone tissues in the early stage of osseointegration. The mouse implant model was established by placing titanium in the femoral head of the mouse and locally injecting NGF in the implant zone. On 1, 2 and 4 weeks after operation, stain samples were collected from animals using hematoxylin-eosin (HE) staining and Masson staining. The effect of NGF on the bone maturation was compared at different time points of early stage osseointegration. The results of HE and Masson staining indicated that the local injection of external NGF can up-regulate bone mass, amount of bone trabecula, and bone maturity in the mouse model. The mature bone rate in treatment group of 1 week and 4 weeks after operation were significantly higher than those in the control group (P<0.05). NGF can shorten the period of bone maturation.

A human visual based binarization technique for histological images

NASA Astrophysics Data System (ADS)

Shreyas, Kamath K. M.; Rajendran, Rahul; Panetta, Karen; Agaian, Sos

2017-05-01

In the field of vision-based systems for object detection and classification, thresholding is a key pre-processing step. Thresholding is a well-known technique for image segmentation. Segmentation of medical images, such as Computed Axial Tomography (CAT), Magnetic Resonance Imaging (MRI), X-Ray, Phase Contrast Microscopy, and Histological images, present problems like high variability in terms of the human anatomy and variation in modalities. Recent advances made in computer-aided diagnosis of histological images help facilitate detection and classification of diseases. Since most pathology diagnosis depends on the expertise and ability of the pathologist, there is clearly a need for an automated assessment system. Histological images are stained to a specific color to differentiate each component in the tissue. Segmentation and analysis of such images is problematic, as they present high variability in terms of color and cell clusters. This paper presents an adaptive thresholding technique that aims at segmenting cell structures from Haematoxylin and Eosin stained images. The thresholded result can further be used by pathologists to perform effective diagnosis. The effectiveness of the proposed method is analyzed by visually comparing the results to the state of art thresholding methods such as Otsu, Niblack, Sauvola, Bernsen, and Wolf. Computer simulations demonstrate the efficiency of the proposed method in segmenting critical information.

Molecular Mechanisms and Treatment Strategies for Obesity-Associated Coronary Artery Disease, an Imminent Military Epidemic

DTIC Science & Technology

2008-12-01

statistically significant. T. Chol indicates total cholesterol ; HDL, high - density lipoprotein . B, Hematoxylin and eosin staining of proximal aortas from...low density lipoprotein receptor null Ldlr/ mice transplanted with Stat1/ bone marrow. Conclusions—STAT1 is critical for endoplasmic reticulum...intracellular accumulation of lipoprotein - derived free cholesterol (FC).11 FC enrichment of macro- phages, like many ER stressors, activates the UPR

A melting method for RNA extraction from the mucosal membrane of the mouse middle ear.

PubMed

Seo, Young Joon; Kim, Sung Huhn; Moon, In Seok; Choi, Jae Young

2015-03-01

There is much confusion surrounding the methods of RNA extraction from the middle ear mucosa of mice. In this study, we worked to develop a "melting method," which is faster, purer, and more reliable than other methods in common use. Thirty-two ears were used for this study. Light microscopy with hematoxylin-eosin staining of the bullae, scanning electron microscopy (SEM), spectrophotometer analysis, and reverse transcription polymerase chain reaction were performed before and after melting the half lateral bullae, which were detached from the temporal bone by using a lateral retroauricular approach. Each resected half bulla contained a well distributed mucosal membrane. After a TRIzol melting duration of 10-30 minutes, only mucosal marker (MUC5AC) was expressed without bony marker (total osteocalcin). The same results were determined from SEM. This melting method, compared with stripping and irrigation methods, is effective and offers an easier, more robust approach to extracting RNA from the middle ear mucosal membranes of mice.

A Precious Diagnostic "Pearl": The Necklace Pattern in Germ Cell Tumors of the Testis.

PubMed

Snow, Justin; Mosquera, Juan Miguel; Scognamiglio, Theresa; Robinson, Brian D; Khani, Francesca

2018-04-01

Diffuse embryoma is a rare pattern of nonseminomatous germ cell tumor of the testis originally described in 1983. We report a case with this predominant pattern in an 18-year-old male with a painless palpable testicular mass. Although it is relatively common to see a diffuse embryoma pattern focally in mixed nonseminomatous germ cell tumors of the testis, it is rarely the predominant pattern and can represent a diagnostic pitfall on routine hematoxylin and eosin stain. We emphasize the importance of recognizing the individual components within the diffuse embryoma pattern, review the literature, and briefly discuss the ancillary immunohistochemical stains that may be utilized to help support the diagnosis.

Rebamipide protects against glaucoma eyedrop-induced ocular surface disorders in rabbits

PubMed Central

Kawaguchi, Ichiro; Higashide, Tomomi; Takeji, Yasuhiro; Sakurai, Kazushi; Kawaguchi, Chiaki; Sugiyama, Kazuhisa

2017-01-01

Purpose This study aimed to determine if rebamipide eyedrops can improve ocular surface damage caused by the use of glaucoma eyedrops. Methods Female Kbl:Dutch rabbits were used to evaluate glaucoma eyedrop-induced ocular surface damage; one eye of each rabbit was untreated and the other was administered glaucoma eyedrops for 30 days. To evaluate the effects of rebamipide on ocular surface damage, one eye of each rabbit was administered vehicle-treated glaucoma eyedrops and the other was administered rebamipide-treated glaucoma eyedrops for 30 days. Corneal and conjunctival epithelial damage was evaluated using fluorescein and rose bengal staining, respectively. Conjunctival inflammation was observed by light microscopy with hematoxylin-eosin staining. Dark cells (in which the corneal microvilli were damaged) were analyzed by scanning electron microscopy. Results There were no significant differences in fluorescein staining between the untreated and glaucoma eyedrop-treated groups; however, rose bengal staining and the number of inflammatory cells in the conjunctiva significantly increased after glaucoma eyedrop treatment. There was a four-fold increase in the number of dark cells in the glaucoma eyedrop-treated group compared to untreated. In contrast, in the conjunctiva of the rebamipide-treated glaucoma eyedrop group, rose bengal staining scores, the number of inflammatory cells, and the number of dark cells were decreased compared to the vehicle-treated glaucoma eyedrop group. Conclusions Results from our in vivo rabbit study demonstrated that short-term use of glaucoma eyedrops induces corneal epithelium disorders at the cellular level, but that simultaneous use of rebamipide has the potential to protect and repair the ocular surface. PMID:29049370

Rapid immunohistochemical detection of tumor cells in gastric carcinoma.

PubMed

Mönig, Stefan P; Luebke, Thomas; Soheili, Afsoon; Landsberg, Stephanie; Dienes, H P; Hölscher, Arnulf H; Baldus, Stephan E

2006-11-01

The detection of single tumor cells or tumor cell clusters represents an important issue in intraoperative frozen section analysis. For example, surgical margins may be evaluated in order to minimize the number of additional operations. Furthermore, intraoperative diagnosis of lymph node micrometastasis (LNM) may help to define the area of appropriate lymph node dissection. In addition to haematoxylin and eosin (H&E)-stained sections, immunohistochemical detection of single tumor cells or cell clusters may be helpful in this context. The aim of this study was to evaluate the clinical significance, reliability and sensitivity of intraoperative rapid immunostaining of frozen sections. Therefore, we compared the results of rapid immunohistochemical staining of frozen sections and paraffin sections applying the EnVision and Histofine(R) detection systems. In a prospective immunohistochemical study, paraffin and frozen sections of 20 gastric cancer specimens were analyzed. Paraffin as well as frozen sections were stained immunohistochemically applying the EnVision and Histofine detection systems. As primary antibodies, AE1/AE3 (anti-cytokeratin), EMA (anti-MUC1) and B lymphocyte marker anti-CD20 were applied. The rapid immunostaining procedure was able to be completed within 10-13 min. Rapid immunohistochemical staining of frozen and paraffin sections of the same tumors resulted in comparable immunoreactivity. The rapid EnVision and Histofine procedures allowed immunostaining of frozen sections in less than 13 min. These methods can represent useful additional tools in routine surgical pathology and research, enabling a more accurate frozen section diagnosis compared to staining with H&E alone. Intraoperative rapid immunostaining can be a simple and useful technique to detect LNM.

Role of FNA and Special Stains in Rapid Cytopathological Diagnosis of Pulmonary Nocardiosis.

PubMed

Sood, Ridhi; Tyagi, Ruchita; Selhi, Pavneet Kaur; Kaur, Gursheen; Kaur, Harpreet; Singh, Akashdeep

2018-01-01

Nocardia, a gram-positive aerobic bacillus of the Actinomycetales family, is a significant opportunistic pathogen in immunocompromised individuals. Clinical and radiological features of pulmonary nocardiosis are nonspecific and can be misdiagnosed as tuberculosis, pneumocystis, staphylococcal or fungal infections, or as malignancy. Aspiration cytology with special stains is a quick and effective approach for accurate diagnosis. We present 7 cases of pulmonary nocardiosis, admitted to the pathology department in a tertiary-care hospital in Punjab. Clinical findings, immune status, laboratory tests, chest radiographs, and computed tomography scans were reviewed. Cytologically, special stains like 1% Ziehl-Neelsen (ZN), 20% ZN, periodic acid-Schiff (PAS), Grocott methenamine silver (GMS), and reticulin stains were studied along with May-Grünwald Giemsa, Papanicolaou, and hematoxylin and eosin. All the patients were immunocompromised. The radiological changes were nonspecific. Cytomorphology showed acute and chronic inflammatory infiltrates with necrosis. None of the cases showed well-defined granulomas. GMS, modified 1% ZN and, Gordon and Sweet reticulin stains highlighted the delicate filamentous bacteria in all cases. PAS and 20% ZN stain for tuberculous bacilli were uniformly negative. FNAC can provide a quick and accurate diagnosis of nocardiosis and thereby facilitate timely medical management. © 2018 S. Karger AG, Basel.

[Sclerostin expression in periodontal ligaments during movement of orthodontic teeth in rats].

PubMed

Yiwen, Chen; Shang, Gao; Tongtong, Xu; Jiahui, Zhang; Jincheng, Li; Huiyan, Zhang; Jinjin, Lu; Min, Hu; Zhihui, Liu

2016-06-01

This study aims to observe the expression of Sclerostin during movement of orthodontic teeth and determine the effect of this protein on remodeling of periodontal tissues. Twenty-four Wistar rats were chosen. Orthodontic forces were applied between the bilateral incisor and first molar to achieve mesial movement. Rats in each group were executed at different time points (0, 1, 3, 5, 7, 14 d). Morphology of periodontal tissue was observed by hematoxylin-eosin (HE) staining. The number of osteoclasts were observed by tartrate-resistant acid phosphatase (TRAP) staining. Sclerostin expression were observed by immunohistochemical staining. HE staining revealed that the resorption of alveolar bone intensified with prolonged movement. Results of immunohistochemical and TRAP staining revealed that Sclerostin expression and number of osteoclasts were related to duration of movement of orthodontic tooth. After staining for 5 days, the number of osteoclasts and Sclerostin expression reached their peak and then began to decline. The numbers of osteoclasts and the expression level of Sclerostin were higher at the compressive side than those at the tensive side. Sclerostin affected orthodontic tooth movement by inhibiting the Wnt signaling pathway and by indirectly or directly controlling bone morphogenetic protein.

Assessment of sperm viability, mitochondrial activity, capacitation and acrosome intactness in extended boar semen during long-term storage.

PubMed

Huo, Li-Jun; Ma, Xing-Hong; Yang, Zeng-Ming

2002-10-15

The purpose of this study was to assess sperm quality in extended boar semen during in vitro storage in order to determine which extender should be used and how long boar semen can be stored. Freshly ejaculated boar semen was diluted with equal volumes of Beltsville thaw solution (BTS), Androhep, KIEV or Zorlesco extenders and stored at 17 degrees C for up to 15 days. Sperm quality was evaluated by examining viability using SYBR-14/PI and Hoechst 33258 staining, mitochondrial activity using 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1) staining, acrosome intactness by Coomassie blue staining, and capacitation status by chlortetracycline (CTC) staining. There were over 50% viable spermatozoa in boar semen extended with Zorlesco and Androhep extenders on Day 13 of storage. The percentage of JC-1-stained spermatozoa was 53.8 +/- 2.1% for Zorlesco and 57.7 +/- 1.60% for Androhep extenders on Day 13 of storage. The percentage of acrosome-intact spermatozoa detected by Coomassie blue staining was higher than that in the SYBR-14PI-, Hoechst 33258-, and JC-1-stained samples in our study. The results from SYBR-14/PI, Hoechst 33258, JC-1, and Coomassie blue staining were highly correlated (r > or = 0.9461). There were less than 15% capacitated spermatozoa in the semen extended with BTS, Androhep and Zorlesco extenders during 9 days of storage. However, most viable boar spermatozoa became capacitated by Day 13 of storage. The rank order of four extenders for maintaining sperm viability and mitochondrial activity was as follows: Androhep, Zorlesco, BTS, KIEV.

Quirks of dye nomenclature. 1. Evans blue.

PubMed

Cooksey, C J

2014-02-01

The history, origin, identity, chemistry and use of Evans blue dye are described along with the first application to staining by Herbert McLean Evans in 1914. In the 1930s, the dye was marketed under the name, Evans blue dye, which was profoundly more acceptable than the ponderous chemical name.

Expression of matrix metalloproteinases 9 and 12 in actinic cheilitis

PubMed Central

Poulopoulos, Athanasios K; Andreadis, Dimitrios; Markopoulos, Anastasios K

2013-01-01

AIM: To investigate the role of matrix-degrading metalloproteinases 9, 12 (MMPs), as mediators of functional connective tissue damage in actinic cheilitis. METHODS: Thirty five formalin-fixed, paraffin embedded specimens of actinic cheilitis, and twelve specimens of normal lower lip vermillion, which were obtained by the archives of the Department of Oral Medicine and Maxillofacial Pathology, were examined. From each block, 5 μm thick sections were cut and routinely stained with Hematoxylin and Eosin. Immunohistochemical studies were performed on 4-μm thick sections of formalin-fixed paraffin embedded actinic cheilitis lesions and of normal lower lip vermillion, for MMP-9 and MMP-12 in serial sections of our specimens. Appropriate positive and negative controls were performed to confirm the specificity of the staining reaction. MMP immunohistochemistry was evaluated using a semiquantitative immunoreactive score. RESULTS: Haematoxylin and eosin staining revealed in actinic cheilitis lesions atrophic stratified squamous cell epithelium, or focally and irregularly hyperplastic of variable thickness, in some areas was observed marked keratin production. Varying degrees of epithelial dysplasia were noticed with a wide spectrum of change within the same specimen. Characteristic was the appearance of chronic inflammatory infiltration, and a band of amorphous acellular, basophilic change like solar elastosis (elastin replacement of collagen). In normal lower lip specimens weak and scanty positive expression of MMP-9 and MMP-12 was observed. Anti-MMP-9 antibody showed a weak reaction, in actinic cheilitis lesions, focal in the elastotic material, in chronic inflammatory cells and mostly in macrophages and neutrophils. Strong and in some cases diffused immunohistochemical expression of MMP-12 was detected in actinic cheilitis lesions in the areas of the fragmented, distorted and thickened elastic fibers. MMP-12 was also expressed in chronic inflammatory cells and mostly macrophages. MMP-12 was significantly higher in actinic cheilitis specimens compared with the normal lower lip specimens (P = 0.0029). CONCLUSION: Our results suggest that especially MMP-12 may play an important role in remodeling events occurring in the connective tissue during long-term exposure to sunlight in the actinic cheilitis lesions. PMID:24520545

[Microbiological testing of the artificial gingival margin in dentures].

PubMed

Hermann, Péter; Klein, Ildikó; Barna, Zsuzsanna; Kaán, Miklós; Fejérdy, Pál

2004-06-01

In everyday practice dental laboratories try to reproduce the natural form of sulcus gingivae at the transitional area between artificial teeth and gingiva of removable dentures, even on esthetically less important areas. Aim of these investigations were to examine how artificial recreation of the sulcus gingivae influences plaque retention, and what is the microbiological relevance of these. Investigations were carried out on the vestibular side of removable dentures of 32 randomly selected patients treated at the Department of Prosthodontics at the Faculty of Dentistry, Semmelweis University. Microbiological samples were taken from each patient using the same method. Samples were taken from the left upper first molars' artificial gingival margin using sterile paper points. Paper points were then transported in Eppendorf-tubes, in 2 ml of physiological saline solution, and processed within a two-hour period of time. Series dilutions were made of the sample solutions, then surface-streaked on Subaraud and Gentamycin, blood-agar, eosin-methylene blue and Mitis Salivarius culture enriched with Bacitracin. Subaraud culture was induced under aerob conditions, at room temperature for two days, then the total amount of fungi quantified. After pure-culturing Candida albicans ID-culture was used for identification, and BioMerieux ATB automatic equipment to identify different Candida species. From pure cultures identification was carried out with Gram-staining, Neisser-staining, catalase, oxidase and also with other biochemical reactions. Blood-agar was used to determine total germ count, and normal commensal pharyngeal and oral bacteria. After collecting the microbiological samples, the conventional shape of the dental margin of gingiva was abolished on one side of the dentures and a smooth transition was created between denture teeth and the artificial gingiva in the molar and premolar region. During our investigations only blastomycetes were found. Besides most common Candida albicans, Candida glabrata, tropicallis, spherica and lambica were also identified. Our patients did not miss artificial sulcus gingivae, had no aesthetic complaints about smooth transition between artificial teeth and gingiva. Microbiological investigations of the samples and the comparative analysis showed, that on the smooth transitional areas compared to conventionally shaped sulci significantly less gram-negative, gram-positive bacteria and oral fungi were found, and there was no plaque formation.

Magnetic resonance and photoacoustic imaging of brain tumor mediated by mesenchymal stem cell labeled with multifunctional nanoparticle introduced via carotid artery injection

NASA Astrophysics Data System (ADS)

Qiao, Yang; Gumin, Joy; MacLellan, Christopher J.; Gao, Feng; Bouchard, Richard; Lang, Frederick F.; Stafford, R. Jason; Melancon, Marites P.

2018-04-01

Objective. To evaluate the feasibility of visualizing bone marrow-derived human mesenchymal stem cells (MSCs) labeled with a gold-coated magnetic resonance (MR)-active multifunctional nanoparticle and injected via the carotid artery for assessing the extent of MSC homing in glioma-bearing mice. Materials and methods. Nanoparticles containing superparamagnetic iron oxide coated with gold (SPIO@Au) with a diameter of ˜82 nm and maximum absorbance in the near infrared region were synthesized. Bone marrow-derived MSCs conjugated with green fluorescent protein (GFP) were successfully labeled with SPIO@Au at 4 μg ml-1 and injected via the internal carotid artery in six mice bearing orthotopic U87 tumors. Unlabeled MSCs were used as a control. The ability of SPIO@Au-loaded MSCs to be imaged using MR and photoacoustic (PA) imaging at t = 0 h, 2 h, 24 h, and 72 h was assessed using a 7 T Bruker Biospec experimental MR scanner and a Vevo LAZR PA imaging system with a 5 ns laser as the excitation source. Histological analysis of the brain tissue was performed 72 h after MSC injection using GFP fluorescence, Prussian blue staining, and hematoxylin-and-eosin staining. Results. MSCs labeled with SPIO@Au at 4 μg ml-1 did not exhibit cell death or any adverse effects on differentiation or migration. The PA signal in tumors injected with SPIO@Au-loaded MSCs was clearly more enhanced post-injection, as compared with the tumors injected with unlabeled MSCs at t = 72 h. Using the same mice, T2-weighted MR imaging results taken before injection and at t = 2 h, 24 h, and 72 h were consistent with the PA imaging results, showing significant hypointensity of the tumor in the presence of SPIO@Au-loaded MSCs. Histological analysis also showed co-localization of GFP fluorescence and iron, thereby confirming that SPIO@Au-labeled MSCs continue to carry their nanoparticle payloads even at 72 h after injection. Conclusions. Our results demonstrated the feasibility of tracking carotid artery-injected SPIO@Au-labeled MSCs in vivo via MR and PA imaging.

Methylene blue MMX® tablets for chromoendoscopy. Bioavailability, colon staining and safety in healthy volunteers undergoing a full colonoscopy.

PubMed

Di Stefano, A F D; Radicioni, M M; Vaccani, A; Fransioli, A; Longo, L; Moro, L; Repici, A

2018-06-01

Methylene blue-MMX® tablets are proposed as an aid for detection and visualisation of adenomas and carcinomas in patients undergoing colonoscopy, by improving their detection rate and highlighting the presence of the intestinal dysplastic lesions. Single total doses of 100 and 200 mg were administered to healthy volunteers undergoing a bowel cleansing preparation and a full colonoscopy to investigate the colonic staining. The pharmacokinetics of methylene blue and the safety after exposure to the tablets were also investigated. With 200 mg, the best staining, assessed as the sum of acceptable and good staining, was achieved in the ascending colon and rectosigmoid (75% subjects each), the transverse and the descending colon (approximately 63% each). Absence of staining or overstaining were reported for no colonic region of interest in any subject. Similar results were observed in the 100 mg dose group. Methylene blue blood concentrations reached a peak (C max ) in a median time (T max ) of 12 h with 100 mg and 16 h with 200 mg. AUC 0-t was 10.7 ± 6.7 μg/mLxh after 100 mg and 25.2 ± 7.4 μg/mLxh after 200 mg. Half-life ranged between 9 and 22 h after the lower dose and between 6 and 26 h after the higher dose. The cumulative excretion was about 28% after 100 mg and about 39% after 200 mg. The overall frequency of adverse events was 39%, but only one was related to the product: abnormal transaminases. The most frequent adverse event was a transient polyuria (17%). One serious adverse event (gastrointestinal haemorrhage) led the subject to study discontinuation and hospitalisation and another subject withdrew the study due to one adverse event (haematemesis). Either event was not related to methylene blue. Copyright © 2017. Published by Elsevier Inc.

ERK1/2/COX-2/PGE2 signaling pathway mediates GPR91-dependent VEGF release in streptozotocin-induced diabetes

PubMed Central

Li, Tingting; Hu, Jianyan; Du, Shanshan; Chen, Yongdong; Wang, Shuai

2014-01-01

Purpose Retinal vascular dysfunction caused by vascular endothelial growth factor (VEGF) is the major pathological change that occurs in diabetic retinopathy (DR). It has recently been demonstrated that G protein-coupled receptor 91 (GPR91) plays a major role in both vasculature development and retinal angiogenesis. In this study, we examined the signaling pathways involved in GPR91-dependent VEGF release during the early stages of retinal vascular change in streptozotocin-induced diabetes. Methods Diabetic rats were assigned randomly to receive intravitreal injections of shRNA lentiviral particles targeting GPR91 (LV.shGPR91) or control particles (LV.shScrambled). Accumulation of succinate was assessed by gas chromatography-mass spectrometry (GC-MS). At 14 weeks, the ultrastructure and function of the retinal vessels of diabetic retinas with or without shRNA treatment were assessed using hematoxylin and eosin (HE) staining, transmission electron microscopy (TEM), and Evans blue dye permeability. The expression of GPR91, extracellular signal-regulated kinases 1 and 2 (ERK1/2) and cyclooxygenase-2 (COX-2) were measured using immunofluorescence and western blotting. COX-2 and VEGF mRNA were determined by quantitative RT–PCR. Prostaglandin E2 (PGE2) and VEGF secretion were detected using an enzyme-linked immunosorbent assay. Results Succinate exhibited abundant accumulation in diabetic rat retinas. The retinal telangiectatic vessels, basement membrane thickness, and Evans blue dye permeability were attenuated by treatment with GPR91 shRNA. In diabetic rats, knockdown of GPR91 inhibited the activities of ERK1/2 and COX-2 as well as the expression of PGE2 and VEGF. Meanwhile, COX-2, PGE2, and VEGF expression was inhibited by ERK1/2 inhibitor U0126 and COX-2 inhibitor NS-398. Conclusions Our data suggest that hyperglycemia causes succinate accumulation and GPR91 activity in retinal ganglion cells, which mediate VEGF-induced retinal vascular change via the ERK1/2/COX-2/PGE2 pathway. This study highlights the signaling pathway as a potential target for intervention in DR. PMID:25324681

Real-time monitoring of radiofrequency ablation and postablation assessment: accuracy of contrast-enhanced US in experimental rat liver model.

PubMed

Wu, Hanping; Wilkins, Luke R; Ziats, Nicholas P; Haaga, John R; Exner, Agata A

2014-01-01

To examine the accuracy of the unenhanced zone at contrast material-enhanced ultrasonography (US) in predicting coagulative necrosis during and 21 days after radiofrequency (RF) ablation by using radiologic-pathologic comparison. Animal studies were approved by the Institutional Animal Care and Use Committee. The livers of 28 rats underwent US-guided RF ablation. In four animals, contrast-enhanced US was performed during ablation and 2 hours and 2, 7, 14, and 21 days after ablation. The unenhanced zone area on US images was measured. DiI-labeled microbubbles were administered during ablation at 2, 4, and 6 minutes or at 2 hours and 2, 7, 14, and 21 days after ablation in the remaining 24 animals (n = 3 at each time point). One minute later, the animal was euthanized, and the ablated liver was harvested. Tissue samples were imaged to quantify total fluorescence, and NADH staining was performed on the same slice. Hematoxylin-eosin staining was also performed. The findings on fluorescence images, NADH-stained images, and hematoxylin-eosin-stained images were compared. The areas of DiI bubble-negative zones, NADH-negative zones, and lightly NADH-staining zones were measured. Data were analyzed by using one-way analysis of variance. The area of the unenhanced zone on contrast-enhanced US images increased during RF ablation and reached a maximum within 2 days after ablation. At histopathologic examination, a transition zone manifested adjacent to the coagulation zone until 2 days after ablation. The DiI-bubble negative zone on fluorescence images and the damaged zone (transition zone plus coagulation zone) on NADH-stained images increased rapidly within 2 hours after ablation, then slowly reached the maximum on day 2. The ratios of the mean areas of these two zones at hour 2 to those at day 2 were 94.6% and 95.6%, respectively. High uniformity between the damaged zone on NADH-stained images and the DiI bubble-negative zone on fluorescence images was noted at all time points. The temporary transition zone in NADH staining is partially damaged and should transition to nonviability 2 days after ablation. These results demonstrate that contrast-enhanced US can help delineate the maximum area of cell damage (to within 5% of the maximum) as early as 2 hours after ablation. Contrast-enhanced US may be a simple and accurate tool for monitoring the effects of RF ablation and quantifying the size of thermal damage after treatment. © RSNA, 2013.

Image analysis assisted study of mitotic figures in oral epithelial dysplasia and squamous cell carcinoma using differential stains.

PubMed

Tandon, Ankita; Singh, Narendra Nath; Brave, V R; Sreedhar, Gadiputi

2016-11-01

Mitosis is a process of cell division resulting in two genetically equivalent daughter cells. Excessive proliferation of cells due to mitosis is the hallmark in pre cancer and cancer. This study was conducted to count the number of mitotic figures in normal oral mucosa, oral epithelial dysplasia and squamous cell carcinoma in both Hematoxylin and Eosin (H&E) and Crystal Violet stained sections. Also the overall number of mitotic figures with both stains were compared along with the evaluation of staining efficacy of both the stains. The present study was conducted on 20 specimens each of the three categories. These were further divided into two groups for staining with H&E and with 1% Crystal Violet respectively. Images were captured and analyzed using image analysis software Dewinter Biowizard 4.1. Comparison of mitotic figure count in three categories in sections stained with both stains showed statistically significant difference ( p  < 0.001). The mean number of mitotic figures seen in Crystal Violet reagent were significantly higher as seen in H&E stain ( p  < 0.001). The overall diagnostic efficacy of Crystal Violet was 87.6%. Crystal Violet scored over H&E stain and also helped to better appreciate metaphases in Squamous cell carcinoma and telophases in dysplasia. Number of mitotic figures progressively increase with the advancement of the pathology. Use of 1% Crystal Violet provides better appreciation of mitotic figures and can be employed as a selective stain in routine histopathology.

7 CFR 201.58d - Fungal endophyte test.

Code of Federal Regulations, 2010 CFR

2010-01-01

... test may be used to determine the amount of fungal endophyte (Acremonium spp.) in certain grasses. (a) Method of preparation of aniline blue stain for use in testing grass seed and plant material for the... grass seed: (1) Take a sub-sample of seed (1 gram is sufficient) from the pure seed portion of the kind...

7 CFR 201.58d - Fungal endophyte test.

Code of Federal Regulations, 2011 CFR

2011-01-01

... test may be used to determine the amount of fungal endophyte (Acremonium spp.) in certain grasses. (a) Method of preparation of aniline blue stain for use in testing grass seed and plant material for the... grass seed: (1) Take a sub-sample of seed (1 gram is sufficient) from the pure seed portion of the kind...

Evaluation of Heterogeneous Metabolic Profile in an Orthotopic Human Glioblastoma Xenograft Model Using Compressed Sensing Hyperpolarized 3D 13C Magnetic Resonance Spectroscopic Imaging

PubMed Central

Park, Ilwoo; Hu, Simon; Bok, Robert; Ozawa, Tomoko; Ito, Motokazu; Mukherjee, Joydeep; Phillips, Joanna J.; James, C. David; Pieper, Russell O.; Ronen, Sabrina M.; Vigneron, Daniel B.; Nelson, Sarah J.

2013-01-01

High resolution compressed sensing hyperpolarized 13C magnetic resonance spectroscopic imaging was applied in orthotopic human glioblastoma xenografts for quantitative assessment of spatial variations in 13C metabolic profiles and comparison with histopathology. A new compressed sensing sampling design with a factor of 3.72 acceleration was implemented to enable a factor of 4 increase in spatial resolution. Compressed sensing 3D 13C magnetic resonance spectroscopic imaging data were acquired from a phantom and 10 tumor-bearing rats following injection of hyperpolarized [1-13C]-pyruvate using a 3T scanner. The 13C metabolic profiles were compared with hematoxylin and eosin staining and carbonic anhydrase 9 staining. The high-resolution compressed sensing 13C magnetic resonance spectroscopic imaging data enabled the differentiation of distinct 13C metabolite patterns within abnormal tissues with high specificity in similar scan times compared to the fully sampled method. The results from pathology confirmed the different characteristics of 13C metabolic profiles between viable, non-necrotic, nonhypoxic tumor, and necrotic, hypoxic tissue. PMID:22851374

Evaluation of heterogeneous metabolic profile in an orthotopic human glioblastoma xenograft model using compressed sensing hyperpolarized 3D 13C magnetic resonance spectroscopic imaging.

PubMed

Park, Ilwoo; Hu, Simon; Bok, Robert; Ozawa, Tomoko; Ito, Motokazu; Mukherjee, Joydeep; Phillips, Joanna J; James, C David; Pieper, Russell O; Ronen, Sabrina M; Vigneron, Daniel B; Nelson, Sarah J

2013-07-01

High resolution compressed sensing hyperpolarized (13)C magnetic resonance spectroscopic imaging was applied in orthotopic human glioblastoma xenografts for quantitative assessment of spatial variations in (13)C metabolic profiles and comparison with histopathology. A new compressed sensing sampling design with a factor of 3.72 acceleration was implemented to enable a factor of 4 increase in spatial resolution. Compressed sensing 3D (13)C magnetic resonance spectroscopic imaging data were acquired from a phantom and 10 tumor-bearing rats following injection of hyperpolarized [1-(13)C]-pyruvate using a 3T scanner. The (13)C metabolic profiles were compared with hematoxylin and eosin staining and carbonic anhydrase 9 staining. The high-resolution compressed sensing (13)C magnetic resonance spectroscopic imaging data enabled the differentiation of distinct (13)C metabolite patterns within abnormal tissues with high specificity in similar scan times compared to the fully sampled method. The results from pathology confirmed the different characteristics of (13)C metabolic profiles between viable, non-necrotic, nonhypoxic tumor, and necrotic, hypoxic tissue. Copyright © 2012 Wiley Periodicals, Inc.

Transparent, biocompatible nanostructured surfaces for cancer cell capture and culture

PubMed Central

Cheng, Boran; He, Zhaobo; Zhao, Libo; Fang, Yuan; Chen, Yuanyuan; He, Rongxiang; Chen, Fangfang; Song, Haibin; Deng, Yuliang; Zhao, Xingzhong; Xiong, Bin

2014-01-01

Circulating tumor cells (CTCs) in the blood which have detached from both the primary tumor and any metastases may be considered as a “liquid biopsy” and are expected to replace tumor biopsies in the monitoring of treatment response and determining patient prognosis. Here, we introduce a facile and efficient CTC detection material made of hydroxyapatite/chitosan (HA/CTS), which is beneficial because of its transparency and excellent biological compatibility. Atomic force microscopy images show that the roughness of the HA/CTS nanofilm (HA/CTSNF) substrates can be controlled by changing the HA:CTS ratio. Enhanced local topographic interactions between nano-components on cancer cell membranes, and the antibody coated nanostructured substrate lead to improved CTC capture and separation. This remarkable nanostructured substrate has the potential for CTC culture in situ and merits further analysis. CTCs captured from artificial blood samples were observed in culture on HA/CTSNF substrates over a period of 14 days by using conventional staining methods (hematoxylin eosin and Wright’s stain). We conclude that these substrates are multifunctional materials capable of isolating and culturing CTCs for subsequent studies. PMID:24904216

Liquid nitrogen vapor is comparable to liquid nitrogen for storage of cryopreserved human sperm: evidence from the characteristics of post-thaw human sperm.

PubMed

Hu, Jingmei; Zhao, Shidou; Xu, Chengyan; Zhang, Lin; Lu, Shaoming; Cui, Linlin; Ma, Jinlong; Chen, Zi-Jiang

2015-11-01

To compare the differences in the characteristics of post-thaw human sperm after storage in either liquid nitrogen (LN2; -196 °C) or LN2 vapor (-167 °C). Experimental study. University hospital. Thirty healthy volunteers who agreed to donate their normal semen samples for infertility or research were included in the study. Semen samples (n = 30) were divided into eight aliquots and frozen. Four aliquots of each human semen sample were stored in LN2 (-196 °C), and the other four aliquots were stored in LN2 vapor (-167 °C). After 1, 3, 6, or 12 months, samples were thawed and analyzed. The motility was evaluated by the manual counting method. The viability was estimated by eosin staining. The morphology was analyzed by Diff-Quik staining. The sperm DNA integrity was determined with acridine orange fluorescent staining, and acrosin activity was assayed by the modified Kennedy method. The characteristics of post-thaw human sperm, including motility, viability, morphology, DNA integrity, and acrosin activity, showed no significant difference between LN2 and LN2 vapor storage for the different time periods. LN2 vapor was comparable to LN2 in post-thaw sperm characteristics, suggesting that LN2 vapor may be substituted for LN2 for the long-term storage of human sperm. Copyright © 2015 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Effects of curcumin plus Soy oligosaccharides on intestinal flora of rats with ulcerative colitis.

PubMed

Huang, G; Ye, L; Du, G; Huang, Y; Wu, Y; Ge, S; Yang, Z; Zhu, G

2017-08-15

To explore the therapeutic effect of curcumin (Cur) and soybean oligosaccharides (SBOS) on ulcerative colitis (UC) through testing the intestinal flora and ulcerative colitis (UC). 80 male SD rats were selected divided into four groups with 20 rats in each group: normal group, sulfasalazine (SASP) group, model group and group of curcumin plus soy oligosaccharide. All animals were treated for 4 weeks. In the fifth week rats were decapitated. Macroscopic damage scores of colonic mucosa were calculated. A 4mL blood sample was taken to detect the contents of serum tumor necrosis factor -α (TNF-α) and interleukin 8 (IL-8) by the double antibody sandwich ABC-ELISA method (enzyme-linked immunosorbent assay). Colonic tissues with the most obvious lesions were obtained using a surgical scissor. A routine hematoxylin-eosin (HE) staining method was used to stain pathological specimens and images of staining results were obtained. Histological injury scores of colonic mucosa were calculated. Ulcerative colitis model rats had the highest macroscopic damage scores and histological injury scores of colonic mucosa. After treatment the contents of TNF-α and IL-8 decreased significantly in the group of curcumin plus soy oligosaccharide compared with the model group with statistical significance (P <0.01) while the contents were close to those in the SASP group. There was no statistical significance (P> 0.05). The treatment could decrease TNF-α and IL- 8 expression and reduce colonic mucosa inflammation and tissue damage.

Histochemical analysis of glycoconjugates in the skin of a catfish (arius tenuispinis, day).

PubMed

Al-Banaw, A; Kenngott, R; Al-Hassan, J M; Mehana, N; Sinowatz, F

2010-02-01

A histochemical study using conventional carbohydrate histochemistry (periodic-acid staining including diastase controls, alcian blue staining at pH 1 and 2.5) as well as using a battery of 14 fluorescein isothiocyanate (FITC)-labelled lectins to identify glycoconjugates present in 10 different areas of the skin of a catfish (Arius tenuispinis) was carried out. The lectins used were: mannose-binding lectins (Con A, LCA and PSA), galactose-binding lectins (PNA, RCA), N-acetylgalactosamine-binding lectins (DBA, SBA, SJA and GSL I), N-acetylglucosamine-binding lectins (WGA and WGAs), fucose-binding lectins (UEA) and lectins which bind to complex carbohydrate configurations (PHA E, PHA L). Conventional glycoconjugate staining (PAS staining, alcian blue at pH 1 and 2.5) showed that the mucous goblet cells contain a considerable amount of glycoconjugates in all locations of the skin, whereas the other unicellular gland type, the club cells, lacked these glycoconjugates. The glycoproteins found in goblet cells are neutral and therefore stain magenta when subjected to PAS staining. Alcian blue staining indicating acid glycoproteins was distinctly positive at pH 1, but gave only a comparable staining at pH 2.5. The mucus of the goblet cells therefore also contains acid glycoproteins rich in sulphate groups. Using FITC-labelled lectins, the carbohydrate composition of the glycoproteins of goblet cells could be more fully characterized. A distinct staining of the mucus of goblet cells was found with the mannose-binding lectins LCA and PSA; the galactosamine-binding lectins DBA, SBA and GLS I; the glucosamine-binding lectin WGA; and PHA E which stains glycoproteins with complex carbohydrate configurations. No reaction occurred with the fucose-binding lectin UEA and the sialic acid-specific lectin SNA. In addition, the galactose-binding lectins PNA and RCA showed only a weak or completely negative staining of the mucus in the goblet cells. The specificity of the lectin staining could be proved by inhibiting binding of the lectins by competitive inhibition with the corresponding sugars. From these data, we can conclude that the mucus produced by the epidermal goblet cells of A. tenuispinis is rich in mannose, N-acetylgalactosamine and N-acetylglucosamine residues.

Immunohistochemical myofiber typing and high-resolution myofibrillar lesion detection in LR white embedded muscle

NASA Technical Reports Server (NTRS)

Thompson, J. L.; Vijayan, K.; Riley, D. A.

2000-01-01

We have developed a method of fixing, embedding, sectioning, and staining that allows high-resolution detection of myofibrillar structure and myosin immunocytochemical muscle fiber typing in serial semithin sections of LR White plastic embedded muscle at the light microscopic level. Traditional approaches, such as cryostat sections, permit fiber typing, but small myofibrillar lesions (1-3 sarcomeres) are difficult to detect because of section thickness. Semithin sections of hydrophobic resins do not stain well either histochemically or immunocytochemically. Electron microscopy can resolve lesions and discriminate fiber types based on morphology, but the sampling area is small. Our goal was to develop a rapid method for defining both fiber type and high-resolution primary myofibrillar lesion damage. Mild fixation (1-4% paraformaldehyde, 0. 05-0.1% glutaraldehyde) and embedment in a hydrophilic resin (LR White) were used. Myofibrillar structure was extremely well preserved at the light microscopic (LM) level, and lesions could be readily resolved in Toluidine blue stained 500-nm sections. Fiber type was defined by LM immunomyosin staining of serial plastic semithin sections, which demonstrated reciprocal staining patterns for "fast (Sigma M4276) and "total" (skeletal muscle) myosins (Sigma M7523). Copyright 2000 Wiley-Liss, Inc.

Induction of Contraception by Intraepididymal Sclerotherapy

PubMed Central

Park, Hyoung Keun; Paick, Sung Hyun; Kim, Hyeong Gon; Lho, Yong Soo

2014-01-01

Purpose The objective of the present study was to evaluate the efficacy of a sclerosing solution for inducing epididymal occlusion in male rats. Materials and Methods Male Sprague-Dawley rats were divided into two groups: an injection group (n=20) and control group (n=20). Before injecting the sclerosing agent, seminal vesiculectomy and sperm identification using electrostimulation were performed in all of the rats. In the injection group, 0.2 mL of 0.1% sodium tetradecyl sulfate solution was injected into the epididymis. In the sham group, only the identification of the epididymis was performed. At 4 and 12 weeks after the injection, semen was collected by electrostimulation and evaluated to assess the contraceptive effect. Epididymis was evaluated by hematoxylin and eosin (H&E) staining. Results After 4 and 12 weeks, semen collection was performed in the two groups. Sperms were not observed in the injection group, while there was no change in the sperms in the sham group. H&E staining showed the obstruction of epididymal tubules and an accumulation of inflammatory cells in the injection group. Conclusions This study showed that the sclerosing agent induced sterilization in male rats. This result suggests that the injection method can replace vasectomy as a contraceptive method. However, a further study of large animals and a clinical study are needed. Further, the long-term effectiveness of this method needs to be studied. PMID:25237657

Parasite detection in patients with post kala-azar dermal leishmaniasis in India: a comparison between molecular and immunological methods

PubMed Central

Salotra, P; Sreenivas, G; Beena, K R; Mukherjee, A; Ramesh, V

2003-01-01

Aims: To evaluate the sensitivity and specificity of serological, immunohistochemical, and molecular methods in the diagnosis of post kala-azar dermal leishmaniasis (PKDL). Methods: Twenty five patients with confirmed PKDL and 25 controls were included in the study. G2D10, a monoclonal antibody against Leishmania, was used for the immunohistochemical (IHC) staining of lesion sections to visualise anti-Leishmania donovani antibodies. The diagnostic usefulness of IHC was compared with enzyme linked immunosorbent assay (ELISA) with a recombinant (rk39) antigen, and a species specific polymerase chain reaction (PCR) assay, amplifying a kinetoplast minicircle DNA sequence. Results: IHC detected 22 of 25 PKDL cases, giving a sensitivity of 88%. The diagnostic sensitivity of both the ELISA and PCR tests was higher (96%). All of the 25 controls examined were negative in PCR, indicating 100% specificity of the test, whereas ELISA showed 96% specificity. Conclusions: IHC with G2D10 significantly enhances the sensitivity of detection of PKDL over routine haematoxylin and eosin staining. ELISA with a recombinant antigen is an economical and practical assay. PCR is the most sensitive and specific diagnostic method for PKDL. The tests described would facilitate the recognition of patients with PKDL, enabling timely treatment, which would contribute greatly to the control of kala-azar. PMID:14600129

Evaluation of Staining-Dependent Colour Changes in Resin Composites Using Principal Component Analysis

PubMed Central

Manojlovic, D.; Lenhardt, L.; Milićević, B.; Antonov, M.; Miletic, V.; Dramićanin, M. D.

2015-01-01

Colour changes in Gradia Direct™ composite after immersion in tea, coffee, red wine, Coca-Cola, Colgate mouthwash, and distilled water were evaluated using principal component analysis (PCA) and the CIELAB colour coordinates. The reflection spectra of the composites were used as input data for the PCA. The output data (scores and loadings) provided information about the magnitude and origin of the surface reflection changes after exposure to the staining solutions. The reflection spectra of the stained samples generally exhibited lower reflection in the blue spectral range, which was manifested in the lower content of the blue shade for the samples. Both analyses demonstrated the high staining abilities of tea, coffee, and red wine, which produced total colour changes of 4.31, 6.61, and 6.22, respectively, according to the CIELAB analysis. PCA revealed subtle changes in the reflection spectra of composites immersed in Coca-Cola, demonstrating Coca-Cola’s ability to stain the composite to a small degree. PMID:26450008

Evaluation of Staining-Dependent Colour Changes in Resin Composites Using Principal Component Analysis.

PubMed

Manojlovic, D; Lenhardt, L; Milićević, B; Antonov, M; Miletic, V; Dramićanin, M D

2015-10-09

Colour changes in Gradia Direct™ composite after immersion in tea, coffee, red wine, Coca-Cola, Colgate mouthwash, and distilled water were evaluated using principal component analysis (PCA) and the CIELAB colour coordinates. The reflection spectra of the composites were used as input data for the PCA. The output data (scores and loadings) provided information about the magnitude and origin of the surface reflection changes after exposure to the staining solutions. The reflection spectra of the stained samples generally exhibited lower reflection in the blue spectral range, which was manifested in the lower content of the blue shade for the samples. Both analyses demonstrated the high staining abilities of tea, coffee, and red wine, which produced total colour changes of 4.31, 6.61, and 6.22, respectively, according to the CIELAB analysis. PCA revealed subtle changes in the reflection spectra of composites immersed in Coca-Cola, demonstrating Coca-Cola's ability to stain the composite to a small degree.

CCD Camera Detection of HIV Infection.

PubMed

Day, John R

2017-01-01

Rapid and precise quantification of the infectivity of HIV is important for molecular virologic studies, as well as for measuring the activities of antiviral drugs and neutralizing antibodies. An indicator cell line, a CCD camera, and image-analysis software are used to quantify HIV infectivity. The cells of the P4R5 line, which express the receptors for HIV infection as well as β-galactosidase under the control of the HIV-1 long terminal repeat, are infected with HIV and then incubated 2 days later with X-gal to stain the infected cells blue. Digital images of monolayers of the infected cells are captured using a high resolution CCD video camera and a macro video zoom lens. A software program is developed to process the images and to count the blue-stained foci of infection. The described method allows for the rapid quantification of the infected cells over a wide range of viral inocula with reproducibility, accuracy and at relatively low cost.

Radiation Resistance of Asporogenous Bacteria in Frozen Beef

DTIC Science & Technology

1976-03-01

Salmonella enteritidis , and Escherichia coli were used. Cultures were grown to the maximum stationary phase for use as an inoculum. Ground beef containing...eosin methylene blue agar, Shigella- Salmonella agar, and growth on plate count agar with 2.5% and 6.5% NaCl was observed. Penicillin susceptibility was...selective media as follows: Staphylococcus Medium No. 110 for S. aureus; Violet Red Bile Agar for E. coli; and Bismuth Sulfite Agar for S. enteritidis

Double immunohistochemical staining with MUC4/p53 is useful in the distinction of pancreatic adenocarcinoma from chronic pancreatitis: a tissue microarray-based study.

PubMed

Bhardwaj, Atul; Marsh, William L; Nash, Jason W; Barbacioru, Catalin C; Jones, Susie; Frankel, Wendy L

2007-04-01

Immunohistochemical stains have been used for the distinction of pancreatic adenocarcinoma from chronic pancreatitis. To determine if a double stain for MUC/p53 improved specificity and sensitivity for distinction of pancreatic adenocarcinoma from chronic pancreatitis by comparing maspin, mucin 4 (MUC4), p53, Smad4, and the double stain MUC4/p53. Seventy-four pancreatic adenocarcinomas and 19 chronic pancreatitis cases were retrieved from archival files. Tissue cores were arrayed to create a tissue microarray of 2-mm cores. Sections were stained with antibodies against maspin, MUC4, p53, and Smad4. Additionally, a 2-color, double stain for MUC4 and p53 was developed and evaluated. Five percent or greater staining in either of the cores was considered positive. Intensity (0, 1, 2) and extent (%) of tumor cells staining was also determined. The sensitivity for distinction of pancreatic adenocarcinoma from chronic pancreatitis with maspin, MUC4, p53, and Smad4 was 90%, 77%, 60%, and 63%, respectively; the specificity was 67%, 78%, 88%, and 88%, respectively. When MUC4 and p53 were combined in a double stain, and positive staining for either considered a positive result, the sensitivity increased to 96% but specificity was 73%. When immunoreactivity for both antibodies was necessary for a positive result, sensitivity fell to 39% but specificity was 100%. No correlation was found between intensity or extent of staining with any of the individual stains and tumor differentiation. The double immunohistochemical stain for MUC4/p53 can be a useful diagnostic tool in conjunction with the hematoxylin-eosin-stained section for pancreatic adenocarcinoma, particularly when limited tumor is available for multiple stains.

A comparative study for selectivity of micronuclei in oral exfoliated epithelial cells.

PubMed

Grover, S; Mujib, Abr; Jahagirdar, A; Telagi, N; Kulkarni, Pg

2012-10-01

Micronucleus (MN) represents small, additional nuclei formed by the exclusion of chromosome fragments or whole chromosomes lagging at mitosis. MN rates, therefore, indirectly reflect chromosome breakage or impairment of the mitotic apparatus. During the last few decades, micronuclei ("MNi") in oral exfoliated epithelial cells are widely used as biomarkers of chromosomal damage, genome instability and cancer risk in humans. However, until now only little attention has been given to the effect of different staining procedures on the results of these MN assays. To compare the MNi frequencies in oral exfoliated epithelial cells using three different stains, i.e.,Feulgen stain, Papanicolaou stain (Pap) and hemotoxylin and eosin stain (H and E). Oral exfoliated cells from 45 cases of potentially malignant disorders (15 oral submucous fibrosis, 15 lichen planus and 15 leukoplakia) and 15 controls with healthy mucosa, were taken and MNi frequencies (No. of MNi/1000 cells) were compared using three different stains. Mean MNi frequency in cases was found to be 3.8 with Feulgen stain, 16.8 with PAP and 25.9 with H and E. In controls, mean MNi frequency was 1.6 with Feulgen stain, 7.7 with PAP and 9.6 with H and E stain. Statistically significant value (P < 0.01) were observed when the three stains were compared together using Kruskal Walli's ANOVA test. Feulgen being a DNA-specific stain gave the least counts, although statistically significant results from the comparison of MNi frequency between cases and controls were obtained with all the three stains.

The extent of intrauterine growth restriction determines the severity of cerebral injury and neurobehavioural deficits in rodents.

PubMed

Ruff, Crystal A; Faulkner, Stuart D; Rumajogee, Prakasham; Beldick, Stephanie; Foltz, Warren; Corrigan, Jennifer; Basilious, Alfred; Jiang, Shangjun; Thiyagalingam, Shanojan; Yager, Jerome Y; Fehlings, Michael G

2017-01-01

Cerebral Palsy (CP) is the most common physical pediatric neurodevelopmental disorder and spastic diplegic injury is its most frequent subtype. CP results in substantial neuromotor and cognitive impairments that have significant socioeconomic impact. Despite this, its underlying pathophysiological mechanisms and etiology remain incompletely understood. Furthermore, there is a need for clinically relevant injury models, which a) reflect the heterogeneity of the condition and b) can be used to evaluate new translational therapies. To address these key knowledge gaps, we characterized a chronic placental insufficiency (PI) model, using bilateral uterine artery ligation (BUAL) of dams. This injury model results in intrauterine growth restriction (IUGR) in pups, and animals recapitulate the human phenotype both in terms of neurobehavioural and anatomical deficits. Effects of BUAL were studied using luxol fast blue (LFB)/hematoxylin & eosin (H&E) staining, immunohistochemistry, quantitative Magnetic Resonance Imaging (MRI), and Catwalk neurobehavioural tests. Neuroanatomical analysis revealed regional ventricular enlargement and corpus callosum thinning in IUGR animals, which was correlated with the extent of growth restriction. Olig2 staining revealed reductions in oligodendrocyte density in white and grey matter structures, including the corpus callosum, optic chiasm, and nucleus accumbens. The caudate nucleus, along with other brain structures such as the optic chiasm, internal capsule, septofimbrial and lateral septal nuclei, exhibited reduced size in animals with IUGR. The size of the pretectal nucleus was reduced only in moderately injured animals. MAG/NF200 staining demonstrated reduced myelination and axonal counts in the corpus callosum of IUGR animals. NeuN staining revealed changes in neuronal density in the hippocampus and in the thickness of hippocampal CA2 and CA3 regions. Diffusion weighted imaging (DWI) revealed regional white and grey matter changes at 3 weeks of age. Furthermore, neurobehavioural testing demonstrated neuromotor impairments in animals with IUGR in paw intensities, swing speed, relative print positions, and phase dispersions. We have characterized a rodent model of IUGR and have demonstrated that the neuroanatomical and neurobehavioural deficits mirror the severity of the IUGR injury. This model has the potential to be applied to examine the pathobiology of and potential therapeutic strategies for IUGR-related brain injury. Thus, this work has potential translational relevance for the study of CP.

Use of calcofluor-blue brightener for the diagnosis of Pneumocystis jirovecii pneumonia in bronchial-alveolar lavage fluids: A single-center prospective study.

PubMed

Desoubeaux, Guillaume; Franck-Martel, Claire; Caille, Agnès; Drillaud, Nicolas; Lestrade Carluer de Kyvon, Marie-Alix; Bailly, Éric; Chandenier, Jacques

2017-04-01

The biological diagnosis of Pneumocystis jirovecii pneumonia (PjP) is based on the investigation of respiratory fluids by conventional staining methods and/or molecular biology. Diagnostic performance of an in-house technique based on calcofluor-blue brightener for the direct detection of P. jirovecii cysts was prospectively assessed in bronchial-alveolar lavage fluids (BALF) from patients with a suspected PjP infection over a three-year period in a single center: the diagnostic yield was compared to that of a commercial kit based on monoclonal immunofluorescence assay (IFA) on replicate smears. May-Grünwald Giemsa (MGG) staining and quantitative Polymerase Chain Reaction (qPCR) were also performed. The gold standard for each patient was the definitive diagnosis of PjP infection by an independent committee based on clinical, radiological, and biological data. Overall, 481 BALF were assessed: 42 were found to be positive for the detection of P. jirovecii by at least one laboratory technique, but only 35 were actually judged to be in agreement with the definitive diagnosis of PjP infection. The sensitivity of the calcofluor-blue brightener technique was 74.3% vs. 60.0%, 34.6%, and 82.9% for IFA, MGG, and qPCR, respectively; and its specificity was 99.6% vs. 99.3%, 100.0%, and 99.4% for IFA, MGG, and qPCR. No technique was shown to be statistically superior to calcofluor-blue brightener. Further validation of the test through multicenter studies is now required, but in light of its low cost and easy preparation, the use of calcofluor-blue brightener in BALF appears to be a valuable alternative method for the routine first-line diagnosis of PjP infection. © The Author 2016. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Opening the Blood-Brain Barrier with MR Imaging-guided Focused Ultrasound: Preclinical Testing on a Trans-Human Skull Porcine Model.

PubMed

Huang, Yuexi; Alkins, Ryan; Schwartz, Michael L; Hynynen, Kullervo

2017-01-01

Purpose To develop and test a protocol in preparation for a clinical trial on opening the blood-brain barrier (BBB) with magnetic resonance (MR) imaging-guided focused ultrasound for the delivery of chemotherapy drugs to brain tumors. Materials and Methods The procedures were approved by the institutional animal care committee. A trans-human skull porcine model was designed for the preclinical testing. Wide craniotomies were applied in 11 pigs (weight, approximately 15 kg). A partial human skull was positioned over the animal's brain. A modified clinical MR imaging-guided focused ultrasound brain system was used with a 3.0-T MR unit. The ultrasound beam was steered during sonications over a 3 × 3 grid at 3-mm spacing. Acoustic power levels of 3-20 W were tested. Bolus injections of microbubbles at 4 μL/kg were tested for each sonication. Levels of BBB opening, hemorrhage, and cavitation signal were measured with MR imaging, histologic examination, and cavitation receivers, respectively. A cavitation safety algorithm was developed on the basis of logistic regression of the measurements and tested to minimize the risk of hemorrhage. Results BBB openings of approximately 1 cm 3 in volume were visualized with gadolinium-enhanced MR imaging after sonication at an acoustic power of approximately 5 W. Gross examination of histologic specimens helped confirm Evans blue (bound to macromolecule albumin) extravasation, and hematoxylin-eosin staining helped detect only scattered extravasation of red blood cells. In cases where cavitation signals were higher than thresholds, sonications were terminated immediately without causing hemorrhage. Conclusion With a trans-human skull porcine model, this study demonstrated BBB opening with a 230-kHz system in preparation for a clinical trial. © RSNA, 2016 Online supplemental material is available for this article.

Gastroprotective effect of alpha-pinene and its correlation with antiulcerogenic activity of essential oils obtained from Hyptis species

PubMed Central

Pinheiro, Marcelo de Almeida; Magalhães, Rafael Matos; Torres, Danielle Mesquita; Cavalcante, Rodrigo Cardoso; Mota, Francisca Sheila Xavier; Oliveira Coelho, Emanuela Maria Araújo; Moreira, Henrique Pires; Lima, Glauber Cruz; Araújo, Pamella Cristina da Costa; Cardoso, José Henrique Leal; de Souza, Andrelina Noronha Coelho; Diniz, Lúcio Ricardo Leite

2015-01-01

Background: Alpha-pinene (α-pinene) is a monoterpene commonly found in essential oils with gastroprotective activity obtained from diverse medicinal plants, including Hyptis species. The genus Hyptis (lamiaceae) consists of almost 400 species widespread in tropical and temperate regions of America. In the north and northeastern Brazil, some Hyptis species are used in traditional medicine to treat gastrointestinal disturbances. Objective: The present study has investigated the gastoprotective effect of purified α-pinene in experimental gastric ulcer induced by ethanol and indomethacin in mice. Materials and Methods: Gastric ulcers were induced in male Swiss mice (20-30 g) by oral administration of absolute ethanol or indomethacin 45 min after oral pretreatment with vehicle, standard control drugs or α-pinene (10, 30, and 100 mg/kg). One hour after the ulcerative challenges, the stomach were removed, and gastric lesions areas measured. The effects of α-pinene on the gastric juice acidity were determined by pylorus ligation model. The gastrointestinal motility and mucus depletion were determined by measuring the gastric levels of phenol red and alcian blue, respectively. Hematoxylin and eosin stained sections of gastric mucosa of the experimental groups were used for histology analysis. Results: α-pinene pretreatment inhibited ethanol-induced gastric lesions, reduced volume and acidity of the gastric juice and increased gastric wall mucus (P < 0.05). Furthermore, we showed an interesting correlation between concentration of α-pinene and gastroprotective effect of Hyptis species (P Pearson = 0.98). Conclusion: Our data showed that the α-pinene exhibited significant antiulcerogenic activity and a great correlation between concentration of α-pinene and gastroprotective effect of Hyptis species was also observed. PMID:25709221

Histological methods to determine blood flow distribution with fluorescent microspheres.

PubMed

Luchtel, D L; Boykin, J C; Bernard, S L; Glenny, R W

1998-11-01

We evaluated several histological methods and determined their advantages and disadvantages for histological studies of tissues and organs perfused with fluorescent microspheres. Microspheres retained their fluorescence in 7-10 microm serial sections with a change in the antimedium from toluene when samples were fixed in formalin and embedded in paraffin. Several antimedia allowed both wax infiltration of tissue and preservation of microsphere fluorescence. Histoclear II was the best substitute for toluene. When samples were fixed in formalin and embedded in glycol methacrylate, thinner (3-5 microm) sections provided greater histological detail but had fewer microspheres per section. Air dried lung tissue followed by Vibratome sectioning provided thick sections (100 microm) that facilitated rapid survey of large volumes of tissue for microspheres but limited histological detail, and the air drying procedure was restricted to lung tissue. Samples fixed in formalin followed by Vibratome sectioning of unembedded tissue provided better histological detail of lung tissue and was also useful for other organs. These sections were more difficult to handle and to mount on slides compared to air dried tissue, whereas fixed tissue embedded in gelatin provided better tissue support for Vibratome sectioning. Rapid freezing followed by cryo-microtome sectioning resulted in frozen sections that were relatively difficult to handle compared to embedded or unembedded tissue; they also deteriorated relatively rapidly with time. Paraffin sections were stained with hematoxylin and eosin or with aqueous methyl green, although tissue autofluorescence by itself was usually sufficient to identify histological features. Methacrylate sections quenched tissue autofluorescence, and Lee's stain or Richardson's stain were used for staining sections. Toluene based mountants such as Cytoseal quenched fluorescence, particularly the red fluorescent microspheres. Aqueous based mountants such as Aquamount, Crystal/Mount, Fluoromount-G were substituted, although such preparations were not as permanent as Cytoseal mounted coverglasses and tended to cause fading of stained sections.

Intraoperative evaluation of sentinel lymph nodes for metastatic melanoma by imprint cytology.

PubMed

Soo, Victoria; Shen, Perry; Pichardo, Rita; Azzazy, Hossam; Stewart, John H; Geisinger, Kim R; Levine, Edward A

2007-05-01

Sentinel lymph node biopsy (SLN) has revolutionized nodal staging. Accurate intraoperative evaluation of SLN permits a single procedure, with lymphadenectomy being performed during the initial operative procedure when the SLN is positive. There is a paucity of literature on intraoperative imprint cytology (IIC) evaluation of the SLN in melanoma. The purpose of this article is to present an update to our experience with IIC for SLN in melanoma. Melanoma patients had SLNs examined by IIC. SLNs were bisected, and imprints were made from each half. Imprints were stained with hematoxylin and eosin and with Diff-Quik. Paraffin-embedded sections were examined with multiple hematoxylin and eosin-stained sections from the SLNs in conjunction with immunohistochemical staining for S-100, Melan-A, and HMB-45 proteins. Metastases were identified in 40 (17%) of 229 patients. Of these, 13 patients were detected by IIC (sensitivity, 33%). The negative predictive value was 88%. No false-positive results were identified (specificity, 100%). The positive predictive value was 100%. The accuracy of IIC was 78%. The sensitivity for detecting macrometastases (>2 mm) was better than that for detecting micrometastases (< or =2 mm): 62% vs. 16% (P < .01). Patients with positive SLNs by IIC had lymphadenectomy under the same anesthetic. A total of 533 nonsentinel lymph nodes were identified in 42 patients. Only two patients (8%) had positive nonsentinel lymph nodes after a negative IIC. IIC is a viable alternative to frozen sectioning when intraoperative evaluation is desired. IIC is significantly more sensitive for macrometastases. IIC evaluation of SLNs in melanoma makes a single operative procedure possible for a significant proportion of patients with regional nodal metastases.

Immunohistochemical findings of the granulomatous reaction associated with tuberculosis.

PubMed

Karimi, Shirin; Shamaei, Masoud; Pourabdollah, Mihan; Sadr, Makan; Karbasi, Mehrdad; Kiani, Arda; Bahadori, Moslem

2016-12-01

The histological diagnosis of Mycobacterium tuberculosis (MTB) has long been a diagnostic challenge in the anatomical pathology field despite availability of different laboratory methods. Immunohistochemistry (IHC) could not only confirm granulomatous tissue involvement but also demonstrate MTB antigen immunolocalization. This study tries to clarify the details of IHC staining for MTB with pAbBCG. A total of 50 patients undergoing simultaneous biopsy and tissue culture with positive tissue culture for MTB during 2005-2009 were selected from the MRC Department at Masih Daneshvari Hospital, Tehran, Iran. Using the archives of the Pathology Department of this hospital, which is a referral center for pathological lung lesions, hematoxylin and eosin slides of the selected patients were evaluated. Twenty-three confirmed TB granulomatous tissue samples with adequate tissue and number of granulomas were chosen and studied by Ziehl-Neelsen and IHC staining with pAbBCG. A total of 23 cases were evaluated, of which 17 (73.9%) were males. The types of tissue obtained from study cases were as follows: pleura (9 cases, 39.1%), lymph node (cervical, axillary, and thoracic [9 cases, 39.1%]), and lung tissues (5 cases, 21.7%). IHC staining was positive in all samples, whereas Ziehl-Neelsen staining was positive in nine cases of 23 (39.1%). IHC showed positive coarse granular cytoplasmic and round, fragmented bacillary staining. In this study, epithelioid cells clearly showed more positive staining at the periphery rather than at the center of granuloma. There is also positive staining in endothelial cells, fibroblasts, plasma cells, macrophages, and lymphocytes outside the granuloma. Detection of TB in tissue slides is still based on the histological pattern of the granuloma, which has several differential diagnoses with different treatments. Presence of mycobacterial antigens and tissue morphology can be evaluated using the IHC technique. Considering the criteria of positive IHC staining of TB granulomatous reactions, this stain not only highlights the presence of mycobacterial antigens for tissue diagnosis, but also could morphologically localize their distribution in different cells. Pathologists must be familiar with adequate staining pattern, elimination of background staining, and type of selected antibody. This method is especially important for application in countries with high prevalence of TB as a technique with early diagnostic value in tissue specimens. Early diagnosis using this technique can reduce related morbidity and mortality and decrease the rate of complications due to misdiagnosis and mistreatment of TB. Copyright © 2016.

Rapid demonstration of Legionella pneumophila in unembedded tissue. An adaptation of the Giménez stain.

PubMed

Greer, P W; Chandler, F W; Hicklin, M D

1980-06-01

The Giménez stain, originally developed for demonstrating rickettsiae, readily stained the Legionnaires' disease bacterium (Legionella pneumophila) in frozen tissue sections and smears of fresh or formalin-fixed lung tissue from patients who had confirmed Legionnaires' disease. With the Giménez procedure, the bacterium stained bright red against a blue-green background. The tissue Gram procedures also stained L. pneumophila in frozen sections and smears, but the staining reaction was weak, and these stains were neither as sensitive nor a consistent as the Giménez procedure.

Iron Stain on Wood

Treesearch

Mark Knaebe

2013-01-01

Iron stain, an unsightly blue–black or gray discoloration, can occur on nearly all woods. Oak, redwood, cypress, and cedar are particularly prone to iron stain because these woods contain large amounts of tannin-like extractives. The discoloration is caused by a chemical reaction between extractives in the wood and iron in steel products, such as nails, screws, and...

THE RELATIVE REACTION WITHIN LIVING MAMMALIAN TISSUES : I. GENERAL FEATURES OF VITAL STAINING WITH LITMUS.

PubMed

Rous, P

1925-02-28

The present paper is the first of a series of reports on the relative reaction of living tissues as determined by vital staining with indicators. It is possible to bring about a localized and a general coloration of living rats and mice with litmus. The animals remain in good health and the coloration of some of the tissues persists for months. Much of the dye is stored in cell granules, especially in those of the reticulo-endothelial elements, but a diffuse staining of certain tissues occurs, notably of bone, epidermis, cartilage, and connective tissue everywhere. In the intensity and localization of the bony coloration litmus has resemblances to madder. Diffuse staining with it renders blue most, if not all, of the tissues affected, while a granular staining causes others to become notably pink, owing to the fact that the indicator, though introduced into the organism in the blue form and circulating as such in the body fluids, is ordinarily red when stored in cells. The polymorphonuclear elements and macrophages of a peritoneal exudate, may become so laden with material colored red by litmus that the blue color of the fluid constituent is masked and the exudate appears a deep, turbid red. The phenomenon is but one manifestation of a notable acidity within cell granules throughout the organism. Like many another in the stained animals it would appear to be of physiological import. Some of the questions suggested by the work will be dealt with in the paper immediately following.

New visible and selective DNA staining method in gels with tetrazolium salts.

PubMed

Paredes, Aaron J; Naranjo-Palma, Tatiana; Alfaro-Valdés, Hilda M; Barriga, Andrés; Babul, Jorge; Wilson, Christian A M

2017-01-15

DNA staining in gels has historically been carried out using silver staining and fluorescent dyes like ethidium bromide and SYBR Green I (SGI). Using fluorescent dyes allows recovery of the analyte, but requires instruments such as a transilluminator or fluorimeter to visualize the DNA. Here we described a new and simple method that allows DNA visualization to the naked eye by generating a colored precipitate. It works by soaking the acrylamide or agarose DNA gel in SGI and nitro blue tetrazolium (NBT) solution that, when exposed to sunlight, produces a purple insoluble formazan precipitate that remains in the gel after exposure to light. A calibration curve made with a DNA standard established a detection limit of approximately 180 pg/band at 500 bp. Selectivity of this assay was determined using different biomolecules, demonstrating a high selectivity for DNA. Integrity and functionality of the DNA recovered from gels was determined by enzymatic cutting with a restriction enzyme and by transforming competent cells after the different staining methods, respectively. Our method showed the best performance among the dyes employed. Based on its specificity, low cost and its adequacy for field work, this new methodology has enormous potential benefits to research and industry. Copyright © 2016 Elsevier Inc. All rights reserved.

[Morphofunctional characteristics of immunocompetent cells in dysplasia of the breast].

PubMed

Dikshteĭn, E A; Burlak, Iu P; Shevchenko, N I

1985-01-01

Immunocompetent cells were studied in the stroma and epithelium of 34 cases of mammary gland dysplasia. The following stainings were used for light microscopy: hematoxylin and eosin, methods of Brachet, van Gieson, Romanovsky-Giemsa, hallocyanine alums, Gomori, PAS-reaction as well as the determination of acid and alkaline phosphatases, glucose-6-phosphate and succinate dehydrogenase were used. 14 cases were studied ultrastructurally. Two types of small B lymphocytes and one type of large lymphocytes, stromal macrophages are described. Their morphofunctional characteristics is given and their properties in proliferating and non-proliferating fibroadenomatosis are shown. Interaction of intraepithelial large granular lymphocytes (normal killers) with immature epithelial cells resulting in the death of these epitheliocytes is described. The results obtained are regarded as a morphological manifestation of the immune surveillance.

Toluidine blue aids in detection of dysplasia and carcinoma in suspicious oral lesions.

PubMed

Chainani-Wu, N; Madden, E; Cox, D; Sroussi, H; Epstein, J; Silverman, S

2015-10-01

Accurate clinical identification of 'higher-risk' oral premalignant lesions or 'higher-risk' areas within lesions is important. Assessment methods that predict their presence have great utility. A cross-sectional, observational study enrolled a consecutive sample of consenting patients diagnosed with oral leukoplakia, erythroleukoplakia, or erythroplakia. Medical history, visual oral examination, ViziLite(®) examination, toluidine blue staining (TBlue(®) ), and finally a biopsy were completed in a single clinic visit. Seventy-seven of 100 examined lesions in 43 patients were biopsied. Sensitivity, specificity, and positive and negative predictive values were computed for visual examination, ViziLite(®) , and TBlue(®) using biopsy results as the gold standard. The sensitivity of TBlue(®) in detecting high-risk lesions (carcinoma in situ or carcinoma) was 94 (71-100, P < 0.0003) and specificity 45 (32-58, P < 0.53), while for carcinoma, sensitivity was 100 (54-100, P < 0.032) and specificity 39 (28-52, P < 0.097). The results of ViziLite(®) testing either by itself or in combination with the information from toluidine blue testing revealed low sensitivity for the detection of high-risk lesions. Clinical examination of leukoplakia, erythroplakia, or erythroleukoplakia lesions combined with toluidine blue staining may aid in the identification of severe dysplasia (carcinoma in situ) or carcinoma. This may help in determining whether, when, and where (the site within a lesion) a biopsy should be taken. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

[Technical recommendations and best practice guidelines for May-Grünwald-Giemsa staining: literature review and insights from the quality assurance].

PubMed

Piaton, Eric; Fabre, Monique; Goubin-Versini, Isabelle; Bretz-Grenier, Marie-Françoise; Courtade-Saïdi, Monique; Vincent, Serge; Belleannée, Geneviève; Thivolet, Françoise; Boutonnat, Jean; Debaque, Hervé; Fleury-Feith, Jocelyne; Vielh, Philippe; Cochand-Priollet, Béatrix; Egelé, Caroline; Bellocq, Jean-Pierre; Michiels, Jean-François

2015-08-01

May-Grünwald-Giemsa (MGG) stain is a Romanowsky-type, polychromatic stain as those of Giemsa, Leishman and Wright. Apart being the reference method of haematology, it has become a routine stain of diagnostic cytopathology for the study of air-dried preparations (lymph node imprints, centrifuged body fluids and fine needle aspirations). In the context of their actions of promoting the principles of quality assurance in cytopathology, the French Association for Quality Assurance in Anatomic and Cytologic Pathology (AFAQAP) and the French Society of Clinical Cytology (SFCC) conducted a proficiency test on MGG stain in 2013. Results from the test, together with the review of literature data allow pre-analytical and analytical steps of MGG stain to be updated. Recommendations include rapid air-drying of cell preparations/imprints, fixation using either methanol or May-Grünwald alone for 3-10minutes, two-step staining: 50% May-Grünwald in buffer pH 6.8 v/v for 3-5minutes, followed by 10% buffered Giemsa solution for 10-30minutes, and running water for 1-3minutes. Quality evaluation must be performed on red blood cells (RBCs) and leukocytes, not on tumour cells. Under correct pH conditions, RBCs must appear pink-orange (acidophilic) or buff-coloured, neither green nor blue. Leukocyte cytoplasm must be almost transparent, with clearly delineated granules. However, staining may vary somewhat and testing is recommended for automated methods (slide stainers) which remain the standard for reproducibility. Though MGG stain remains the reference stain, Diff-Quik(®) stain can be used for the rapid evaluation of cell samples. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Comparison of two methods used to prepare smears of mouse lung tissue for detection of Pneumocystis carinii.

PubMed Central

Thomson, R B; Smith, T F; Wilson, W R

1982-01-01

The laboratory diagnosis of Pneumocystis carinii pneumonia in humans includes the identification of cysts in stained lung tissue impression smears. By using a mouse model, we compared the number of cysts in lung tissue impression smears with those contained in a concentrate of homogenized lung tissue. Eleven C3H/HEN mice developed P. carinii infection after corticosteroid injections, a low protein (8%) diet, and tetracycline administered in drinking water. Impression smears were prepared with freshly bisected lung tissue. Smears of concentrates were prepared with sediment from centrifuged lung tissue homogenates. All smears were made in duplicate, stained with toluidine blue O or methenamine silver, coded, randomized, and examined. The concentrate preparations contained more cysts per microscopic field than the impression preparations (P less than 0.01). Concentrates prepared by grinding with a mortar and pestle contained more cysts than concentrates prepared by blending with a Stomacher (P less than 0.05). Cysts were detected equally well with either the toluidine blue O or silver stain (not significant). Lung tissue concentrates were superior to lung tissue impressions for detecting P. carinii cysts in mice. Use of lung tissue concentrates should be considered for the diagnosis of human P. carinii infection. PMID:6181088

[Pneumocystis pneumonia biological diagnosis at Fann Teaching Hospital in Dakar, Senegal].

PubMed

Dieng, Y; Dieng, T; Sow, D; Wlouhou, S; Sylla, K; Tine, R; Ndiaye, M; Ndiaye, J L; Faye, B; Faye, O; Gaye, O

2016-03-01

Data relative to Pneumocystis pneumonia in sub-Saharan Africa are not well known. Weakness of the technical material and use of little sensitive biological tools of diagnosis are among the evoked reasons. The objective of this study is to update the data of the disease at the Fann Teaching Hospital in Dakar and to estimate biological methods used in diagnosis. A descriptive longitudinal study was carried out from January 5th, 2009 to October 31st, 2011 in the parasitology and mycology laboratory of the Fann Teaching Hospital in Dakar. The bronchoalveolar lavages received in the laboratory were examined microscopically for Pneumocystis jirovecii by indirect fluorescent assay or after Giemsa or toluidine blue O staining. One hundred and eighty-three bronchoalveolar lavages withdrawn from 183 patients were received in the laboratory. Sixteen were positive for P. jirovecii at 9% frequency. Four among these patients were HIV positive. Indirect fluorescent assay allowed finding of P. jirovecii among 16 patients while Giemsa staining discovered P. jirovecii only in a single patient. No case was diagnosed by toluidine blue O staining. Pneumocystis pneumonia in Parasitology and Mycology Laboratory of Fann Teaching Hospital at Dakar was mainly diagnosed among HIV patients. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Exposure to As, Cd and Pb-mixture impairs myelin and axon development in rat brain, optic nerve and retina

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rai, Nagendra Kumar; Ashok, Anushruti; Developmental Toxicology, Council of Scientific and Industrial Research-Indian Institute of Toxicology Research

Arsenic (As), lead (Pb) and cadmium (Cd) are the major metal contaminants of ground water in India. We have reported the toxic effect of their mixture (metal mixture, MM), at human relevant doses, on developing rat astrocytes. Astrocyte damage has been shown to be associated with myelin disintegration in CNS. We, therefore, hypothesized that the MM would perturb myelinating white matter in cerebral cortex, optic nerve (O.N.) and retina. We observed modulation in the levels of myelin and axon proteins, such as myelin basic protein (MBP), proteolipid protein, 2′-, 3′-cyclic-nucleotide-3′-phosphodiesterase, myelin-associated glycoprotein and neurofilament (NF) in the brain of developingmore » rats. Dose and time-dependent synergistic toxic effect was noted. The MBP- and NF-immunolabeling, as well as luxol-fast blue (LFB) staining demonstrated a reduction in the area of intact myelin-fiber, and an increase in vacuolated axons, especially in the corpus-callosum. Transmission electron microscopy (TEM) of O.N. revealed a reduction in myelin thickness and axon-density. The immunolabeling with MBP, NF, and LFB staining in O.N. supported the TEM data. The hematoxylin and eosin staining of retina displayed a decrease in the thickness of nerve-fiber, plexiform-layer, and retinal ganglion cell (RGC) count. Investigating the mechanism revealed a loss in glutamine synthetase activity in the cerebral cortex and O.N., and a fall in the brain derived neurotrophic factor in retina. An enhanced apoptosis in MBP, NF and Brn3b-containing cells justified the diminution in myelinating axons in CNS. Our findings for the first time indicate white matter damage by MM, which may have significance in neurodevelopmental-pediatrics, neurotoxicology and retinal-cell biology. - Highlights: • As, Cd and Pb-mixture, at human relevant dose, demyelinate developing rat CNS. • The attenuation in myelin and axon is synergistic. • The optic nerve and brain demonstrate reduced glutamine synthetase. • The retina exhibits diminished neurotrophin levels and cellular differentiation. • The toxic effect is apoptotic.« less

Upfront immunohistochemistry improves specificity of Helicobacter pylori diagnosis. A French pathology laboratory point of view.

PubMed

Ginestet, Florent; Guibourg, Briac; Doucet, Laurent; Théreaux, Jérémie; Robaszkiewicz, Michel; Marcorelles, Pascale; Uguen, Arnaud

2017-10-01

There is no consensus about the histopathologic methods to detect Helicobacter pylori in gastric biopsies to date. We aimed to question about the value of upfront anti-H. pylori immunohistochemistry in this field. We led a retrospective study about the rate of H. pylori-positive gastric biopsies before and after the implementation of upfront immunohistochemistry, the inter-rater and intermethods agreements in H. pylori identification about Hematoxylin-Eosin Saffron (HES), Giemsa, and immunohistochemistry stains and the histopathologic features associated with low amounts of H. pylori. First, the rate of H. pylori-positive gastric biopsies significantly diminished after the implementation of upfront immunohistochemistry (from 21.15% to 12.56%, P<.0001), suggesting potential overdiagnosis of H. pylori infection before the use of immunohistochemistry. Secondly, immunohistochemistry was the most reproducible and performing stain (kappa values >0.80), but HES and Giemsa stains also presented good-to-very good agreements. Finally, less than 1% of gastric biopsies with inconspicuous H. pylori infection showed no mucosal injury pointing out that any HES-detected mucosal injury could help to preselect the gastric biopsies requiring ancillary stains for the detection of H. pylori. Albeit being considered as a gold standard in the detection of H. pylori, the interest of using immunohistochemistry as an upfront stain on gastric biopsies is still debated. In our opinion, its use in second line in case of ambiguous HE/HES-Giemsa result is more appropriate. Further effort is needed to optimize the inexpensive but feasible HE/HES-based detection of H. pylori. © 2017 John Wiley & Sons Ltd.

Histopathological characteristics of cutaneous lesions caused by Leishmania Viannia panamensis in Panama

PubMed Central

González, Kadir; Diaz, Rosendo; Ferreira, Aurea F.; García, Víctor; Paz, Héctor; Calzada, José E.; Ruíz, Michelle; Laurenti, Márcia; Saldaña, Azael

2018-01-01

ABSTRACT Cutaneous leishmaniasis (CL) is an endemic disease in the Republic of Panama, caused by Leishmania (Viannia) parasites, whose most common clinical manifestation is the presence of ulcerated lesions on the skin. These lesions usually present a chronic inflammatory reaction, sometimes granulomatous, with the presence of lymphocytes, plasma cells and macrophages. This study describes the histopathological characteristics found in the skin lesions of patients with CL caused by Leishmania (V.) panamensis in Panama. We analyzed 49 skin biopsy samples from patients with clinical suspicion of CL, by molecular tests (PCR for subgenus Viannia and HSP-70) and by Hematoxylin-Eosin staining. Samples were characterized at the species level by PCR-HSP-70/RFLP. From the 49 samples studied, 46 (94%) were positive by PCR and were characterized as Leishmania (V.) panamensis. Of these, 48% were positive by Hematoxylin-Eosin staining with alterations being observed both, in the epidermis (85%) and in the dermis (100%) of skin biopsies. The inflammatory infiltrate was characterized according to histopathological patterns: lymphohistiocytic (50%), lymphoplasmacytic (61%) and granulomatous (46%) infiltration, being the combination of these patterns frequently found. The predominant histopathological characteristics observed in CL lesions caused by L. (V.) panamensis in Panama were: an intense inflammatory reaction in the dermis with a combination of lymphohistiocytic, lymphoplasmacytic and granulomatous presentation patterns and the presence of ulcers, acanthosis, exocytosis and spongiosis in the epidermis. PMID:29451596

Inflammatory Mediators in Xanthelasma Palpebrarum: Histopathologic and Immunohistochemical Study.

PubMed

Govorkova, Maria S; Milman, Tatyana; Ying, Gui-Shuang; Pan, Wei; Silkiss, Rona Z

To evaluate the expression of inflammatory mediators in xanthelasma palpebrarum. In this retrospective histopathologic case-control study, xanthelasma specimens obtained from the private practice and pathology archives of 1 author (R.Z.S.) were analyzed and compared with the blepharoplasty tissues from age- and sex-matched controls. Paraffin-embedded tissue sections were stained with hematoxylin-eosin and CD3, CD20, CD163, cyclooxygenase-1, inducible nitric oxide synthase, matrix metallopeptidase-9, and myeloperoxidase antibodies. Immunostaining was quantified by light microscopy and with a computerized image analysis system of scanned images. Hematoxylin-eosin-stained preparations of xanthelasma specimens demonstrated significantly more intense chronic lymphocytic infiltrate when compared with the control blepharoplasty tissues (p < 0.001). Immunohistochemical studies revealed more intense CD3+ T cell and CD163+ histiocytic infiltrate (11% vs. 5%; p = 0.02 and 28% vs. 5%; p = 0.003, respectively) and increased expression of cyclooxygenase-1 (44% vs. 20% expressing cells; p < 0.001 and 21% vs. 9% strongly expressing cells; p = 0.008) and inducible nitric oxide synthase (43% vs. 26% expressing cells; p = 0.03 and 42% vs. 25% strongly expressing cells; p = 0.02) in xanthelasma specimens compared with control tissues. The inflammatory milieu in xanthelasma appears to be analogous to descriptions of the early stages of cardiac atherosclerotic plaque formation. These findings may contribute to the understanding of xanthelasma pathogenesis and to the development of potential targeted therapies.

Fibrinogen Demonstration in Oral Lichen Planus: An Immunofluorescence Study on Archival Tissues.

PubMed

Shirol, Pallavi D; Naik, Veena; Kale, Alka

2015-10-01

Lichen planus is a premalignant condition with minimal diagnostic aids. This study is an attempt to use paraffin embedded sections of lichen planus with immunofluorescein stain and to evaluate the immunofluorescent sections to establish pattern of fibrinogen deposition. Thirty-five paraffin embedded sections of old and new cases of oral lichen planus (study group) and five normal oral mucosa (control group) were chosen. Two sections of each (H & E) case were taken, one was stained with hematoxylin and eosin and another with fluorescein isothiocynate conjugate (FITC) polyclonal rabbit antibody against fibrinogen. Fluorescent findings were examined with a fluorescent microscope. A high statistical significant correlation was found in respect to fluorescence positivity, intensity of fluorescence and distribution of fluorescence each with p < 0.0001 and fluorescence at blood vessel walls (p = 0.0003). This study suggested that paraffin embedded sections can be successfully used in direct immunofluorescence staining in routine set up where only formalin fixed tissues are received. Paraffin embedded sections can be successfully used in direct immunofluorescence staining when only formalin fixed tissues are received.

[Method of determination of fat deposit in chickens].

PubMed

Bakhnev, D

1975-01-01

The method described is based on the histochemical demonstration of fats with the use of Sudan stains and the clearing of tissues in an alkaline medium. It is employed to investigate whole birds, thick sections or individual organs to determine the morphotopographic localization of the fat depots. The procedure comprises the alcohol fixation of tissues, the staining of lipids with Sudan black, the clearing of tissues in a solution of potassium hydroxide, and the final mounting of the cleared materials in glycerine. This treatment results in that the sites of fat deposition assume a blue coloration, the bones are grey, and all remaining tissues and organs (if properly bled) are colorless and transparent. The method can also be successfully used with materials that have been preliminary fixed with formalin but their clearing has been considerably worse.

Antimicrobial resistant coliform bacteria in the Gomti river water and determination of their tolerance level.

PubMed

Akhter, Asma; Imran, Mohd; Akhter, Firoz

2014-01-01

The distribution of resistance to ampicillin, chloramphenicol, sulfonamides, tetracycline, and streptomycin among coliform in the Gomti river water samples was investigated. The coliform populations were isolated on Mac Conky and eosin methylene blue (EMB) agar plates supplemented with antibiotics. The incidence of resistance among the coliform population varied considerably in different drug and water sampling sites. Coliform bacteria showed lower drug resistant viable count in sampling site-III (receiving treated wastewater) as compared to more polluted site-I and site-II. Viable count of coliform population obtained on both medium was recorded higher against erythromycin from sampling site-III. Lower viable count of coliforms was recorded against tetracycline in site-II and III. Similar resistance pattern was obtained in the frequency of E. coli and Enterobacter species from all the three sampling sites. Percentage of antibiotic resistant E. coli was observed higher than Enterobacter spp among the total coliforms against all antibiotics tested without Erythromycin and penicillin in site-I and II respectively. Isolates of E. coli and Enterobacter spp. showed their tolerance level (MIC) in the range of 2-100 against the antibiotics tested. Maximum number of isolates of both genus exhibited their MICs at lower concentration range 2-5µg/ml against ciprofloxacin, tetracyclin and amoxycillin. EMB - Eosin methylene blue, IMViC tests - Indole, Methyl Red, Voges Proskauer and Citrate Utilization Tests, MIC - Minimum inhibitory concentration.

Antimicrobial resistant coliform bacteria in the Gomti river water and determination of their tolerance level

PubMed Central

Akhter, Asma; Imran, Mohd; Akhter, Firoz

2014-01-01

The distribution of resistance to ampicillin, chloramphenicol, sulfonamides, tetracycline, and streptomycin among coliform in the Gomti river water samples was investigated. The coliform populations were isolated on Mac Conky and eosin methylene blue (EMB) agar plates supplemented with antibiotics. The incidence of resistance among the coliform population varied considerably in different drug and water sampling sites. Coliform bacteria showed lower drug resistant viable count in sampling site-III (receiving treated wastewater) as compared to more polluted site-I and site-II. Viable count of coliform population obtained on both medium was recorded higher against erythromycin from sampling site-III. Lower viable count of coliforms was recorded against tetracycline in site-II and III. Similar resistance pattern was obtained in the frequency of E. coli and Enterobacter species from all the three sampling sites. Percentage of antibiotic resistant E. coli was observed higher than Enterobacter spp among the total coliforms against all antibiotics tested without Erythromycin and penicillin in site-I and II respectively. Isolates of E. coli and Enterobacter spp. showed their tolerance level (MIC) in the range of 2-100 against the antibiotics tested. Maximum number of isolates of both genus exhibited their MICs at lower concentration range 2-5µg/ml against ciprofloxacin, tetracyclin and amoxycillin. Abbreviations EMB - Eosin methylene blue, IMViC tests - Indole, Methyl Red, Voges Proskauer and Citrate Utilization Tests, MIC - Minimum inhibitory concentration. PMID:24966515

Influence of Light Emitting Diode-Derived Blue Light Overexposure on Mouse Ocular Surface.

PubMed

Lee, Hyo Seok; Cui, Lian; Li, Ying; Choi, Ji Suk; Choi, Joo-Hee; Li, Zhengri; Kim, Ga Eon; Choi, Won; Yoon, Kyung Chul

2016-01-01

To investigate the influence of overexposure to light emitting diode (LED)-derived light with various wavelengths on mouse ocular surface. LEDs with various wavelengths were used to irradiate C57BL/6 mice at an energy dose of 50 J/cm2, twice a day, for 10 consecutive days. The red, green, and blue groups represented wavelengths of 630 nm, 525 nm, and 410 nm, respectively. The untouched group (UT) was not exposed to LED light and served as the untreated control. Tear volume, tear film break-up time (TBUT), and corneal fluorescein staining scores were measured on days 1, 3, 5, 7, and 10. Levels of interferon (IFN)-γ, interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α were measured in the cornea and conjunctiva using a multiplex immunobead assay at day 10. Levels of malondialdehyde (MDA) were measured with an enzyme-linked immunosorbent assay. Flow cytometry, 2'7'-dichlorofluorescein diacetate (DCF-DA) assay, histologic analysis, immunohistochemistry with 4-hydroxynonenal, and terminal deoxynucleotidyl transferase-mediated dUTP-nick end labeling (TUNEL) staining were also performed. TBUT of the blue group showed significant decreases at days 7 and 10, compared with the UT and red groups. Corneal fluorescein staining scores significantly increased in the blue group when compared with UT, red, and green groups at days 5, 7, and 10. A significant increase in the corneal levels of IL-1β and IL-6 was observed in the blue group, compared with the other groups. The blue group showed significantly increased reactive oxygen species production in the DCF-DA assay and increased inflammatory T cells in the flow cytometry. A significantly increased TUNEL positive cells was identified in the blue group. Overexposure to blue light with short wavelengths can induce oxidative damage and apoptosis to the cornea, which may manifest as increased ocular surface inflammation and resultant dry eye.

Effects of normal saline and selenium-enriched hot spring water on experimentally induced rhinosinusitis in rats.

PubMed

Kim, Dong-Hyun; Yeo, Sang Won

2013-01-01

This prospective, randomized, and controlled study examined the effects of normal saline and selenium-enriched hot spring water on experimentally induced rhinosinusitis in rats. The study comprised two control groups (untreated and saline-treated) and three experimental groups of Sprague Dawley rats. The experimental groups received an instillation of lipopolysaccharide (LPS) only, LPS+normal saline (LPS/saline), or LPS+selenium-enriched hot spring water (LPS/selenium). Histopathological changes were identified using hematoxylin-eosin staining. Leakage of exudate was identified using fluorescence microscopy. Microvascular permeability was measured using the Evans blue dye technique. Expression of the Muc5ac gene was measured using reverse transcription-polymerase chain reaction. Mucosal edema and expression of the Muc5ac gene were significantly lower in the LPS/saline group than in the LPS group. Microvascular permeability, mucosal edema, and expression of the Muc5ac gene were significantly lower in the LPS/selenium group than in the LPS group. Mucosal edema was similar in the LPS/selenium group and LPS/saline group, but capillary permeability and Muc5ac expression were lower in the LPS/selenium group. This study shows that normal saline and selenium-enriched hot spring water reduce inflammatory activity and mucus hypersecretion in LPS-induced rhinosinusitis in rats. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Celiac disease and intestinal metaplasia of the esophagus (Barrett's esophagus).

PubMed

Maieron, Roberto; Elli, Luca; Marino, Marco; Floriani, Irene; Minerva, Francesco; Avellini, Claudio; Falconieri, Giovanni; Pizzolitto, Stefano; Zilli, Maurizio

2005-01-01

Previous studies on celiac patients demonstrated that exposure to gliadin alters the motility of the upper gastrointestinal tract, leading to increased acid reflux. No literature is available regarding the possible presence of specialized intestinal metaplasia of the esophagus as a consequence of chronic reflux in adult celiac patients. Our purpose was to evaluate endoscopically and histologically the esophagi of a group of untreated celiac patients. We studied 60 celiac patients, 13 men and 47 women (mean age, 40 +/- 14 [SD] years; range, 18-80 years), at their first endoscopy (following a normal diet). The distal esophagus was evaluated and multiple biopsies were taken. Hematoxylin-eosin and alcian blue stainings were performed. A group of nonceliac, age- and sex-matched patients was used as a control. We found intestinal metaplasia in the distal esophagus of 16 of 60 (26.6%) celiacs (mean age, 45 +/- 13 years; range, 27-75 years), in comparison with a control-group prevalence of 10.9% (OR, 3.9; 95% CI, 1.4-11.2%). Among the celiac group with metaplasia, only one patient had reflux-like symptoms. None had esophagitis. In conclusion, we observed an increased prevalence of esophageal metaplasia in patients with celiac disease. This finding could be the result of motor abnormalities leading to chronic acid reflux, combined with a mucosa which is sensitive to gliadin.

Specificity of dermal mucin in the diagnosis of lupus erythematosus: comparison with other dermatitides and normal skin.

PubMed

Vincent, Jeremy G; Chan, May P

2015-10-01

Increased dermal mucin is a feature of lupus erythematosus (LE); however, its amount and distribution have not been well characterized. The differentiation of LE from other forms of dermatitis can be challenging when other features of LE are subtle or equivocal. One hundred and thirty-five skin specimens showing LE, graft vs. host disease, erythema multiforme/fixed drug eruption, lichen planus, polymorphous light eruption (PMLE), urticaria, eczematous dermatitis and psoriasis and normal skin with and without photodamage were collected. The amounts of mucin in the papillary, superficial reticular and deep reticular dermis were scored from 0 to 3 on hematoxylin-eosin (H&E) and alcian blue (AB) stains, and compared between groups. The mean scores in the reticular dermis were significantly higher in LE than in other categories except PMLE and eczematous dermatitis. A combined H&E + AB score of ≥5 in the superficial reticular dermis gave an overall specificity of 85.7% for LE. Mucin in the papillary dermis failed to distinguish among entities. Normal photodamaged skin showed significantly more mucin in the superficial reticular dermis compared to non-photodamaged skin. While LE is associated with increased mucin deposition, scant to moderate amount of mucin alone has limited specificity and is common in other dermatitides or photodamaged skin. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

A Histopathological Scheme for the Quantitative Scoring of Intervertebral Disc Degeneration and the Therapeutic Utility of Adult Mesenchymal Stem Cells for Intervertebral Disc Regeneration

PubMed Central

Shu, Cindy C.; Smith, Margaret M.; Smith, Susan M.; Dart, Andrew J.; Little, Christopher B.; Melrose, James

2017-01-01

The purpose of this study was to develop a quantitative histopathological scoring scheme to evaluate disc degeneration and regeneration using an ovine annular lesion model of experimental disc degeneration. Toluidine blue and Haematoxylin and Eosin (H&E) staining were used to evaluate cellular morphology: (i) disc structure/lesion morphology; (ii) proteoglycan depletion; (iii) cellular morphology; (iv) blood vessel in-growth; (v) cell influx into lesion; and (vi) cystic degeneration/chondroid metaplasia. Three study groups were examined: 5 × 5 mm lesion; 6 × 20 mm lesion; and 6 × 20 mm lesion plus mesenchymal stem cell (MSC) treatment. Lumbar intervertebral discs (IVDs) were scored under categories (i–vi) to provide a cumulative score, which underwent statistical analysis using STATA software. Focal proteoglycan depletion was associated with 5 × 5 mm annular rim lesions, bifurcations, annular delamellation, concentric and radial annular tears and an early influx of blood vessels and cells around remodeling lesions but the inner lesion did not heal. Similar features in 6 × 20 mm lesions occurred over a 3–6-month post operative period. MSCs induced a strong recovery in discal pathology with a reduction in cumulative histopathology degeneracy score from 15.2 to 2.7 (p = 0.001) over a three-month recovery period but no recovery in carrier injected discs. PMID:28498326

Study on Mucin in Normal-Appearing Leg Skin.

PubMed

Fernandez-Flores, Angel

2017-03-01

Dermal deposits of mucin in the legs have been described associated with venous insufficiency. However, some degree of stasis dermatitis is generally common in aged individuals. Therefore, some amount of mucin is expected a priori in the reticular dermis of aged patients, even in the absence of clinical lesions. To test this hypothesis, the authors investigated the mucin in the legs of aged individuals without any dermatologic disease. Cutaneous samples were taken from the legs of 15 autopsy cases. A sample of the skin of the legs (either from the left or the right leg without any distinction being made) was randomly taken (without selecting any specific area or attending to macroscopical features). The skin samples were fixed in formaldehyde, and sections obtained from all samples were stained with hematoxylin and eosin, iron, and Alcian blue. Iron deposits were graded as 0/4 in 7 cases, as 1/4 in 4 cases, as 2/4 in 2 cases, and as 4/4 in 2 cases. Cases with greater deposits of iron also had other signs of stasis, such as neovascularization. All the samples scored 0 for dermal mucin deposits in the reticular dermis. The authors conclude that mucin deposits in the legs are not inherent to aging. Therefore, any mucin deposit in the reticular dermis, as well as expansion of the periadnexal dermis by mucin deposits, should be considered abnormal.

Therapeutic effects of full spectrum light on the development of atopic dermatitis-like lesions in NC/Nga mice.

PubMed

Kwon, Tae-Rin; Mun, Seog Kyun; Oh, Chang Taek; Hong, Hyuckki; Choi, Yeon Shik; Kim, Bong-Jun; Kim, Beom Joon

2014-01-01

Full spectrum light (FSL) includes UVA, visible light and infrared light. Many studies have investigated the application of FSL in severe cases of atopic dermatitis (AD) in humans; however, FSL has not yet been studied in an animal model. The purpose of this study was to evaluate the therapeutic effects of FSL on AD-like skin lesions using NC/Nga mice, with the aim of mitigating itching and attenuating the expression of adhesion molecules. We examined the effects of FSL on mite allergen-treated NC/Nga mice by assessing skin symptom severity, ear thickness, serum IgE levels, and the cytokine expression. We examined the histology of lesions using hematoxylin-eosin, toluidine blue and immunohistochemical staining. Our findings suggest that FSL phototherapy exerts positive therapeutic effects on Dermatophagoides farinae (Df)-induced AD-like skin lesions in NC/Nga mice by reducing IgE levels, thus promoting recovery of the skin barrier. The mechanisms by which FSL phototherapy exerts its effects may also involve the inhibition of scratching behavior, reduction of IL-6 levels and reductions in adhesion molecule expression. The present study indicates that FSL phototherapy inhibits the development of AD in NC/Nga mice by suppressing cytokine, chemokine and adhesion molecule expression, and thus, could potentially be useful in treating AD. © 2014 The American Society of Photobiology.

Ex vivo brain tumor analysis using spectroscopic optical coherence tomography

NASA Astrophysics Data System (ADS)

Lenz, Marcel; Krug, Robin; Welp, Hubert; Schmieder, Kirsten; Hofmann, Martin R.

2016-03-01

A big challenge during neurosurgeries is to distinguish between healthy tissue and cancerous tissue, but currently a suitable non-invasive real time imaging modality is not available. Optical Coherence Tomography (OCT) is a potential technique for such a modality. OCT has a penetration depth of 1-2 mm and a resolution of 1-15 μm which is sufficient to illustrate structural differences between healthy tissue and brain tumor. Therefore, we investigated gray and white matter of healthy central nervous system and meningioma samples with a Spectral Domain OCT System (Thorlabs Callisto). Additional OCT images were generated after paraffin embedding and after the samples were cut into 10 μm thin slices for histological investigation with a bright field microscope. All samples were stained with Hematoxylin and Eosin. In all cases B-scans and 3D images were made. Furthermore, a camera image of the investigated area was made by the built-in video camera of our OCT system. For orientation, the backsides of all samples were marked with blue ink. The structural differences between healthy tissue and meningioma samples were most pronounced directly after removal. After paraffin embedding these differences diminished. A correlation between OCT en face images and microscopy images can be seen. In order to increase contrast, post processing algorithms were applied. Hence we employed Spectroscopic OCT, pattern recognition algorithms and machine learning algorithms such as k-means Clustering and Principal Component Analysis.

Preparation of Partial-Thickness Burn Wounds in Rodents Using a New Experimental Burning Device.

PubMed

Sakamoto, Michiharu; Morimoto, Naoki; Ogino, Shuichi; Jinno, Chizuru; Kawaguchi, Atsushi; Kawai, Katsuya; Suzuki, Shigehiko

2016-06-01

The manual application of hot water or hot metal to an animal's skin surface is often used to prepare burn wound models. However, manual burn creation is subject to human variability. We developed a new device that can control the temperature, time, and pressure of contact to produce precise and reproducible animal burn wounds and investigated the conditions required to prepare various burn wounds using our new device. We prepared burn wounds on F344 rats using 3 contact times 2, 4, and 10 seconds using a stamp heated to 80°C. We observed the wound-healing process macroscopically and histologically and evaluated the burn depth using a laser speckle contrast-imaging device, which evaluated the blood flow of the wound. The changes in the burned area over time, tissue perfusion of the burn wounds, histological evaluation of the burn depth by hematoxylin-eosin and azocarmine and aniline blue staining, and the epithelialization rate (the ratio of the epithelialized area to the wound length) were evaluated on histological sections. Results indicated that the burn wounds prepared with contact times of 2, 4, and 10 seconds corresponded to superficial dermal burns, deep dermal burns, and full-thickness burns, respectively. We demonstrated that partial- and full-thickness burn wounds can be precisely and reproducibly created with our new automated burning device.

Role of Mast Cells in Oral Lichen Planus and Oral Lichenoid Reactions.

PubMed

Ramalingam, Suganya; Malathi, Narasimhan; Thamizhchelvan, Harikrishnan; Sangeetha, Narasimhan; Rajan, Sharada T

2018-01-01

Oral lichen planus (OLP) is a chronic T cell mediated disease of oral mucosa, skin, and its appendages with a prevalence of 0.5 to 2.6% worldwide. Oral lichenoid reactions (OLR) are a group of lesions with diverse aetiologies but have clinical and histological features similar to OLP, thereby posing a great challenge in differentiating both lesions. Mast cells are multifunctional immune cells that play a major role in the pathogenesis of lichen planus by release of certain chemical mediators. Increased mast cell densities with significant percentage of degranulation have been observed as a consistent finding in pathogenesis of oral lichen planus. The current study was aimed at quantifying the mast cells in histopathological sections of OLP and OLR thereby aiding a means of distinguishing these lesions. The study group involved 21 cases of oral lichen planus, 21 cases of oral lichenoid reactions, and 10 control specimens of normal buccal mucosa. All the cases were stained with Toluidine Blue and routine haematoxylin and eosin and the mast cells were quantified. The results were analyzed using the Kruskal-Wallis test and an intergroup analysis was performed using Mann-Whitney U test. The number of mast cells showed an increased value in oral lichen planus when compared to oral lichenoid reaction and thus an estimation of mast cells count could aid in distinguishing OLP from OLR histopathologically.

A Histopathological Scheme for the Quantitative Scoring of Intervertebral Disc Degeneration and the Therapeutic Utility of Adult Mesenchymal Stem Cells for Intervertebral Disc Regeneration.

PubMed

Shu, Cindy C; Smith, Margaret M; Smith, Susan M; Dart, Andrew J; Little, Christopher B; Melrose, James

2017-05-12

The purpose of this study was to develop a quantitative histopathological scoring scheme to evaluate disc degeneration and regeneration using an ovine annular lesion model of experimental disc degeneration. Toluidine blue and Haematoxylin and Eosin (H&E) staining were used to evaluate cellular morphology: (i) disc structure/lesion morphology; (ii) proteoglycan depletion; (iii) cellular morphology; (iv) blood vessel in-growth; (v) cell influx into lesion; and (vi) cystic degeneration/chondroid metaplasia. Three study groups were examined: 5 × 5 mm lesion; 6 × 20 mm lesion; and 6 × 20 mm lesion plus mesenchymal stem cell (MSC) treatment. Lumbar intervertebral discs (IVDs) were scored under categories (i-vi) to provide a cumulative score, which underwent statistical analysis using STATA software. Focal proteoglycan depletion was associated with 5 × 5 mm annular rim lesions, bifurcations, annular delamellation, concentric and radial annular tears and an early influx of blood vessels and cells around remodeling lesions but the inner lesion did not heal. Similar features in 6 × 20 mm lesions occurred over a 3-6-month post operative period. MSCs induced a strong recovery in discal pathology with a reduction in cumulative histopathology degeneracy score from 15.2 to 2.7 ( p = 0.001) over a three-month recovery period but no recovery in carrier injected discs.

Acetylated sialic acid residues and blood group antigens localise within the epithelium in microvillous atrophy indicating internal accumulation of the glycocalyx

PubMed Central

Phillips, A D; Brown, A; Hicks, S; Schüller, S; Murch, S H; Walker-Smith, J A; Swallow, D M

2004-01-01

Background: Microvillous atrophy, a disorder of intractable diarrhoea in infancy, is characterised by the intestinal epithelial cell abnormalities of abnormal accumulation of periodic acid-Schiff (PAS) positive secretory granules within the apical cytoplasm and the presence of microvillous inclusions. The identity of the PAS positive material is not known, and the aim of this paper was to further investigate its composition. Methods: Formaldehyde fixed sections were stained with alcian blue/PAS to identify the acidic or neutral nature of the material, phenylhydrazine blocking was employed to stain specifically for sialic acid, and saponification determined the presence of sialic acid acetylation. The specificity of sialic acid staining was tested by digestion with mild sulphuric acid. Expression of blood group related antigens was tested immunochemically. Results: Alcian blue/PAS staining identified a closely apposed layer of acidic material on the otherwise neutral (PAS positive) brush border in controls. In microvillous atrophy, a triple layer was seen with an outer acidic layer, an unstained brush border region, and accumulation within the epithelium of a neutral glycosubstance that contained acetylated sialic acid. Blood group antigens were detected on the brush border, in mucus, and within goblet cells in controls. In microvillous atrophy they were additionally expressed within the apical cytoplasm of epithelial cells mirroring the PAS abnormality. Immuno electron microscopy localised expression to secretory granules. Conclusions: A neutral, blood group antigen positive, glycosubstance that contains acetylated sialic acid accumulates in the epithelium in microvillous atrophy. Previous studies have demonstrated that the direct and indirect constitutive pathways are intact in this disorder and it is speculated that the abnormal staining pattern reflects accumulation of glycocalyx related material. PMID:15542511

Characterisation of myofibroblasts in fibrovascular tissues of primary and recurrent pterygia

PubMed Central

Touhami, A; Di Pascuale, M A; Kawatika, T; Valle, M Del; Rosa, R H; Dubovy, S; Tseng, S C G

2005-01-01

Aims: To determine the presence and origin of myofibroblasts in pterygia. Methods: 86 specimens including head, body, and fibrovascular tissue from 52 primary and 34 recurrent pterygia and five exenterated eyes without pterygia were searched for the origin of myofibroblasts. All tissues were subjected to haematoxylin and eosin staining, immunohistochemistry using antibodies against alpha smooth muscle actin (α-SMA), desmin, vimentin, and caldesmon, and transmission electron microscopy (TEM). The phenotype of fibroblasts subcultured in a serum free medium from pterygium fibrovascular tissues was characterised by the above antibodies. Bundles of dense fibrous tissues were noted in 86% of the fibrovascular tissue specimens evaluated. Cells within these bundles were characterised as myofibroblasts based on positive staining to α-SMA, but negative to desmin and caldesmon, markers for smooth muscle cells. Interestingly, positive α-SMA staining was also found in the periorbital fibroadipose tissue posterior to Tenon’s capsule near the nasal conjunctiva in all exenterated specimens. All first passage fibroblasts expressed vimentin, some were positive to α-SMA, but all were negative to desmin or caldesmon. Cells in pterygium fibrovascular tissues showed ultrastructural features of intracytoplasmic bundles of microfilaments, consistent with myofibroblastic differentiation. Conclusion: These studies collectively demonstrate the presence of contractile myofibroblasts bundle in pterygia and in the periorbital fibroadipose tissue posterior to Tenon’s capsule of exenterated eyes without pterygium. PMID:15722301

THE RELATIVE REACTION WITHIN LIVING MAMMALIAN TISSUES

PubMed Central

Rous, Peyton

1925-01-01

The present paper is the first of a series of reports on the relative reaction of living tissues as determined by vital staining with indicators. It is possible to bring about a localized and a general coloration of living rats and mice with litmus. The animals remain in good health and the coloration of some of the tissues persists for months. Much of the dye is stored in cell granules, especially in those of the reticulo-endothelial elements, but a diffuse staining of certain tissues occurs, notably of bone, epidermis, cartilage, and connective tissue everywhere. In the intensity and localization of the bony coloration litmus has resemblances to madder. Diffuse staining with it renders blue most, if not all, of the tissues affected, while a granular staining causes others to become notably pink, owing to the fact that the indicator, though introduced into the organism in the blue form and circulating as such in the body fluids, is ordinarily red when stored in cells. The polymorphonuclear elements and macrophages of a peritoneal exudate, may become so laden with material colored red by litmus that the blue color of the fluid constituent is masked and the exudate appears a deep, turbid red. The phenomenon is but one manifestation of a notable acidity within cell granules throughout the organism. Like many another in the stained animals it would appear to be of physiological import. Some of the questions suggested by the work will be dealt with in the paper immediately following. PMID:19868995

Method for long-term preservation of thin-layer polyacrylamide gels by producing a gelatine coating.

PubMed

Hofmann, K

1991-02-01

Thin-layer polyacrylamide gels can be preserved and stored for unlimited periods by covering them with a gelatine coating. The method is inexpensive and simple. After air-drying, the gel is immersed in an aqueous 10% solution of highly viscous gelatine between 55 and 60 degrees C. The coated gel is dried by hanging it in air. The method was checked successfully with gels of different thicknesses (0.15-0.50 mm) and after using different staining methods, e.g., with silver, Coomassie Brilliant Blue and pseudoperoxidase.

An easy and inexpensive method for quantitative analysis of endothelial damage by using vital dye staining and Adobe Photoshop software.

PubMed

Saad, Hisham A; Terry, Mark A; Shamie, Neda; Chen, Edwin S; Friend, Daniel F; Holiman, Jeffrey D; Stoeger, Christopher

2008-08-01

We developed a simple, practical, and inexpensive technique to analyze areas of endothelial cell loss and/or damage over the entire corneal area after vital dye staining by using a readily available, off-the-shelf, consumer software program, Adobe Photoshop. The purpose of this article is to convey a method of quantifying areas of cell loss and/or damage. Descemet-stripping automated endothelial keratoplasty corneal transplant surgery was performed by using 5 precut corneas on a human cadaver eye. Corneas were removed and stained with trypan blue and alizarin red S and subsequently photographed. Quantitative assessment of endothelial damage was performed by using Adobe Photoshop 7.0 software. The average difference for cell area damage for analyses performed by 1 observer twice was 1.41%. For analyses performed by 2 observers, the average difference was 1.71%. Three masked observers were 100% successful in matching the randomized stained corneas to their randomized processed Adobe images. Vital dye staining of corneal endothelial cells can be combined with Adobe Photoshop software to yield a quantitative assessment of areas of acute endothelial cell loss and/or damage. This described technique holds promise for a more consistent and accurate method to evaluate the surgical trauma to the endothelial cell layer in laboratory models. This method of quantitative analysis can probably be generalized to any area of research that involves areas that are differentiated by color or contrast.

Characterization of atherosclerosis by histochemical and immunohistochemical methods in African grey parrots (Psittacus erithacus) and Amazon parrots (Amazona spp.).

PubMed

Fricke, Cornelia; Schmidt, Volker; Cramer, Kerstin; Krautwald-Junghanns, Maria-Elisabeth; Dorrestein, Gerry M

2009-09-01

The aim of the study was to characterize atherosclerotic changes in African grey parrots (Psittacus erithacus) and Amazon parrots (Amazona spp.) by histochemical and immunohistochemical methods. Samples of the aorta ascendens and trunci brachiocephalici from 62 African grey parrots and 35 Amazon parrots were stained by hematoxylin and eosin and Elastica van Gieson for grading of atherosclerosis in these birds. Four different stages were differentiated. The incidence of atherosclerosis in the examined parrots was 91.9% in African grey parrots and 91.4% in Amazon parrots. To evaluate the pathogenesis in birds, immunohistochemical methods were performed to demonstrate lymphocytes, macrophages, smooth muscle cells, and chondroitin sulfate. According to the missing lymphocytes and macrophages and the absence of invasion and proliferation of smooth muscle cells in each atherosclerotic stage, "response-to-injury hypothesis" seems inapplicable in parrots. Additionally, we found alterations of vitally important organs (heart, lungs) significantly correlated with atherosclerosis of the aorta ascendens.

Computer-assisted bladder cancer grading: α-shapes for color space decomposition

NASA Astrophysics Data System (ADS)

Niazi, M. K. K.; Parwani, Anil V.; Gurcan, Metin N.

2016-03-01

According to American Cancer Society, around 74,000 new cases of bladder cancer are expected during 2015 in the US. To facilitate the bladder cancer diagnosis, we present an automatic method to differentiate carcinoma in situ (CIS) from normal/reactive cases that will work on hematoxylin and eosin (H and E) stained images of bladder. The method automatically determines the color deconvolution matrix by utilizing the α-shapes of the color distribution in the RGB color space. Then, variations in the boundary of transitional epithelium are quantified, and sizes of nuclei in the transitional epithelium are measured. We also approximate the "nuclear to cytoplasmic ratio" by computing the ratio of the average shortest distance between transitional epithelium and nuclei to average nuclei size. Nuclei homogeneity is measured by computing the kurtosis of the nuclei size histogram. The results show that 30 out of 34 (88.2%) images were correctly classified by the proposed method, indicating that these novel features are viable markers to differentiate CIS from normal/reactive bladder.

Mucosal barrier in ulcerative colitis and Crohn's disease.

PubMed

Dorofeyev, A E; Vasilenko, I V; Rassokhina, O A; Kondratiuk, R B

2013-01-01

Background. The mucus layer in the gastrointestinal tract plays important role in host innate defense, regulation of secretion, and absorption processes, maintaining colonization resistance, which composes the integrity of protective mucus barrier in the large intestine. Investigations of mucin expression in the colon mucosa can improve the understanding of protective function of mucosal barrier in ulcerative colitis (UC) and Crohn's disease (CD). Materials and Methods. 77 patients with UC and CD were examined. Histological analysis of colon mucosa was done by standard method (haematoxylin-eosin, alcian blue at pH 1.0 and 2.5 to determine sulfated and nonsulfated glycosaminoglycans and glycoproteins, and goblet cells). To characterize the mucus production the PAS-reaction was performed. Immunohistochemistry was performed using monoclonal mouse antibodies raised against MUC2, MUC3, MUC4, and TFF3 (USBiological, USA). Results. The moderate expression of MUC2 and MUC3 (50.0% and 32.1%, P = 0.03) and high expression of MUC4 and TFF3 in the colon mucosa were observed in all patients with CD. The intensive labeling of MUC4 and TFF3 occurred more often (42.9% and 57.1%, P = 0.03) in patients with CD. The level of expression of secretory MUC2 and transmembrane MUC3 and MUC4 in all patients with UC was low, up to its complete absence (59.2% and 53.1% cases, P = 0.05). TFF3 expression had high and medium staining intensity in patients with UC. Conclusions. Different types of mucins synthesis, secretion, and expression were found in patients with UC and CD. The expression of mucin MUC2, MUC3, MUC4, and TFF3 correlated with the activity of disease and the extent of the inflammatory process in the large intestine. The most pronounced alteration of mucins expression was observed in patients with severe UC and CD.

Optimization of a Histopathological Biomarker for Sphingomyelin Accumulation in Acid Sphingomyelinase Deficiency

PubMed Central

Johnson, Jennifer; Maloney, Colleen L.; Yandl, Emily; Griffiths, Denise; Thurberg, Beth L.; Ryan, Susan

2012-01-01

Niemann-Pick disease (types A and B), or acid sphingomyelinase deficiency, is an inherited deficiency of acid sphingomyelinase, resulting in intralysosomal accumulation of sphingomyelin in cells throughout the body, particularly within those of the reticuloendothelial system. These cellular changes result in hepatosplenomegaly and pulmonary infiltrates in humans. A knockout mouse model mimics many elements of human ASMD and is useful for studying disease histopathology. However, traditional formalin-fixation and paraffin embedding of ASMD tissues dissolves sphingomyelin, resulting in tissues with a foamy cell appearance, making quantitative analysis of the substrate difficult. To optimize substrate fixation and staining, a modified osmium tetroxide and potassium dichromate postfixation method was developed to preserve sphingomyelin in epon-araldite embedded tissue and pulmonary cytology specimens. After processing, semi-thin sections were incubated with tannic acid solution followed by staining with toluidine blue/borax. This modified method provides excellent preservation and staining contrast of sphingomyelin with other cell structures. The resulting high-resolution light microscopy sections permit digital quantification of sphingomyelin in light microscopic fields. A lysenin affinity stain for sphingomyelin was also developed for use on these semi-thin epon sections. Finally, ultrathin serial sections can be cut from these same tissue blocks and stained for ultrastructural examination by electron microscopy. PMID:22614361

Continuous 900-megahertz electromagnetic field applied in middle and late-adolescence causes qualitative and quantitative changes in the ovarian morphology, tissue and blood biochemistry of the rat.

PubMed

Okatan, Derya Öztürk; Kaya, Haydar; Aliyazıcıoğlu, Yüksel; Demir, Selim; Çolakoğlu, Serdar; Odacı, Ersan

2018-02-01

The purpose of this study was to use histological and biochemical methods in order to evaluate changes taking place in the ovarian of rats exposed to the effect of a 900-megahertz (MHz) electromagnetic field (EMF) in middle and late adolescence. Twenty-four 34-d-old female Sprague-Dawley rats were assigned equally to control, sham and EMF groups. EMF group rats were exposed to the effect of a 900-MHz EMF for 1 h a day, at the same time every day between postnatal days 35 and 59, while inside an EMF cage. Sham group rats were kept inside the EMF cage for the same time between postnatal days 35 and 59 without being exposed to any EMF effect. At the end of the study, rats' ovarian were removed and blood specimens were taken. Right ovarium tissues were subjected to routine histological procedures and stained with hematoxylin and eosin, periodic acid shift and Masson's trichrome. Follicles were counted in ovarian sections stained with hematoxylin and eosin. The TUNEL method was used to evaluate apoptosis. Left ovarian tissue and blood specimens were investigated biochemically. Histopathological examination of EMF group ovarian tissue revealed thinning in the zona granulosa and theca layers, shrinking in granulosa cells, reduced mitotic activity and leukocyte infiltration in the follicles and stroma. Secondary follicle numbers in the EMF group were significantly lower than in the other groups. In terms of biochemistry, EMF and sham group superoxide dismutase, catalase and anti-Mullerian hormone levels and EMF group 3-nitrotyrosine values increased significantly compared to the control group. EMF and sham group serum catalase and 8-hydroxy-deoxiguanosine values increased significantly compared to the control group, and EMF group total oxidant status and oxidative stress index values were significantly higher compared to the sham and control groups. A total of 900-MHz EMF applied in middle and late adolescence may cause changes in the morphology and biochemistry of the rat ovarium.

Destructive effect of HIFU on rabbit embedded endometrial carcinoma tissues and their vascularities

PubMed Central

Guan, Liming; Xu, Gang

2017-01-01

Objectives To evaluate damage effect of High-intensity focused ultrasound on early stage endometrial cancer tissues and their vascularities. Materials and Methods Rabbit endometrial cancer models were established via tumor blocks implantation for a prospective control study. Ultrasonic ablation efficacy was evaluated by pathologic and imaging changes. The target lesions of experimental rabbits before and after ultrasonic ablation were observed after autopsy. The slides were used for hematoxylin-eosin staining, elastic fiber staining and endothelial cell staining; the slides were observed by optical microscopy. One slide was observed by electron microscopy. Then the target lesions of experimental animals with ultrasonic ablation were observed by vascular imaging, one group was visualized by digital subtract angiography, one group was quantified by color Doppler flow imaging, and one group was detected by dye perfusion. SPSS 19.0 software was used for statistical analyses. Results Histological examination indicated that High-intensity focused ultrasound caused the tumor tissues and their vascularities coagulative necrosis. Tumor vascular structure components including elastic fiber, endothelial cells all were destroyed by ultrasonic ablation. Digital subtract angiography showed tumor vascular shadow were dismissed after ultrasonic ablation. After ultrasonic ablation, gray-scale of tumor nodules enhanced in ultrasonography, tumor peripheral and internal blood flow signals disappeared or significantly reduced in color Doppler flow imaging. Vascular perfusion performed after ultrasonic ablation, tumor vessels could not filled by dye liquid. Conclusion High-intensity focused ultrasound as a noninvasive method can destroy whole endometrial cancer cells and their supplying vascularities, which maybe an alternative approach of targeted therapy and new antiangiogenic strategy for endometrial cancer. PMID:28121624

A Tissue-Specific Approach to the Analysis of Metabolic Changes in Caenorhabditis elegans

PubMed Central

Pujol, Claire; Ipsen, Sabine; Brodesser, Susanne; Mourier, Arnaud; Tolnay, Markus; Frank, Stephan; Trifunović, Aleksandra

2011-01-01

The majority of metabolic principles are evolutionarily conserved from nematodes to humans. Caenorhabditis elegans has widely accelerated the discovery of new genes important to maintain organismic metabolic homeostasis. Various methods exist to assess the metabolic state in worms, yet they often require large animal numbers and tend to be performed as bulk analyses of whole worm homogenates, thereby largely precluding a detailed studies of metabolic changes in specific worm tissues. Here, we have adapted well-established histochemical methods for the use on C. elegans fresh frozen sections and demonstrate their validity for analyses of morphological and metabolic changes on tissue level in wild type and various mutant strains. We show how the worm presents on hematoxylin and eosin (H&E) stained sections and demonstrate their usefulness in monitoring and the identification of morphological abnormalities. In addition, we demonstrate how Oil-Red-O staining on frozen worm cross-sections permits quantification of lipid storage, avoiding the artifact-prone fixation and permeabilization procedures of traditional whole-mount protocols. We also adjusted standard enzymatic stains for respiratory chain subunits (NADH, SDH, and COX) to monitor metabolic states of various C. elegans tissues. In summary, the protocols presented here provide technical guidance to obtain robust, reproducible and quantifiable tissue-specific data on worm morphology as well as carbohydrate, lipid and mitochondrial energy metabolism that cannot be obtained through traditional biochemical bulk analyses of worm homogenates. Furthermore, analysis of worm cross-sections overcomes the common problem with quantification in three-dimensional whole-mount specimens. PMID:22162770

Diagnosis of breast cancer biopsies using quantitative phase imaging

NASA Astrophysics Data System (ADS)

Majeed, Hassaan; Kandel, Mikhail E.; Han, Kevin; Luo, Zelun; Macias, Virgilia; Tangella, Krishnarao; Balla, Andre; Popescu, Gabriel

2015-03-01

The standard practice in the histopathology of breast cancers is to examine a hematoxylin and eosin (H&E) stained tissue biopsy under a microscope. The pathologist looks at certain morphological features, visible under the stain, to diagnose whether a tumor is benign or malignant. This determination is made based on qualitative inspection making it subject to investigator bias. Furthermore, since this method requires a microscopic examination by the pathologist it suffers from low throughput. A quantitative, label-free and high throughput method for detection of these morphological features from images of tissue biopsies is, hence, highly desirable as it would assist the pathologist in making a quicker and more accurate diagnosis of cancers. We present here preliminary results showing the potential of using quantitative phase imaging for breast cancer screening and help with differential diagnosis. We generated optical path length maps of unstained breast tissue biopsies using Spatial Light Interference Microscopy (SLIM). As a first step towards diagnosis based on quantitative phase imaging, we carried out a qualitative evaluation of the imaging resolution and contrast of our label-free phase images. These images were shown to two pathologists who marked the tumors present in tissue as either benign or malignant. This diagnosis was then compared against the diagnosis of the two pathologists on H&E stained tissue images and the number of agreements were counted. In our experiment, the agreement between SLIM and H&E based diagnosis was measured to be 88%. Our preliminary results demonstrate the potential and promise of SLIM for a push in the future towards quantitative, label-free and high throughput diagnosis.

Fluoro jade-C staining in the assessment of brain injury after deep hypothermia circulatory arrest.

PubMed

Wang, Ren; Ma, Wei-Guo; Gao, Guo-Dong; Mao, Qun-Xia; Zheng, Jun; Sun, Li-Zhong; Liu, Ying-Long

2011-02-04

To evaluate the efficacy of Fluoro Jade-C staining (FJC) in the assessment of brain injury after deep hypothermia circulatory arrest (DHCA). Six healthy adult miniature male pigs underwent DHCA, the rectal temperature was down to 18°C, circulation was stopped , circulatory arrest was maintained for 60 minutes. On postoperative day 1, perfusion-fixation was performed on brain tissue. Cerebral cortex, hippocampus, cerebellum were taken for sampling. FJC, hematoxylin-eosin staining (HE), nissl staining (NISSL), terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) were performed to detect the histological and pathological changes. Histological scores of all slices were ranked. Comparison between the FJC and other techniques was done by analysis of variance (ANOVA) according to histological scores. All animals survived the operation. On the cerebral cortex, in comparison of FJC between HE, NISSL and TUNEL, the p value was 0.90, 0.40, 0.16 respectively (p>0.05). On the hippocampus, the comparison of FJC with HE, NISSL and TUNEL had a p value of 0.12, 0.23, 0.62 respectively (p>0.05). On the cerebellum, in comparing FJC with HE, NISSL and TUNEL, the p value was 0.96, 0.77, 0.96 respectively (p>0.05). On representative regions, the results of FJC were in accordance with that of TUNEL, NISSL and HE. Furthermore, ascertainment of brain injury is easier with FJC. FJC is a reliable and convenient method to assess brain injury after DHCA. Copyright © 2010 Elsevier B.V. All rights reserved.

Automated robust registration of grossly misregistered whole-slide images with varying stains

NASA Astrophysics Data System (ADS)

Litjens, G.; Safferling, K.; Grabe, N.

2016-03-01

Cancer diagnosis and pharmaceutical research increasingly depend on the accurate quantification of cancer biomarkers. Identification of biomarkers is usually performed through immunohistochemical staining of cancer sections on glass slides. However, combination of multiple biomarkers from a wide variety of immunohistochemically stained slides is a tedious process in traditional histopathology due to the switching of glass slides and re-identification of regions of interest by pathologists. Digital pathology now allows us to apply image registration algorithms to digitized whole-slides to align the differing immunohistochemical stains automatically. However, registration algorithms need to be robust to changes in color due to differing stains and severe changes in tissue content between slides. In this work we developed a robust registration methodology to allow for fast coarse alignment of multiple immunohistochemical stains to the base hematyoxylin and eosin stained image. We applied HSD color model conversion to obtain a less stain color dependent representation of the whole-slide images. Subsequently, optical density thresholding and connected component analysis were used to identify the relevant regions for registration. Template matching using normalized mutual information was applied to provide initial translation and rotation parameters, after which a cost function-driven affine registration was performed. The algorithm was validated using 40 slides from 10 prostate cancer patients, with landmark registration error as a metric. Median landmark registration error was around 180 microns, which indicates performance is adequate for practical application. None of the registrations failed, indicating the robustness of the algorithm.

Alpha2-Adrenergic Receptors and Breast Tumor Stroma: A Novel Pathway Driving Breast Cancer Growth and Metastasis

DTIC Science & Technology

2015-10-01

sectioned into 5-micron sections. Three serial sections were mounted onto each slide and stained using hematoxylin and eosin (H&E). Five sets of... serial sections were taken from each lung, 100 µm distance between each set. This spacing allows surveillance of metastatic lesions throughout the lung...functions that promote tumor growth may be altered by DEX treatment. For example, IFN- is associated with effector function of natural killer cells

Modulation of Stem Cell Differentiation and Myostatin as an Approach to Counteract Fibrosis in Muscle Dystrophy and Regeneration After Injury. Addendum

DTIC Science & Technology

2012-03-01

considerable increase in central nuclei in the regenerating myofibers, and molsidomine supplementation appears to have upregulated the overall stem cell...the increase of central nuclei as indicator of muscle repair observed in the SC group in comparison to the UT group, in frozen tissue sections...treatments on myofiber repair will be better defined by the counting of central nuclei on hematoxylin/eosin stained frozen sections as in Fig 3, and the

Quantitative immunohistochemistry of factor VIII-related antigen in breast carcinoma: a comparison of computer-assisted image analysis with established counting methods.

PubMed

Kohlberger, P D; Obermair, A; Sliutz, G; Heinzl, H; Koelbl, H; Breitenecker, G; Gitsch, G; Kainz, C

1996-06-01

Microvessel density in the area of the most intense neovascularization in invasive breast carcinoma is reported to be an independent prognostic factor. The established method of enumeration of microvessel density is to count the vessels using an ocular raster (counted microvessel density [CMVD]). The vessels were detected by staining endothelial cells using Factor VIII-related antigen. The aim of the study was to compare the CMVD results with the percentage of factor VIII-related antigen-stained area using computer-assisted image analysis. A true color red-green-blue (RGB) image analyzer based on a morphologically reduced instruction set computer processor was used to evaluate the area of stained endothelial cells. Sixty invasive breast carcinomas were included in the analysis. There was no significant correlation between the CMVD and the percentage of factor VIII-related antigen-stained area (Spearman correlation coefficient = 0.24, confidence interval = 0.02-0.46). Although high CMVD was significantly correlated with poorer recurrence free survival (P = .024), percentage of factor VIII-related antigen-stained area showed no prognostic value. Counted microvessel density and percentage of factor VIII-related antigen-stained area showed a highly significant correlation with vessel invasion (P = .0001 and P = .02, respectively). There was no correlation between CMVD and percentage of factor VIII-related antigen-stained area with other prognostic factors. In contrast to the CMVD within malignant tissue, the percentage of factor VIII-related antigen-stained area is not suitable as an indicator of prognosis in breast cancer patients.

Modified use of methylene blue in the tissue compression technique to detect sarcocysts in meat-producing animals.

PubMed

Ng, Yit Han; Subramaniam, Vellayan; Lau, Yee Ling

2015-11-30

Sarcocystosis in meat-producing animals is a major cause of reduced productivity in many countries, especially those that rely on agriculture. Although several diagnostic methods are available to detect sarcocystosis, many are too time-consuming for routine use in abattoirs and meat inspection centers, where large numbers of samples need to be tested. This study aimed to compare the sensitivity of the methylene blue tissue preparation, unstained tissue preparation and nested PCR in the detection of sarcocysts in tissue samples. Approximately three-fold more sarcocysts were detected in methylene blue-stained tissue compared to unstained controls (McNemar's test: P<0.01). Test sensitivity was comparable to that of the gold standard for sarcocyst detection, nested polymerase chain reaction. These results suggest that methylene blue can be used in tissue compression as a rapid, safe, and inexpensive technique for the detection of ruminant sarcocystosis in abattoirs. Copyright © 2015 Elsevier B.V. All rights reserved.

The effects of intradermal and topical mitomycin C on wound healing.

PubMed

Porter, Glen T; Gadre, Swarupa A; Calhoun, Karen H

2006-07-01

To determine the effect of intradermal and topical mitomycin C (MMC) on skin wound healing. A prospective, controlled study in a rat wound model performed in an academic medical center. Intradermal and topical MMC application decreased wound integrity when compared with saline-treated animals at 1 week, 2 weeks, 1 month, and 6 months. Skin necrosis occurred in animals that received intradermal MMC. Hemotoxylin and eosin and immunohistochemical staining showed no consistent difference between treatment arms. Fibrosis and collagen deposition were reduced in MMC-treated wounds on trichrome staining. MMC-treated wounds showed decreased wound strength compared with controls. Intradermal MMC can cause skin necrosis. Histologic findings did not always correspond with clinical data. The data suggest cautious use of MMC in clinical situations when wound breaking strength is critical.

Expression of GAT1 in male reproductive system and its effects on reproduction in mice.

PubMed

Zhang, JinFu; Gui, YaPing; Yuan, Tao; Bian, CuiDong; Guo, LiHe

2009-12-01

The present study was carried out to identify GABA (gamma-aminobutyric acid) transport protein I (GAT1) in male reproductive organs and to study the effect of GAT1 overexpression on the male reproductive system in GAT1 transgenic mice (TG). Expression and location of GAT1 in testes, epididymis, and sperm of wild-type (WT) mice were identified by immunohistochemistry and western-blot. Histological changes of testes, epididymis, and sperm of transgenic mice overexpressing GAT1 were detected by immunofluorenscent staining and haematoxylin and eosin (HE) staining. GAT1 expression was detected in the testes, epididymis, and sperm of non-transgenic mice. Vacuolization and deformity of spermatogenic cells were observed in the transgenic mice, but the epididymis was unremarkable. Immunofluorenscent staining showed that the number of diastrophic and decapitated sperm increased significantly in transgenic mice to 46.9% from 7.3% in nontransgenic mice. These results suggest that abnormal expression of GAT1 could result in spermiogenesis function injury, sperm paramorphia and dysgenesis.

Breast cancer metastasizing to the stomach mimicking primary gastric cancer: A case report

PubMed Central

Yim, Kwangil; Ro, Sang Mi; Lee, Jieun

2017-01-01

Breast cancer with stomach metastasis rare with an incidence of 1% or less among metastatic breast cancer patients. We experienced a case of breast cancer metastasizing to the stomach in 65-year-old female patient. She experienced dyspepsia and poor oral intake before visiting the clinic. Diffuse infiltration with nodular mucosal thickening of the stomach wall was observed, suggesting advanced gastric cancer based on gross endoscopic finding. Spread of poorly cohesive tumor cells in the gastric mucosa observed upon hematoxylin and eosin stain resembled signet ring cell carcinoma, but diffuse positive staining for GATA3 in immunohistochemical stain allowed for a conclusive diagnosis of breast cancer metastasizing to the stomach. Based on the final diagnosis, systemic chemotherapy was administered instead of primary surgical resection. After 2 cycles of docetaxel administration, she showed a partial response based on abdominal computed tomography scan. This case is an unusual presentation of breast cancer metastasizing to the gastrointestinal tract. PMID:28405154

Width of thermal damage after using the YAG contact laser for cutting biological tissue: animal experimental investigation.

PubMed

Mecke, H; Schünke, M; Schnaidt, S; Freys, I; Semm, K

1991-01-01

At the University Women's Clinic in Kiel, the YAG contact laser has been used as a cutting instrument in pelviscopic operations since 1987. When the laser cuts, it produces only a scant amount of mechanical trauma. The determining factor is the amount of thermal damage produced along the wound margins and in direct neighboring tissue. The extent of the tissue change seen in the uterus and liver parenchyma of rats and the striated muscle of rabbits after application of the YAG contact laser was demonstrated using various staining techniques and stains. Liver parenchyma proved to be the most sensitive to thermal damage. In the uterine horn, enzyme-histochemical ATPase and alkaline phosphatase demonstrations showed a significantly wider zone of thermal damage after laser incision than did hematoxylin-eosin and Goldner staining techniques. A good understanding of the extent of thermal damage is essential for atraumatic pelviscopic operations using the YAG contact laser and also for the preventing of complications.

Breast cancer metastasizing to the stomach mimicking primary gastric cancer: A case report.

PubMed

Yim, Kwangil; Ro, Sang Mi; Lee, Jieun

2017-03-28

Breast cancer with stomach metastasis rare with an incidence of 1% or less among metastatic breast cancer patients. We experienced a case of breast cancer metastasizing to the stomach in 65-year-old female patient. She experienced dyspepsia and poor oral intake before visiting the clinic. Diffuse infiltration with nodular mucosal thickening of the stomach wall was observed, suggesting advanced gastric cancer based on gross endoscopic finding. Spread of poorly cohesive tumor cells in the gastric mucosa observed upon hematoxylin and eosin stain resembled signet ring cell carcinoma, but diffuse positive staining for GATA3 in immunohistochemical stain allowed for a conclusive diagnosis of breast cancer metastasizing to the stomach. Based on the final diagnosis, systemic chemotherapy was administered instead of primary surgical resection. After 2 cycles of docetaxel administration, she showed a partial response based on abdominal computed tomography scan. This case is an unusual presentation of breast cancer metastasizing to the gastrointestinal tract.

Altering the axial light gradient affects photomorphogenesis in emerging seedlings of Zea mays L

NASA Technical Reports Server (NTRS)

Parks, B. M.; Poff, K. L.

1986-01-01

The axial (longitudinal) red light gradient (632 nanometers) of 4 day old dark-grown maize seedlings is increased by staining the peripheral cells of the coleoptile. The magnitude of increase in the light gradient is dependent solely on the light-absorbing qualities of the stain used. Metanil yellow has no effect on the axial red-light gradient, while methylene blue causes a large increase in this light gradient. These stains did not affect growth in darkness or the sensitivity of mesocotyl elongation to red light. However, mesocotyl elongation was altered for the dark-grown seedlings stained with methylene blue when these seedlings were transplanted, covered with soil, and permitted to emerge under natural lighting conditions. These observations are consistent with the idea that there is a single perceptive site below the coleoptilar node, and suggest that this perceptive site gives the actinic light which has traveled downward through the length of the shoot from an entry point in the plant tip region.

Effect of autologous platelet-rich plasma on the chondrogenic differentiation of rabbit adipose-derived stem cells in vitro

PubMed Central

TANG, XIAO-BO; DONG, PEI-LONG; WANG, JIAN; ZHOU, HAI-YANG; ZHANG, HAI-XIANG; WANG, SHAN-ZHENG

2015-01-01

This study aimed to isolate rabbit adipose-derived stem cells (ADSCs) and explore the potential of platelet-rich plasma (PRP) in the chondrogenic differentiation of ADSCs, thereby potentially providing a new approach for the repair and regeneration of cartilage injury. Rabbit ADSCs were isolated and characterized by induction towards adipogenic, osteogenic and chondrogenic lineages in vitro. The isolated ADSCs were also cultured with or without 10% PRP. Immunofluorescence staining, toluidine blue staining and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) were used to detect type II collagen (Col II) and aggrecan (AGC) expression. Col II immunofluorescence staining and toluidine blue staining indicated that following induction by autologous PRP, ADSCs manifested Col II and AGC expression. The expression of Col II and AGC mRNA was significantly upregulated in the PRP-treated cells when compared with that in control cells. Autologous PRP produced by laboratory centrifugation was able to promote the chondrogenic differentiation of rabbit ADSCs in vitro. PMID:26622340

Comparative study of efficacy, rapidity of detection, and cost-effectiveness of potassium hydroxide, calcofluor white, and Chicago sky blue stains in the diagnosis of dermatophytoses.

PubMed

Prakash, R; Prashanth, Hosakere Veerappa; Ragunatha, Shivanna; Kapoor, Meenakshi; Anitha, T K; Krishnamurthy, Veena

2016-04-01

The diagnosis of superficial mycosis such as dermatophytosis is often done clinically. However, in difficult cases, a rapid test with high sensitivity and specificity helps in the immediate confirmation and administration of treatment. The efficacy, rapidity of detection, and cost-effectiveness of KOH preparation, calcofluor white (CW) stain, and Chicago sky blue (CSB) stain in the identification of fungal elements were assessed in patients with dermatophytoses attending the dermatology clinic of a tertiary care hospital. All three tests were performed on each sample collected from 73 patients according to standard procedure. The slides were examined after 5 and 30 minutes in × 10 and × 40 magnifications. The sensitivity and specificity of CW and CSB at 5 and 30 minutes were calculated using KOH preparation as the standard test. CSB stain showed highest positivity (94.5%) within 5 minutes when compared to KOH (75.3%) and CW (83.5%). After 30 minutes, positivity increased in KOH (84.9%) and CW stains (89%), but it remained the same in CSB stain. Both CW and CSB stains when compared to 10% KOH are equally sensitive (100%), but CW was more specific (72.7%), particularly at 30 minutes. When cost of performing tests on 100 specimens is considered, KOH, CW, and CSB stains cost Rs 5, 100, and 15, respectively. CSB stain is a better stain for rapid diagnosis of dermatophytoses because of ease of performance, rapidity of detection, better appreciation of morphology of fungal elements, and cost effectiveness. © 2016 The International Society of Dermatology.

Comparison of three protein extraction procedures from toxic and non-toxic dinoflagellates for proteomics analysis.

PubMed

Jiang, Xi-Wen; Wang, Jing; Chan, Leo Lai; Lam, Paul Kwan Sing; Gu, Ji-Dong

2015-08-01

Three methods for extraction and preparation of high-quality proteins from both toxic and non-toxic dinoflagellates for proteomics analysis, including Trizol method, Lysis method and Tris method, were compared with the subsequent protein separation profiles using 2-D differential gel electrophoresis (2-D DIGE), Coomassie Blue and silver staining. These methods showed suitability for proteins with different pIs and molecular weights. Tris method was better for low molecular weight and low pI protein isolation; whereas both Lysis and Trizol method were better for high-molecular weight and high pI protein purification. Trizol method showed good results with Alexandrium species and Gynodinium species, and the background in gel was much clearer than the other two methods. At the same time, only Lysis method caused breaking down of the target proteins. On the other hand, Trizol method obtained higher concentration of ribulose-1,5-bisphosphate carboxylase/oxygenase proteins by Western-blotting, while Tris method was the best for peridinin-chlorophyll-protein complexes protein and T1 protein preparation. DIGE was better than Coomassie Blue and silver staining, except for some limitations, such as the high cost of the dyes, relatively short shelf life and the requirements for extensive and special image capturing equipment. Some proteins related to PSTs synthesis in dinoflagellates are hydrophobic with high molecular weight or binding on membranes and Trizol method performed better than Tris method for these proteins. The Trizol method and 2-D DIGE were effective combination for proteomics investigations of dinoflagellates. This procedure allows reliable and high recovery efficiency of proteins from dinoflagellates for better understanding on their occurrence and toxin-production for physiological and biochemical information.

Breast cancer diagnosis using spatial light interference microscopy

NASA Astrophysics Data System (ADS)

Majeed, Hassaan; Kandel, Mikhail E.; Han, Kevin; Luo, Zelun; Macias, Virgilia; Tangella, Krishnarao; Balla, Andre; Popescu, Gabriel

2015-11-01

The standard practice in histopathology of breast cancers is to examine a hematoxylin and eosin (H&E) stained tissue biopsy under a microscope to diagnose whether a lesion is benign or malignant. This determination is made based on a manual, qualitative inspection, making it subject to investigator bias and resulting in low throughput. Hence, a quantitative, label-free, and high-throughput diagnosis method is highly desirable. We present here preliminary results showing the potential of quantitative phase imaging for breast cancer screening and help with differential diagnosis. We generated phase maps of unstained breast tissue biopsies using spatial light interference microscopy (SLIM). As a first step toward quantitative diagnosis based on SLIM, we carried out a qualitative evaluation of our label-free images. These images were shown to two pathologists who classified each case as either benign or malignant. This diagnosis was then compared against the diagnosis of the two pathologists on corresponding H&E stained tissue images and the number of agreements were counted. The agreement between SLIM and H&E based diagnosis was 88% for the first pathologist and 87% for the second. Our results demonstrate the potential and promise of SLIM for quantitative, label-free, and high-throughput diagnosis.

Histological Evaluation of Selected Organs of the Eurasian Beavers (Castor fiber) Inhabiting Poland.

PubMed

Dolka, I; Giżejewska, A; Giżejewski, Z; Kluciński, W; Kołodziejska, J

2015-10-01

There is a general scarcity of data on the histological structure of major organs in the Eurasian beaver (Castor fiber). This study presents the histological characteristics of beaver organs such as the liver, spleen, cardiac muscle, lungs and kidneys. Tissue samples were collected from 21 beavers and analysed. Selected samples of tail tissue were additionally examined. Tissue samples were placed in neutral buffered formalin and embedded in paraffin. 4-μm-thick sections were stained with haematoxylin and eosin, and other staining techniques were also used. Scant amounts of inter-lobular connective tissue were found in the liver. Ion or copper deposition was not observed, but scattered cytoplasmic glycogen deposits were present in hepatocytes. Our results suggest that beavers have defensive rather than storage spleens. Interestingly, the presence of melanin in splenic red pulp was noted. The histological structure of the examined organs closely resembled that of other rodent species. According to our knowledge, this is the first study evaluating the histological structure of beaver organs. Nevertheless, precise characterization of the evaluated organs requires further work with the involvement of accurate and reliable techniques, such as molecular biology or electron microscopy methods. © 2014 Blackwell Verlag GmbH.

Nasopharyngeal carcinoma heterogeneity of DNA content identified on cytologic preparations.

PubMed

Maohuai, C; Chang, A R; Lo, D

2001-06-01

To evaluate tumor heterogeneity of DNA content in nasopharyngeal carcinoma (NPC) performed on cytologic specimens. Image cytometric analysis of DNA ploidy status of 40 NPCs was performed on nasopharyngeal brushing smears stained with the Feulgen method after hematoxylin eosin staining. If the DNA distribution pattern from the same tumor exhibited diploid, aneuploid or/and tetraploid peaks or some combination of these patterns, the presence of tumor heterogeneity of DNA content was identified. Thirty-four cases (85%) had a nondiploid DNA pattern among the 40 NPCs. Twenty-eight cases exhibited tumor heterogeneity of DNA content (70%). Of the 28 tumors, 13 (46%) had a combination of diploid and tetraploid patterns, 10 (37%) had a combination of diploid and aneuploid patterns, 3 cases (11%) had a combination of tetraploid and aneuploid patterns, and 2 cases had two aneuploid stem lines. The relationship between DNA ploidy pattern and tumor histologic and cytologic morphology was also examined. There is a high incidence of DNA content heterogeneity in NPC. The relevance of tumor heterogeneity to the biologic behavior of NPC awaits further study. DNA quantification with image cytometry on destained cytologic preparations is feasible and reliable.

[Reproduction of the fish Brotula clarkae (Pisces: Ophidiidae) in the Colombian Pacific].

PubMed

Acevedo, Jenny; Angulo, Wilberto; Ramírez, Manuel; Zapata, Luis A

2007-01-01

We studied the reproductive ecology of the fish Brotula clarkae based on 754 individuals obtained from the industrial and artisanal fisheries in the Colombian Pacific (March 1994 to December 1996). Histological sections (paraplast embedding and Harris's hematoxylin-eosin stain) were made from 151 females (26-100 cm total length). Additionally, mean diameter and particular features are described for oocyte phases and ovarian development stages. The mean size at initial sexual maturity of females was determined with two methods (graphic and statistic) in 61.3 cm and 62.3 cm respectively. The general sex ratio was 1:1. There are several spawn pulses between May and October. The mean fecundity by spawn pulse was 1,005,657 eggs. We recommend a fishing ban during the reproduction period and a minimal catch size above 62.3 cm.

A magnetic induction heating system with multi-cascaded coils and adjustable magnetic circuit for hyperthermia.

PubMed

Huang, Chi-Fang; Chao, Hsuan-Yi; Chang, Hsun-Hao; Lin, Xi-Zhang

2016-01-01

Based on the characteristics of cancer cells that cannot survive in an environment with temperature over 42 °C, a magnetic induction heating system for cancer treatment is developed in this work. First, the methods and analyses for designing the multi-cascaded coils magnetic induction hyperthermia system are proposed, such as internal impedance measurement of power generator, impedance matching of coils, and analysis of the system. Besides, characteristics of the system are simulated by a full-wave package for engineering optimization. Furthermore, by considering the safety factor of patients, a two-sectional needle is designed for hyperthermia. Finally, this system is employed to test the liver of swine in ex-vivo experiments, and through Hematoxylin and Eosin (H&E) stain and NADPH oxidase activity assay, the feasibility of this system is verified.

Characterization of the Tumor Secretome from Tumor Interstitial Fluid (TIF).

PubMed

Gromov, Pavel; Gromova, Irina

2016-01-01

Tumor interstitial fluid (TIF) surrounds and perfuses bodily tumorigenic tissues and cells, and can accumulate by-products of tumors and stromal cells in a relatively local space. Interstitial fluid offers several important advantages for biomarker and therapeutic target discovery, especially for cancer. Here, we describe the most currently accepted method for recovering TIF from tumor and nonmalignant tissues that was initially performed using breast cancer tissue. TIF recovery is achieved by passive extraction of fluid from small, surgically dissected tissue specimens in phosphate-buffered saline. We also present protocols for hematoxylin and eosin (H&E) staining of snap-frozen and formalin-fixed, paraffin-embedded (FFPE) tumor sections and for proteomic profiling of TIF and matched tumor samples by high-resolution two-dimensional gel electrophoresis (2D-PAGE) to enable comparative analysis of tumor secretome and paired tumor tissue.

Cell-block procedure in endoscopic ultrasound-guided-fine-needle-aspiration of gastrointestinal solid neoplastic lesions

PubMed Central

Ieni, Antonio; Barresi, Valeria; Todaro, Paolo; Caruso, Rosario Alberto; Tuccari, Giovanni

2015-01-01

In the present review we have analyzed the clinical applications of endoscopic ultrasound-guided-fine-needle-aspiration (EUS-FNA) and the methodological aspects obtained by cell-block procedure (CBP) in the diagnostic approach to the gastrointestinal neoplastic pathology. CBP showed numerous advantages in comparison to the cytologic routine smears; in particular, better preservation of cell architecture, achievement of routine haematoxylin-eosin staining equivalent to histological slides and possibility to perform immunohistochemistry or molecular analyses represented the most evident reasons to choose this method. Moreover, by this approach, the differential diagnosis of solid gastrointestinal neoplasias may be more easily achieved and the background of contaminant non-neoplastic gastrointestinal avoided. Finally, biological samples collected by EUS-FNA CBP-assisted should be investigated in order to identify and quantify further potential molecular markers. PMID:26322154

Microplasma effect on skin scaffold for melanoma cancer treatment

NASA Astrophysics Data System (ADS)

Abdullah, Zulaika; Zaaba, S. K.; Mustaffa, M. T.; Mohamad, C. W. S. R.; Zakaria, A.

2017-03-01

An atmospheric plasma system using Helium gas was developed. The effect of helium plasma treatment on skin scaffold surface was studied by scanning electron microscopy (SEM). The changes of skin scaffold surfaces before and after helium plasma treatment was recorded. The surface of skin scaffold changed with the prolonged of helium plasma treatment time. The depth of helium plasma penetration was studied using methylene blue dye staining method. The methylene blue will detect the presence or absence of an oxygen that was induced from plasma excitation. The presence of the oxygen indicated on the depth of helium plasma penetration. Results showed plasma are able to penetrate 4mm of skin scaffold after 1200 seconds of exposure.

Effect of biological characteristics of different types of uterine fibroids, as assessed with T2-weighted magnetic resonance imaging, on ultrasound-guided high-intensity focused ultrasound ablation.

PubMed

Zhao, Wen-Peng; Chen, Jin-Yun; Chen, Wen-Zhi

2015-02-01

The aims of this study were to assess the effects of the biological characteristics of different types of uterine fibroids, as assessed with T2-weighted magnetic resonance imaging (MRI), on ultrasound-guided high-intensity focused ultrasound (USgHIFU) ablation. Thirty-five patients with 39 symptomatic uterine fibroids who underwent myomectomy or hysterectomy were enrolled. Before surgery, the uterine fibroids were subdivided into hypo-intense, iso-intense, heterogeneous hyper-intense and homogeneous hyper-intense categories based on signal intensity on T2-weighted MRI. Tissue density and moisture content were determined in post-operative samples and normal uterine tissue, the isolated uterine fibroids were subjected to USgHIFU, and the extent of ablation was measured using triphenyltetrazolium chloride. Hematoxylin and eosin staining and sirius red staining were undertaken to investigate the organizational structure of the uterine fibroids. Estrogen and progesterone receptor expression was assayed via immunohistochemical staining. The mean diameter of uterine fibroids was 6.9 ± 2.8 cm. For all uterine fibroids, the average density and moisture content were 10.7 ± 0.7 mg/mL and 75.7 ± 2.4%, respectively; and for the homogeneous hyper-intense fibroids, 10.3 ± 0.5 mg/mL and 76.6 ± 2.3%. The latter subgroup had lower density and higher moisture content compared with the other subgroups. After USgHIFU treatment, the extent of ablation of the hyper-intense fibroids was 102.7 ± 42.1 mm(2), which was significantly less than those of the hypo-intense and heterogeneous hyper-intense fibroids. Hematoxylin and eosin staining and sirius red staining revealed that the homogeneous hyper-intense fibroids had sparse collagen fibers and abundant cells. Immunohistochemistry results revealed that estrogen and progesterone receptors were highly expressed in the homogeneous hyper-intense fibroids. This study revealed that lower density, higher moisture content, sparse collagen fibers, abundant cells and overexpression of estrogen and progesterone receptors are important biological characteristics that resulted in poor efficacy in the treatment of homogeneous hyper-intense fibroids. Copyright © 2015 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.

Determination of oestrous cycle of the rats by direct examination: how reliable?

PubMed

Yener, T; Turkkani Tunc, A; Aslan, H; Aytan, H; Cantug Caliskan, A

2007-02-01

For determination of the oestrous cycle in rats classical Papanicolaou technique has long been used successfully. Instead of using many stains in Papanicolaou, staining the vaginal secretions with only methylene blue has also been defined. Recently a new technique in which vaginal samples are directly examined under light microscope has been introduced. The aim of this study was to assess the reliability of this new technique by comparing it with the classical staining techniques. From 20 Wistar rats 60 vaginal samples were collected with a micropipette, three from each. Briefly, the vagina was flushed two to three times then the fluid was placed onto a glass slide. The fluid was equally distributed onto three glass slides. The glass slides were coded. Two samples were stained with Papanicolaou and methylene blue while the other one was examined directly. Determination of the phases of the oestrous cycle was made by the same histologist who was blinded to the groups and coding system. After determination of the oestrous phase in all samples, the results were compared and it was found that the results were matching. In conclusion, the same results can be obtained with the direct examination technique and this technique is reliable, so there is no need to use relatively time-consuming, less practical and more expensive techniques such as Papanicolaou or methylene blue.

The Ratio and Concentration of Two Monoterpenes Mediate Fecundity of the Pinewood Nematode and Growth of Its Associated Fungi

PubMed Central

Niu, Hongtao; Zhao, Lilin; Lu, Min; Zhang, Shuai; Sun, Jianghua

2012-01-01

The pinewood nematode (PWN) Bursaphelenchus xylophilus, vectored primarily by the sawyer beetle, Monochamus alternatus, is an important invasive pest and causal agent of pine wilt disease of Chinese Masson pine, Pinus massoniana. Previous work demonstrated that the ratios and concentrations of α-pinene∶β-pinene differed between healthy trees and those trees containing blue-stain fungus (and M. alternatus pupae). However, the potential influence of the altered monoterpene ratios and concentrations on PWN and associated fungi remained unknown. Our current results show that low concentrations of the monoterpenes within petri dishes reduced PWN propagation, whereas the highest concentration of the monoterpenes increased PWN propagation. The propagation rate of PWN treated with the monoterpene ratio representative of blue-stain infected pine (α-pinene∶β-pinene = 1∶0.8, 137.6 mg/ml) was significantly higher than that (α-pinene∶β-pinene = 1∶0.1, 137.6 mg/ml) representative of healthy pines or those damaged by M. alternatus feeding, but without blue stain. Furthermore, inhibition of mycelial growth of associated fungi increased with the concentration of the monoterpenes α-pinene and β-pinene. Additionally, higher levels of β-pinene (α-pinene∶β-pinene = 1∶0.8) resulted in greater inhibition of the growth of the associated fungi Sporothrix sp.2 and Ophiostoma ips strains, but had no significant effects on the growth of Sporothrix sp.1, which is the best food resource for PWN. These results suggest that host monoterpenes generally reduce the reproduction of PWN. However, PWN utilizes high monoterpene concentrations and native blue-stain fungus Sporothrix sp.1 to improve its own propagation and overcome host resistance, which may provide clues to understanding the ecological mechanisms of PWN's successful invasion. PMID:22363713

Questions on unusual Mimivirus-like structures observed in human cells.

PubMed

Lusi, Elena Angela; Maloney, Dan; Caicci, Federico; Guarascio, Paolo

2017-01-01

Background: Mimiviruses or giant viruses that infect amoebas have the ability to retain the Gram stain, which is usually used to colour bacteria. There is some evidence suggesting that Mimiviruses can also infect human cells. Guided by these premises, we performed a routine Gram stain on a variety of human specimens to see if we could detect the same Gram positive blue granules that identify Mimiviruses in the amoebas.  Methods: We analysed 24 different human specimens (liver, brain, kidney, lymph node and ovary) using Gram stain histochemistry, electron microscopy immunogold, high resolution mass spectrometry and protein identification.  Results: We detected in the human cells Gram positive granules that were distinct from bacteria. The fine blue granules displayed the same pattern of the Gram positive granules that diagnose Mimiviruses in the cytoplasm of the amoebas. Electron microscopy confirmed the presence of human Mimiviruses-like structures and mass spectrometry identified histone H4 peptides, which had the same footprints as giant viruses. However, some differences were noted: the Mimivirus-like structures identified in the human cells were ubiquitous and manifested a distinct mammalian retroviral antigenicity.  Conclusions: Our main hypotheses are that the structures could be either giant viruses having a retroviral antigenicity or ancestral cellular components having a viral origin. However, other possible alternatives have been proposed to explain the nature and function of the newly identified structures.

An in vitro analysis model for investigating the staining effect of various chlorhexidine-based mouthwashes.

PubMed

Kouadio, Alain-Ayepa; Struillou, Xavier; Bories, Céline; Bouler, Jean-Michel; Badran, Zahi; Soueidan, Assem

2017-03-01

There are different mouthwashes containing chlorhexidine in different concentrations, as well as various excipients. Chlorhexidine induce stains or discoloration in teeth and mucous membranes. The aim of this work was to design a model to reproduce in vitro staining associated with the use of different mouthwashes containing chlorhexidine. We used as substrates of natural teeth and elephant ivory slices. Different incubation baths were conducted over 21 days in culture dishes at 37°C. At the beginning of experiment before incubation (D0) and after 21 days (D21) of incubation with different mouthwashes, pictures of substrates were taken in a standardized manner and an image analysis software was used to analyse and quantify the staining under the various conditions by using the 3 main colours (Red, Green, Blue, RGB). The results of this work demonstrate a very good reproducibility of the protocol, and secondly, a different expression statistically significant of the primary blue colour. We suggest that for a given concentration of chlorhexidine, the staining effects may vary depending on the excipients used. This replicable model, easy to implement over a relatively short duration, can be used for evaluation of existing mouthwashes, and to test the excipients anti discoloration proposed by manufacturers. Key words: In vitro, chlorhexidine, mouthwashes, dental stain, tooth discoloration.

Assessment of the Dutch organ-culture system of corneal preservation within the Eye Bank of South Australia.

PubMed

Williams, K A; Noack, L M; Alfrich, S J; Danz, R; Erickson, S A; Coster, D J

1988-02-01

Thirteen per cent of all corneas harvested by the Eye Bank of South Australia during 1986 were discarded because storage time in McCarey-Kaufman medium exceeded four days. We have therefore examined the suitability of the Dutch method of long-term corneal storage for our purposes. Twenty-two human corneas that had been discarded from the Eye Bank were assessed using the trypan blue-sucrose staining technique, and then placed into long-term storage for 15 to 17 days. They were then reassessed by vital dye staining before permanent flat-mounts were prepared for silver staining of the endothelium. A good correlation (albeit subjective) was found between the non-destructive and destructive techniques of endothelial cell assessment. Those corneas that failed to survive organ culture storage were easily detected. The Dutch system of corneal preservation and post-storage assessment seems well-suited to Australian eye-banking.

Ultrastructural visualisation of proteoglycans in early unmineralised dentine of rat tooth germs stained with cuprolinic blue.

PubMed Central

Tenorio, D; Reid, A R; Katchburian, E

1990-01-01

The ultrastructural distribution and localisation of proteoglycans (PGs) of early developing rat dentine were examined using cuprolinic blue in a critical electrolyte concentration procedure. Results show that the cuprolinic blue method produces images of higher morphological quality than other cationic dyes. PGs appeared as ribbon-like electron-opaque precipitates of various sizes, ranging between 1.4 and 0.2 microns in length, distributed throughout the matrix and in close association with well preserved matrix vesicles and collagen fibrils. Matrix vesicles revealed tightly packed PG filaments which appeared to be attached to their membrane. It is possible that the close association of PG filaments with matrix vesicles and collagen indicates that PGs are related to the process of mineralisation of dentine. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 Fig. 8 PMID:2384338

Different immunohistochemical and ultrastructural phenotypes of squamous differentiation in bladder cancer.

PubMed

Gaisa, Nadine T; Braunschweig, Till; Reimer, Nina; Bornemann, Jörg; Eltze, Elke; Siegert, Sabine; Toma, Marieta; Villa, Luigi; Hartmann, Arndt; Knuechel, Ruth

2011-03-01

Besides worse prognosis of bladder cancer with squamous differentiation (pure squamous cell carcinoma (SCC) or mixed urothelial carcinoma (UC/SCC)), high-grade non-keratinising squamous differentiation is difficult to identify in haematoxylin-eosin stainings. This study aims to validate routine immunohistochemical markers for squamous differentiation in a larger cohort of patients. Tissue microarrays of 89 pure SCCs and mixed UC/SCCs, 66 urothelial carcinomas (UC), precursor lesions and normal urothelium were stained for cytokeratin (CK) 5/6, CK 5/14, CK 7, CK 20 and uroplakin III. Electron microscopy was performed to confirm the differentiation. Pure SCCs displayed staining throughout the epithelium for CK 5/6 (76.6% (36/47)) and CK 5/14 (95.8% (46/48)), focal staining for CK 7 (28.9% (13/45)) and no staining for CK 20 and uroplakin III (both 0% (0/48)). UCs exhibited a basal or diffuse staining for CK 5/6 (30.2% (16/53)) and CK 5/14 (57.1% (32/56)), focal positivity for CK 7 (83.6% (46/55)), CK 20 (50.9% (29/57)) and uroplakin III (21.8% (12/55)). Each marker discriminated SCC and UC significantly (p < 0.01). A third subgroup rarely showed full epithelial staining for CK 5/6 (14.3% (1/7)) and CK 5/14 (28.6% (2/7)), focal staining for CK 7 (85.7% (6/7)) and no staining for CK 20 and uroplakin III (both 0% (0/7)). Electron microscopy could prove both, SCC and UC characteristics, revealing a transient type. A staining pattern with CK 5/6- and CK 5/14-positivity plus CK 20- and uroplakin III-negativity identified squamous differentiation in bladder tumours and revealed a third type of squamous transdifferentiation.

Histological staining can enhance the performance of spectroscopic microscopy on sensing nanoarchitectural alterations of biological cells (Conference Presentation)

NASA Astrophysics Data System (ADS)

Zhang, Di; Cherkezyan, Lusik; Li, Yue; Capoglu, Ilker; Subramanian, Hariharan; Taflove, Allen; Backman, Vadim

2017-02-01

Our group had previously established that nanoscale three-dimensional refractive index (RI) fluctuations of a linear, dielectric, label-free medium can be sensed in the far field through spectroscopic microscopy, regardless of the diffraction limit of optical microscopy. Adopting this technique, Partial Wave Spectroscopic (PWS) Microscopy was able to sense nanoarchitectural alterations in early-stage cancers. With the success of PWS on detecting cancer from healthy clinical samples, we further investigated whether and how histological staining can enhance the performance of PWS by both finite difference time domain (FDTD) simulations and experiments. In this investigation, the dispersion models of hematoxylin and eosin were extracted from the absorption spectra of H and E stained cells. Using these models, the effect of staining were studied via FDTD simulations of unstained versus stained samples with various nanostructures. We observed that, the spectral variance was increased and the spectral variance difference between two samples with distinct nanostructures was enhanced in stained samples by over 200%. Furthermore, we investigated with FDTD whether molecule-specific staining can be used to enhance signals from a medium composing of the desired molecule. Samples with two mixed random media were created and the desired medium was either stained or unstained. Our results showed that the difference between the nanostructures of only the desired medium was enhanced in stained samples. We concluded that, with molecule-specific staining, PWS can selectively target the nanoarchitecture of a desired molecule. Lastly, these results were validated by experiments using human buccal cells from healthy or lung cancer patients. This study has significant impact in improving the performance of PWS on quantifying nanoarchitectural alterations during cancer.

Necrosulfonamide Attenuates Spinal Cord Injury via Necroptosis Inhibition.

PubMed

Wang, Yongxiang; Wang, Jingcheng; Wang, Hua; Feng, Xinmin; Tao, Yuping; Yang, Jiandong; Cai, Jun

2018-06-01

Spinal cord injury (SCI) is a serious trauma without efficient treatment currently. Necroptosis can be blocked post injury by special inhibitors. This study is to investigate the effects, mechanism, and potential benefit of necrosulfonamide (NSA) for SCI therapy. Pathologic condition was detected using hematoxylin-eosin staining on injured spinal cord and other major organs. Necroptosis-related factors-RIP1, RIP3, and MLKL-were detected using Western blot. Detections on mitochondrial functions such as adenosine triphosphate generation and activities of superoxide dismutase and caspase-3 were also performed. Finally, ethologic performance was detected using a 21-point open-field locomotion test. Reduced lesions and protected neurons were found in the injured spinal cord after treatment with NSA using hematoxylin-eosin staining for pathologic detection. No obvious toxicity on rat liver, kidney, heart, and spleen was detected. Rather than RIP1 and RIP3, MLKL was significantly inhibited by the NSA using Western blot detection. Adenosine triphosphate generation was obviously decreased post injury but slightly increased after the NSA treatment, especially 24 hours post injury. No significant changes were found on activities of superoxide dismutase and caspase-3 after the treatment of NSA. Ethologic performance was significantly improved using a 21-point, open-field locomotion test. Our research indicates NSA attenuates the spinal cord injury via necroptosis inhibition. It might be a potential and safe chemical benefit for SCI therapy. To our knowledge, this is the first study on the effects of NSA as treatment of traumatic SCI. Copyright © 2018 Elsevier Inc. All rights reserved.

Clinicopathological characteristics of Xp11.2 translocation renal cell carcinoma in adolescents and adults: Diagnosis using immunostaining of transcription factor E3 and fluorescence in situ hybridization analysis.

PubMed

Hirobe, Megumi; Masumori, Naoya; Tanaka, Toshiaki; Kitamura, Hiroshi; Tonooka, Akiko; Hasegawa, Tadashi; Tsukamoto, Taiji

2016-02-01

To determine the rate and clinicopathological features of Xp11.2 translocation carcinoma using immunostaining of transcription factor E3 and fluorescence in situ hybridization analysis. We evaluated 638 patients with renal cell carcinoma treated at Sapporo Medical University Hospital, Sapporo, Japan, from 1990 to 2009 by reviewing all hematoxylin-eosin-stained sections and carrying out immunostaining of transcription factor E3 for all cases. Fluorescence in situ hybridization analysis was carried out for patients with positive immunostaining or with findings suspicious for Xp11.2 translocation carcinoma on hematoxylin-eosin-stained sections. In this analysis, we set a cut-off level for split signals of at least 10% of nuclei. Of the 631 patients, 20 (3.2%) were positive for immunostaining. Finally, five patients were diagnosed with Xp11.2 translocation carcinoma (0.8%). Four of these patients were female and aged less than 50 years, and three cases were diagnosed as stage IV with multiple regional lymph nodal or visceral metastases. The positive predictive value of immunostaining was 25%. Patients with Xp11 translocation renal cell carcinoma tend to be younger, more frequently female and diagnosed at a more advanced stage. Immunostaining followed by fluorescence in situ hybridization analysis is an accurate and cost-effective approach for diagnosis of Xp11 translocation renal cell carcinoma. © 2015 The Japanese Urological Association.

Near-Infrared Confocal Laser Reflectance Cytoarchitectural Imaging of the Substantia Nigra and Cerebellum in the Fresh Human Cadaver.

PubMed

Cheyuo, Cletus; Grand, Walter; Balos, Lucia L

2017-01-01

Cytoarchitectural neuroimaging remains critical for diagnosis of many brain diseases. Fluorescent dye-enhanced, near-infrared confocal in situ cellular imaging of the brain has been reported. However, impermeability of the blood-brain barrier to most fluorescent dyes limits clinical utility of this modality. The differential degree of reflectance from brain tissue with unenhanced near-infrared imaging may represent an alternative technique for in situ cytoarchitectural neuroimaging. We assessed the utility of unenhanced near-infrared confocal laser reflectance imaging of the cytoarchitecture of the cerebellum and substantia nigra in 2 fresh human cadaver brains using a confocal near-infrared laser probe. Cellular images based on near-infrared differential reflectance were captured at depths of 20-180 μm from the brain surface. Parts of the cerebellum and substantia nigra imaged using the probe were subsequently excised and stained with hematoxylin and eosin for histologic correlation. Near-infrared reflectance imaging revealed the 3-layered cytoarchitecture of the cerebellum, with Purkinje cells appearing hyperreflectant. In the substantia nigra, neurons appeared hyporeflectant with hyperreflectant neuromelanin cytoplasmic inclusions. Cytoarchitecture of the cerebellum and substantia nigra revealed on near-infrared imaging closely correlated with the histology on hematoxylin-eosin staining. We showed that unenhanced near-infrared reflectance imaging of fresh human cadaver brain can reliably identify and distinguish neurons and detailed cytoarchitecture of the cerebellum and substantia nigra. Copyright © 2016 Elsevier Inc. All rights reserved.

The response of visible/near infrared absorbance to wood-staining fungi

Treesearch

Brian K. Via; Lori G. Eckhardt; Chi-Leung So; Todd F. Shupe; Leslie H. Groom; Michael Stine

2006-01-01

The influence of blue-stain fungi [Ophiostoma minus (Hedgcock) H. and P. Sydow and Leptographium serpens (Goid.) Siemaszko] on absorbance at the visible and near infrared wavelengths was investigated. Forty trees were sampled at breast height from longleaf pine (Pinus palustris Mill.). One half of each increment...

Identification criteria of the rare multi-flagellate Lophomonas blattarum: comparison of different staining techniques.

PubMed

Alam-Eldin, Yosra Hussein; Abdulaziz, Amany Mamdouh

2015-09-01

Bronchopulmonary lophomoniasis (BPL) is an emerging disease of potential importance. BPL is presented by non-specific clinical picture and is usually accompanied by immunosuppression. Culture of Lophomonas blattarum is difficult and its molecular diagnosis has not yet been developed. Therefore, microscopic examination of respiratory samples, e.g., bronchoalveolar lavage (BAL) or sputum, is the mainstay of BPL diagnosis. Creola bodies and ciliocytophthoria are two forms of bronchial cells which occur in chest diseases with non-specific clinical picture like that of BPL. Both forms could be misrecognized as multi-flagellates because of their motile cilia in the wet mounts and due to shape variability of L. blattarum in stained smears. The aim of the study is to compare different staining techniques for visualizing L. blattarum to improve the recognition and diagnosis of BPL, to distinguish respiratory epithelial cells from L. blattarum and to decide which stain is recommended in suspected cases of BPL. BAL samples from patients which contain L. blattarum, creola bodies, and ciliocytophthoria were collected then wet mounts were examined. The BAL samples were also stained by Papanicolaou (PAP), Giemsa, hematoxylin and eosin (H & E), trichrome, Gram, and Diff-Quik (DQ) stains. The different staining techniques were compared regarding the stain quality. In wet mounts, the ciliary movement was coordinate and synchronous while the flagellar movement was wavy and leaded to active swimming of L. blattarum. In stained slides, bronchial cells were characterized by the presence of basal nucleus and the terminal bar from which the cilia arise. Trichrome was the best stain in demonstration of cellular details of L. blattarum. H & E, PAP, and Giemsa stains showed good quality of stains. Gram and DQ stains showed only pale hues of L. blattarum. We recommended adding Wheatley's trichrome staining to the differential diagnosis workup of cases of non-specific chest infections, especially when BPL is suspected, to avoid overdiagnosis or underdiagnosis of it.

Improvement of malaria diagnostic system based on acridine orange staining.

PubMed

Kimura, Masatsugu; Teramoto, Isao; Chan, Chim W; Idris, Zulkarnain Md; Kongere, James; Kagaya, Wataru; Kawamoto, Fumihiko; Asada, Ryoko; Isozumi, Rie; Kaneko, Akira

2018-02-07

Rapid diagnosis of malaria using acridine orange (AO) staining and a light microscope with a halogen lamp and interference filter was deployed in some malaria-endemic countries. However, it has not been widely adopted because: (1) the lamp was weak as an excitation light and the set-up did not work well under unstable power supply; and, (2) the staining of samples was frequently inconsistent. The halogen lamp was replaced by a low-cost, blue light-emitting diode (LED) lamp. Using a reformulated AO solution, the staining protocol was revised to make use of a concentration gradient instead of uniform staining. To evaluate this new AO diagnostic system, a pilot field study was conducted in the Lake Victoria basin in Kenya. Without staining failure, malaria infection status of about 100 samples was determined on-site per one microscopist per day, using the improved AO diagnostic system. The improved AO diagnosis had both higher overall sensitivity (46.1 vs 38.9%: p = 0.08) and specificity (99.0 vs 96.3%) than the Giemsa method (N = 1018), using PCR diagnosis as the standard. Consistent AO staining of thin blood films and rapid evaluation of malaria parasitaemia with the revised protocol produced superior results relative to the Giemsa method. This AO diagnostic system can be set up easily at low cost using an ordinary light microscope. It may supplement rapid diagnostic tests currently used in clinical settings in malaria-endemic countries, and may be considered as an inexpensive tool for case surveillance in malaria-eliminating countries.

Morphological and molecular analysis of the collagen fibers in inflammatory process

NASA Astrophysics Data System (ADS)

de Carvalho, Luis Felipe das Chagas e. Silva; Alves, Mônica Ghislaine Oliveira; Soares, Carlos Alexandre; Almeida, Janete Dias; da Silva Martinho, Herculano

2011-07-01

Collagen makes up one third of the total protein in humans, being formed by the connection of three polypeptide chains arranged in a triple helix. This protein has fundamental importance in the formation of extracellular matrix of connective tissue. This study aimed to analyze the structural changes of collagen, which are resulting from inflammatory processes in oral mucosa, and to make the comparative analysis between the histopathology and the Raman spectra. The samples of tissues with inflammatory fibrous hyperplasia (IFH) and normal mucosa (NM) were evaluated by Raman Spectroscopy, hematoxylin-eosin and Massons trichrome stain. The histological analysis in both stains showed differences in collagen fibers, which was presented as thin fibers and arranged in parallel direction in NM and as collagen fibers are thick, mature and not organized, showing that these types of stain show morphological changes of collagen in IFH. The Raman Spectroscopy discriminate the groups of NM and IFH based on vibrational modes of proline, hydroxiproline and CH3, CH2. The histological stains only shows information from morphological data, and can be complemented by Raman spectra. This technique could demonstrate that inflammatory process caused some changes in collagen structure which is related to aminoacids such as proline and hidroxyproline.

Histopathological vascular investigation of the peritumoral brain zone of glioblastomas.

PubMed

Tamura, Ryota; Ohara, Kentaro; Sasaki, Hikaru; Morimoto, Yukina; Yoshida, Kazunari; Toda, Masahiro

2018-01-01

To date, no histopathological vascular investigation focusing on peritumoral brain zone (PBZ) has been reported for glioblastoma. We analyzed 10 newly diagnosed cases of glioblastomas. For these PBZs, histopathological investigation was performed by hematoxylin-eosin (H&E) staining and immunohistochemistry was analyzed for CD31, CD34, Factor VIII, VEGF, VEGFR-1/2, Ki67, p53 and nestin. Although it was difficult to identify PBZ by H&E, Ki67 and p53 staining, there were apparent differences in nestin staining among PBZ, tumor core (TC), and normal zone (NZ). Therefore, in this study, we divided PBZ from TC and NZ by nestin staining. Differences in histological features, microvessel density, expression of VEGF and its receptors were assessed for PBZ, TC and NZ. The microvessel density, as determined by counting CD31, CD34 and VEGF receptors, and VEGF-A expression were lower in PBZ than TC. The expression patterns for CD31, CD34 and VEGF receptors in vessels show dissociation in PBZ. In addition, the vascular characteristics of the PBZ may correlate with findings of radiographic imaging. We provide the first clinicopathological evidence that PBZ exhibits unique angiogenic characteristics. These in situ observations will help to elucidate the mechanisms of tumor recurrence.

Tri-modal confocal mosaics detect residual invasive squamous cell carcinoma in Mohs surgical excisions

NASA Astrophysics Data System (ADS)

Gareau, Dan; Bar, Anna; Snaveley, Nicholas; Lee, Ken; Chen, Nathaniel; Swanson, Neil; Simpson, Eric; Jacques, Steve

2012-06-01

For rapid, intra-operative pathological margin assessment to guide staged cancer excisions, multimodal confocal mosaic scan image wide surgical margins (approximately 1 cm) with sub-cellular resolution and mimic the appearance of conventional hematoxylin and eosin histopathology (H&E). The goal of this work is to combine three confocal imaging modes: acridine orange fluorescence (AO) for labeling nuclei, eosin fluorescence (Eo) for labeling cytoplasm, and endogenous reflectance (R) for marking collagen and keratin. Absorption contrast is achieved by alternating the excitation wavelength: 488 nm (AO fluorescence) and 532 nm (Eo fluorescence). Superposition and false-coloring of these modes mimics H&E, enabling detection of cutaneous squamous cell carcinomas (SCC). The sum of mosaic Eo+R is false-colored pink to mimic the appearance of eosin, while the AO mosaic is false-colored purple to mimic the appearance of hematoxylin in H&E. In this study, mosaics of 10 Mohs surgical excisions containing invasive SCC, and five containing only normal tissue were subdivided for digital presentation equivalent to 4× histology. Of the total 50 SCC and 25 normal sub-mosaics presented, two reviewers made two and three type-2 errors (false positives), respectively. Limitations to precisely mimic H&E included occasional elastin staining by AO. These results suggest that confocal mosaics may effectively guide staged SCC excisions in skin and other tissues.

Tri-modal confocal mosaics detect residual invasive squamous cell carcinoma in Mohs surgical excisions

PubMed Central

Bar, Anna; Snaveley, Nicholas; Lee, Ken; Chen, Nathaniel; Swanson, Neil; Simpson, Eric; Jacques, Steve

2012-01-01

Abstract. For rapid, intra-operative pathological margin assessment to guide staged cancer excisions, multimodal confocal mosaic scan image wide surgical margins (approximately 1 cm) with sub-cellular resolution and mimic the appearance of conventional hematoxylin and eosin histopathology (H&E). The goal of this work is to combine three confocal imaging modes: acridine orange fluorescence (AO) for labeling nuclei, eosin fluorescence (Eo) for labeling cytoplasm, and endogenous reflectance (R) for marking collagen and keratin. Absorption contrast is achieved by alternating the excitation wavelength: 488 nm (AO fluorescence) and 532 nm (Eo fluorescence). Superposition and false-coloring of these modes mimics H&E, enabling detection of cutaneous squamous cell carcinomas (SCC). The sum of mosaic Eo+R is false-colored pink to mimic the appearance of eosin, while the AO mosaic is false-colored purple to mimic the appearance of hematoxylin in H&E. In this study, mosaics of 10 Mohs surgical excisions containing invasive SCC, and five containing only normal tissue were subdivided for digital presentation equivalent to 4× histology. Of the total 50 SCC and 25 normal sub-mosaics presented, two reviewers made two and three type-2 errors (false positives), respectively. Limitations to precisely mimic H&E included occasional elastin staining by AO. These results suggest that confocal mosaics may effectively guide staged SCC excisions in skin and other tissues. PMID:22734774

Epiretinal membrane negative staining and double peeling in a single block with Brilliant Blue G.

PubMed

Martins, David; Neves, Pedro

2018-01-01

To describe a surgical technique for combined peeling of epiretinal and internal limiting membranes. The authors present their procedure of choice for epiretinal membrane surgery: negative staining effect using Brilliant Blue G and single block removal of the epiretinal and internal limiting membranes in a single step. A total of 26 eyes were operated with the described technique. In all cases, the peeling was performed successfully and with no complications. Minimum postoperative follow-up was 12 months. There were no recurrences of epiretinal membranes. The ideal surgical approach for epiretinal membranes should attempt to reduce mechanical trauma, light exposure, and dye toxicity.

A randomized, prospective cross-over trial comparing methylene blue-directed biopsy and conventional random biopsy for detecting intestinal metaplasia and dysplasia in Barrett's esophagus.

PubMed

Ragunath, K; Krasner, N; Raman, V S; Haqqani, M T; Cheung, W Y

2003-12-01

The value of methylene blue-directed biopsies (MBDB) in detecting specialized intestinal metaplasia and dysplasia in Barrett's esophagus remains unclear. The aim of this study was to compare the accuracy of MBDB with random biopsy in detecting intestinal metaplasia and dysplasia in patients with Barrett's esophagus. A prospective, randomized, cross-over trial was undertaken to compare MBDB with random biopsy in patients with Barrett's esophagus segments 3 cm or more in length without macroscopic evidence of dysplasia or cancer. Dysplasia was graded as: indefinite for dysplasia, low-grade dysplasia, high-grade dysplasia, or carcinoma, and was reported in a blinded fashion. Fifty-seven patients were recruited, 44 of whom were male. A total of 1,269 biopsies were taken (MBDB-651, random biopsie-618). Analysis of the results by per-biopsy protocol showed that the MBDB technique diagnosed significantly more specialized intestinal metaplasia (75 %) compared to the random biopsy technique (68 %; P = 0.032). The sensitivity and specificity rates of MBDB for diagnosing specialized intestinal metaplasia were 91 % (95 % CI, 88 - 93 %) and 43 % (95 % CI, 36 - 51 %), respectively. The sensitivity and specificity rates of MBDB for diagnosing dysplasia or carcinoma were 49 % (95 % CI, 38 - 61 %) and 85 % (95 % CI, 82 - 88 %), respectively. There were no significant differences in the diagnosis of dysplasia and carcinoma - MBDB 12 %, random biopsy 10 %. The methylene blue staining pattern appeared to have an influence on the detection of specialized intestinal metaplasia and dysplasia/carcinoma. Dark blue staining was associated with increased detection of specialized intestinal metaplasia (P < 0.0001), and heterogeneous staining (P = 0.137) or no staining (P = 0.005) were associated with dysplasia and/or carcinoma detection. The MBDB technique prolonged the endoscopy examination by an average of 6 min. The diagnostic accuracy of the MBDB technique was superior to that of the random biopsy technique for identifying specialized intestinal metaplasia, but not dysplasia or carcinoma. The intensity of methylene blue staining has an influence on the detection of specialized intestinal metaplasia and dysplasia or carcinoma, which may help in targeting the biopsies. Although MBDB prolongs the endoscopy procedure slightly, it is a safe and well-tolerated procedure. Further clinical studies on the MBDB technique exclusively in endoscopically normal dysplastic Barrett's esophagus are needed.