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Sample records for labelled red cell

  1. Use of indium-111 as a red cell label

    SciTech Connect

    AuBuchon, J.P.; Brightman, A.

    1989-02-01

    To select the most promising 111In chelate for use as a second red cell (RBC) label for comparison of the survival of autologous and allogeneic cells, 49 normal RBC samples were studied in vitro after being labeled with 111In-8-hydroxyquinolinol (111In-oxine) prepared by three different methods, 111In-tropolone, and 111In-acetylacetone. Labeling efficiencies reached 99 percent and did not decline when the amount of 111In used was increased from 1.75 to 50 muCi per ml of RBCs. Storage of labeled RBCs in normal AB plasma at 4, 22, and 37 degrees C for up to 48 hours resulted in a similar rate of loss of the label from the RBCs with all labeling methods. These rates were time- and temperature-dependent and were accurate predictions of the rates found in later in vivo experimentation. Fresh RBCs from 11 subjects were labeled with 111In chelated with oxine in the presence of the RBCs or chelated with tropolone just prior to the labeling. RBC mass determinations using these autologous RBCs labeled with 111In accurately reflected the subjects' RBC masses as predicted through standard morphometric formulae. The rate of disappearance of the radionuclide after reinfusion of the autologous RBCs decreased with time. At 24 hours after reinfusion, 89.5 +/- 1.29 percent (mean +/- SEM) of the 111In-tropolone and 87.3 +/- 1.25 percent of the 111In-oxine continued in circulation. 111In is a simple and efficient agent for the labeling of RBCs for blood volume determinations and short-term survivals.

  2. In vivo red blood cell compatibility testing using indium-113m tropolone-labeled red blood cells

    SciTech Connect

    Morrissey, G.J.; Gravelle, D.; Dietz, G.; Driedger, A.A.; King, M.; Cradduck, T.D.

    1988-05-01

    In vivo radionuclide crossmatch is a method for identifying compatible blood for transfusion when allo- or autoantibodies preclude the use of conventional crossmatching techniques. A technique for labeling small volumes of donor red blood cells with (/sup 113m/In)tropolone is reported. The use of /sup 113m/In minimizes the accumulation of background radioactivity and the radiation dose especially so when multiple crossmatches are performed. Labeling red cells with (/sup 113m/In)tropolone is faster and easier to perform than with other radionuclides. Consistently high labeling efficiencies are obtained and minimal /sup 113m/In activity elutes from the labeled red blood cells. A case study involving 22 crossmatches is presented to demonstrate the technique. The radiation dose equivalent from /sup 113m/In is significantly less than with other radionuclides that may be used to label red cells.

  3. Kit for the selective labeling of red blood cells in whole blood with .sup.9 TC

    DOEpatents

    Srivastava, Suresh C.; Babich, John W.; Straub, Rita; Richards, Powell

    1992-01-01

    Disclosed herein are a method and kit for the preparation of .sup.99m Tc labeled red blood cells using whole blood in a closed sterile system containing stannous tin in a form such that it will enter the red blood cells and be available therein for reduction of technetium.

  4. Kit for the selective labeling of red blood cells in whole blood with [sup 99]Tc

    DOEpatents

    Srivastava, S.C.; Babich, J.W.; Straub, R.; Richards, P.

    1992-05-26

    Disclosed herein are a method and kit for the preparation of [sup 99m]Tc labeled red blood cells using whole blood in a closed sterile system containing stannous tin in a form such that it will enter the red blood cells and be available therein for reduction of technetium. No Drawings

  5. Gallbladder visualization during technetium-99m-labeled red cell scintigraphy for gastrointestinal bleeding

    SciTech Connect

    Brill, D.R.

    1985-12-01

    Localization of radionuclide activity in the gallbladder was seen on delayed views following injection of 99mTc-labeled red blood cells for gastrointestinal bleeding in five patients. The mechanism for this unusual finding probably relates to labeling of heme, the biochemical precursor of bilirubin. All patients had had prior transfusions. All but one had severe renal impairment, probably an important predisposing factor.

  6. Tc-99m-labeled red blood cells for the measurement of red cell mass in newborn infants: concise communication

    SciTech Connect

    Linderkamp, O.; Betke, K.; Fendel, H.; Klemm, J.; Lorenzen, K.; Riegel, K.P.

    1980-07-01

    In vitro and in vivo investigations were performed to examine the binding of Tc-99m to neonatal red blood cells (RBC). Labeling efficiency was about 90%, and unbound Tc-99m less than 3% after one washing, in premature and full-term newborns and in children. Thus presence of high percentages of fetal hemoglobin (Hb F) did not influence the labeling of RBCs with Tc-99m. RBCs of 11 newborns were hemolysed and the distribution of Tc-99m on RBC components was analyzed. Although Hb F percentage averaged (60.0 +- 8.10)% (s.d.), only (11.9 +- 3.7)% of Tc-99m was bound by Hb F, whereas (45.0 +- 6.1)% was associated with Hb A. RBC membranes bound (13.7 +- 4.3)% and (29.3 +- 4.0)% were found unbound in hemolysates. These results indicate that Tc-99m preferentially binds to beta chains. In vivo equilibration of Tc-99m RBCs and of albumin labeled with Evans blue was investigated in five newborn infants. Tc-99m RBCs were stable in each case during the first hour after injection. Elution of Tc-99m from RBCs was (3.4 +- 1.5)% per h. Body-to-venous hematocrit ratio averaged 0.86 +- 0.03.

  7. Accuracy of blood volume estimations in critically ill children using 125I-labelled albumin and 51Cr-labelled red cells.

    PubMed

    Linderkamp, O; Holthausen, H; Seifert, J; Butenandt, I; Riegel, K P

    1977-06-01

    Blood volume was estimated using 51chromium labelled red cells and 125iodinated human serum albumin in 5 children with sepsis, in 6 burned children and 7 children with acute lymphoblastic leukaemia. Studies of the equilibration pattern demonstrated that the mixing time of labelled red cells was prolonged to 40 minutes or more in 5 children, indicating the existence of slowly circulating red cells. Mixing of labelled albumin was complete within 10 minutes in 15 patients and within 20 minutes in all the children studied. In a burned patient with severe sepsis, exchange transfusion improved the clinical state and normalized the equilibration pattern of labelled red cells. The mean body/venous haematocrit ratio was 0.893+/-0.018 (SD) in the children with sepsis, 0.859+/-0.052 in the burned patients, and 0.916+/-0.078 in the children with acute lymphoblastic leukaemia, increasing with spleen size in the latter group. PMID:267010

  8. New Red-Emitting Tetrazine-Phenoxazine Fluorogenic Labels for Live-Cell Intracellular Bioorthogonal Labeling Schemes.

    PubMed

    Knorr, Gergely; Kozma, Eszter; Herner, András; Lemke, Edward A; Kele, Péter

    2016-06-20

    The synthesis of a set of tetrazine-bearing fluorogenic dyes suitable for intracellular labeling of proteins in live cells is presented. The red excitability and emission properties ensure minimal autofluorescence, while through-bond energy-transfer-based fluorogenicity reduces nonspecific background fluorescence of unreacted dyes. The tetrazine motif efficiently quenches fluorescence of the phenoxazine core, which can be selectively turned on chemically upon bioorthogonal inverse-electron-demand Diels-Alder reaction with proteins modified genetically with strained trans-cyclooctenes. PMID:27218228

  9. Hyperemic peripheral red marrow in a patient with sickle cell anemia demonstrated on Tc-99m labeled red blood cell venography

    SciTech Connect

    Heiden, R.A.; Locko, R.C.; Stent, T.R. )

    1991-03-01

    A 25-year-old gravid woman, homozygous for sickle cell anemia, with a history of recent deep venous thrombosis, was examined using Tc-99m labeled red blood cell venography for recurrent thrombosis. Although negative for thrombus, the study presented an unusual incidental finding: the patient's peripheral bone marrow was hyperemic in a distribution consistent with peripheral red bone marrow expansion. Such a pattern has not been documented before using this technique. This report supports other literature that has demonstrated hyperemia of peripheral red bone marrow in other hemolytic anemias. This finding may ultimately define an additional role of scintigraphy in assessing the pathophysiologic status of the sickle cell patient.

  10. Method and kit for the selective labeling of red blood cells in whole blood with Tc-99m

    DOEpatents

    Srivastava, S.C.; Babich, J.W.; Straub, R.; Richards, P.

    1988-07-05

    Disclosed herein are a method and kit for the preparation of [sup 99m]Tc labeled red blood cells using whole blood in a closed sterile system containing stannous tin in a form such that it will enter the red blood cells and be available for the reduction of technetium. No Drawings

  11. Method and kit for the selective labeling of red blood cells in whole blood with TC-99M

    DOEpatents

    Srivastava, Suresh C.; Babich, John W.; Straub, Rita; Richards, Powell

    1988-01-01

    Disclosed herein are a method and kit for the preparation of .sup.99m Tc labeled red blood cells using whole blood in a closed sterile system containing stannous tin in a form such that it will enter the red blood cells and be available therein for the reduction of technetium.

  12. An enzyme-linked immunoabsorbent assay for estimating red cell survival of transfused red cells-validation using CR-51 labeling

    SciTech Connect

    Drew, H.; Kickler, T.; Smith, B.; LaFrance, N.

    1984-01-01

    The survival time of transfused red cells antigenically distinct from the recipient's red cells was determined using an indirect enzyme linked antiglobulin test. These results were then compared to those determined by Cr-51 labeling. Three patients with hypoproliferative anemias and one patient (2 studies) with traumatic hemolytic anemia caused by a prosthetic heart valve were studied. Survival times were performed by transfusing a 5cc aliquot of Cr-51 labeled cells along with the remaining unit. One hour post transfusion, a blood sample was drawn and used as the 100% value. Subsequent samples drawn over a 2-3 week period were then compared to the initial sample to determine percent survival for both methods. The ELISA method for measuring red cell survival in antigenically distinct cells is in close agreement with the Cr-51 method. Although CR-51 labeling is the accepted method for red cell survival determination the ELISA method can be used when radioisotopes are unavailable or contraindicated or when the decision to estimate red cell survival is made after transfusion.

  13. Gallbladder Activity on 99mTc-Labeled Red Cell Scintigraphy Confirmed by SPECT/CT Imaging.

    PubMed

    Wang, Ling; Jing, Hongli; Chen, Libo; Wang, Zhenghua; Li, Fang

    2016-09-01

    Tc-labeled red cell (Tc-RBC) scintigraphy is commonly used to detect gastrointestinal bleeding. Gallbladder visualization on Tc-RBC scintigraphy is not common. We present a case of gallbladder visualization on Tc-RBC scintigraphy confirmed by SPECT/CT imaging in a patient with chronic renal failure and anemia. PMID:27405034

  14. AUR memorial award--1988. MRI enhancement of perfused tissues using chromium labeled red blood cells as an intravascular contrast agent

    SciTech Connect

    Eisenberg, A.D.; Conturo, T.E.; Price, R.R.; Holburn, G.E.; Partain, C.L.; James, A.E. Jr. )

    1989-10-01

    It has been demonstrated that chromium (Cr) labeling significantly decreases the relaxation times of packed red blood cells (RBCs). In this study, the spin-lattice relaxation time (T1) of human red cells was shortened from 836 ms to 29 ms and the spin-spin relaxation time (T2) shortened from 134 ms to 18 ms, when the cells were labeled at a Cr incubation concentration of 50 mM. Labeling of canine cells at 50 mM resulted in a T1 of 36 ms and a T2 of 26 ms. A labeling concentration of 10 mM produced similar relaxation enhancement, with uptake of 47% of the available Cr, and was determined to be optimal. The enhancement of longitudinal and transverse relaxation rates (1/T1,-1/T2) per amount of hemoglobin-bound Cr are 6.9 s-1 mM-1 and 9.8 s-1 mM-1 respectively, different from those of a pure Cr+3 solution. Labeling cells at 10 mM decreased the survival half-time in vivo from 16.6 days to 4.7 days in dogs. No difference in red cell survival was found with the use of hetero-transfusion versus auto-transfusion of labeled RBCs. Significant shortening of the T1 (912 ms to 266 ms, P = .03) and T2 (90 ms to 70 ms, P = .006) of spleen and the T1 (764 ms to 282 ms, P = .005) and the T2 (128 ms to 86 ms, P = .005) of liver occurred when 10% of the RBC mass of dogs was exchanged with Cr labeled cells. Liver and spleen spin density changes (P greater than 0.23) and muscle spin density and relaxation changes (P greater than 0.4) were insignificant. The in vivo T1 of a canine spleen which had been infarcted did not change following transfusion with labeled cells, where the T1 of liver did shorten. We believe this preliminary study suggests that Cr labeled red cells may have the potential to become an intravascular magnetic resonance imaging contrast agent.

  15. Assessment of soft tissue hemangiomas in children utilizing Tc-99m labelled red blood cells

    SciTech Connect

    Miller, J.H.

    1984-01-01

    Hemangiomas may present in infancy as soft tissue masses. Occasionally these lesions may be extensive or may not be clinically recognized as a hemangioma, often causing concern for the presence of a malignant lesion. In later childhood these lesions, which may be occult, may cause overgrowth of an extremity. Evaluation of soft tissue masses suspected of being a hemangioma utilizing Technetium 99m labelled red blood cells has been very valuable. This method allows a dynamic evaluation of first pass blood flow. Subsequent static scintiphotos allow an assessment of the lesion itself. These scintiphotos may be obtained sequentially to evaluate therapy. Twenty patients were evaluated by this method ranging in age from two months to eleven years. There were 13 females and seven males. Lesions evaluated by this method include six hemangiomas of the head and neck: parotic region (2), facial (3), and tongue (1). Extremity lesions were evaluated in six children including both upper extremity (1) and lower extremity (5). Torso lesions evaluated include chest wall (2), abdominal wall (2), and one hemangioma of the gut. This procedure is quickly performed on an outpatient basis, has high anatomic resolution, provides and assessment of these lesions in a manner not available by any other imaging procedure and usually requires no sedation. The radiation exposure for this procedure is low (approximately, a 400mR total body dose) and has been well tolerated by both patients and their parents. Scintigraphic evaluation should be the first diagnostic method utilized in the evaluation of these lesions.

  16. Measurement of Retinal Blood Flow Using Fluorescently Labeled Red Blood Cells1,2,3

    PubMed Central

    Kornfield, Tess E.

    2015-01-01

    Abstract Blood flow is a useful indicator of the metabolic state of the retina. However, accurate measurement of retinal blood flow is difficult to achieve in practice. Most existing optical techniques used for measuring blood flow require complex assumptions and calculations. We describe here a simple and direct method for calculating absolute blood flow in vessels of all sizes in the rat retina. The method relies on ultrafast confocal line scans to track the passage of fluorescently labeled red blood cells (fRBCs). The accuracy of the blood flow measurements was verified by (1) comparing blood flow calculated independently using either flux or velocity combined with diameter measurements, (2) measuring total retinal blood flow in arterioles and venules, (3) measuring blood flow at vessel branch points, and (4) measuring changes in blood flow in response to hyperoxic and hypercapnic challenge. Confocal line scans oriented parallel and diagonal to vessels were used to compute fRBC velocity and to examine velocity profiles across the width of vessels. We demonstrate that these methods provide accurate measures of absolute blood flow and velocity in retinal vessels of all sizes. PMID:26082942

  17. Quantitative label-free redox proteomics of reversible cysteine oxidation in red blood cell membranes.

    PubMed

    Zaccarin, Mattia; Falda, Marco; Roveri, Antonella; Bosello-Travain, Valentina; Bordin, Luciana; Maiorino, Matilde; Ursini, Fulvio; Toppo, Stefano

    2014-06-01

    Reversible oxidation of cysteine residues is a relevant posttranslational modification of proteins. However, the low activation energy and transitory nature of the redox switch and the intrinsic complexity of the analysis render quite challenging the aim of a rigorous high-throughput screening of the redox status of redox-sensitive cysteine residues. We describe here a quantitative workflow for redox proteomics, where the ratio between the oxidized forms of proteins in the control vs treated samples is determined by a robust label-free approach. We critically present the convenience of the procedure by specifically addressing the following aspects: (i) the accurate ratio, calculated from the whole set of identified peptides rather than just isotope-tagged fragments; (ii) the application of a robust analytical pipeline to frame the most consistent data averaged over the biological variability; (iii) the relevance of using stringent criteria of analysis, even at the cost of losing potentially interesting but statistically uncertain data. The pipeline has been assessed on red blood cell membrane challenged with diamide as a model of a mild oxidative condition. The cluster of identified proteins encompassed components of the cytoskeleton more oxidized. Indirectly, our analysis confirmed the previous observation that oxidized hemoglobin binds to membranes while oxidized peroxiredoxin 2 loses affinity. PMID:24642086

  18. Splenic scintigraphy using Tc-99m-labeled heat-denatured red blood cells in pediatric patients: concise communication

    SciTech Connect

    Ehrlich, C.P.; Papanicolaou, N.; Treves, S.; Hurwitz, R.A.; Richards, P.

    1982-03-01

    Ten children underwent splenic imaging with heat-denatured red blood cells labeled with technetium-99m (Tc-99m DRBC). The presenting problems included the heterotaxia syndrome, recurrent idiopathic thrombocytopenic purpura following splenectomy, mass in the left posterior hemithorax, and blunt abdominal trauma. In nine patients, the presence or absence of splenic tissue was established. A splenic hematoma was identified in the tenth patient. All patients were initially scanned with Tc-99m sulfur colloid (Tc-99m SC), and were selected for Tc-99m DRBC scintigraphy only after the results of the SC scans failed to establish the clinical problem beyond doubt. The availability of kits containing stannous ions, essential for efficient and stable labeling of red blood cells with Tc-99m and requiring only a small volume of blood, make splenic scintigraphy in children a relatively simple and definitive diagnostic procedure, when identification of splenic tissue is of clinical importance.

  19. Design of an automated algorithm for labeling cardiac blood pool in gated SPECT images of radiolabeled red blood cells

    SciTech Connect

    Hebert, T.J. |; Moore, W.H.; Dhekne, R.D.; Ford, P.V.; Wendt, J.A.; Murphy, P.H.; Ting, Y.

    1996-08-01

    The design of an automated computer algorithm for labeling the cardiac blood pool within gated 3-D reconstructions of the radiolabeled red blood cells is investigated. Due to patient functional abnormalities, limited resolution, and noise, certain spatial and temporal features of the cardiac blood pool that one would anticipate finding in every study are not present in certain frames or with certain patients. The labeling of the cardiac blood pool requires an algorithm that only relies upon features present in all patients. The authors investigate the design of a fully-automated region growing algorithm for this purpose.

  20. Recurrent gastrointestinal bleeding diagnosed by delayed scintigraphy with Tc-99m-labeled red blood cells.

    PubMed

    Nwakanma, Lois; Meyerrose, Gary; Kennedy, Shalyn; Rakvit, Ariwan; Bohannon, Todd; Silva, Micheal

    2003-08-01

    A 56-year-old woman presented with bright-red blood from the rectum. Esophagogastroduodenoscopy revealed mild gastritis. Colonoscopy demonstrated diverticulosis without active bleeding, and in vitro tagged red blood cell scintigraphy was unremarkable. There was no further evidence of bleeding and the patient was discharged home. The patient returned with recurrent bright-red blood from the rectum. Although delayed scintigraphic images seldom demonstrate the site of bleeding, delayed images at 12 hours demonstrated active bleeding near the hepatic flexure in this patient. This was confirmed with selective mesenteric angiography, and was treated with coil embolization of the tertiary branches of the right middle colic artery. PMID:12897664

  1. Rapid and Label-Free Separation of Burkitt's Lymphoma Cells from Red Blood Cells by Optically-Induced Electrokinetics

    PubMed Central

    Liang, Wenfeng; Zhao, Yuliang; Liu, Lianqing; Wang, Yuechao; Dong, Zaili; Li, Wen Jung; Lee, Gwo-Bin; Xiao, Xiubin; Zhang, Weijing

    2014-01-01

    Early stage detection of lymphoma cells is invaluable for providing reliable prognosis to patients. However, the purity of lymphoma cells in extracted samples from human patients' marrow is typically low. To address this issue, we report here our work on using optically-induced dielectrophoresis (ODEP) force to rapidly purify Raji cells' (a type of Burkitt's lymphoma cell) sample from red blood cells (RBCs) with a label-free process. This method utilizes dynamically moving virtual electrodes to induce negative ODEP force of varying magnitudes on the Raji cells and RBCs in an optically-induced electrokinetics (OEK) chip. Polarization models for the two types of cells that reflect their discriminate electrical properties were established. Then, the cells' differential velocities caused by a specific ODEP force field were obtained by a finite element simulation model, thereby established the theoretical basis that the two types of cells could be separated using an ODEP force field. To ensure that the ODEP force dominated the separation process, a comparison of the ODEP force with other significant electrokinetics forces was conducted using numerical results. Furthermore, the performance of the ODEP-based approach for separating Raji cells from RBCs was experimentally investigated. The results showed that these two types of cells, with different concentration ratios, could be separated rapidly using externally-applied electrical field at a driven frequency of 50 kHz at 20 Vpp. In addition, we have found that in order to facilitate ODEP-based cell separation, Raji cells' adhesion to the OEK chip's substrate should be minimized. This paper also presents our experimental results of finding the appropriate bovine serum albumin concentration in an isotonic solution to reduce cell adhesion, while maintaining suitable medium conductivity for electrokinetics-based cell separation. In short, we have demonstrated that OEK technology could be a promising tool for efficient and

  2. Rapid and label-free separation of Burkitt's lymphoma cells from red blood cells by optically-induced electrokinetics.

    PubMed

    Liang, Wenfeng; Zhao, Yuliang; Liu, Lianqing; Wang, Yuechao; Dong, Zaili; Li, Wen Jung; Lee, Gwo-Bin; Xiao, Xiubin; Zhang, Weijing

    2014-01-01

    Early stage detection of lymphoma cells is invaluable for providing reliable prognosis to patients. However, the purity of lymphoma cells in extracted samples from human patients' marrow is typically low. To address this issue, we report here our work on using optically-induced dielectrophoresis (ODEP) force to rapidly purify Raji cells' (a type of Burkitt's lymphoma cell) sample from red blood cells (RBCs) with a label-free process. This method utilizes dynamically moving virtual electrodes to induce negative ODEP force of varying magnitudes on the Raji cells and RBCs in an optically-induced electrokinetics (OEK) chip. Polarization models for the two types of cells that reflect their discriminate electrical properties were established. Then, the cells' differential velocities caused by a specific ODEP force field were obtained by a finite element simulation model, thereby established the theoretical basis that the two types of cells could be separated using an ODEP force field. To ensure that the ODEP force dominated the separation process, a comparison of the ODEP force with other significant electrokinetics forces was conducted using numerical results. Furthermore, the performance of the ODEP-based approach for separating Raji cells from RBCs was experimentally investigated. The results showed that these two types of cells, with different concentration ratios, could be separated rapidly using externally-applied electrical field at a driven frequency of 50 kHz at 20 Vpp. In addition, we have found that in order to facilitate ODEP-based cell separation, Raji cells' adhesion to the OEK chip's substrate should be minimized. This paper also presents our experimental results of finding the appropriate bovine serum albumin concentration in an isotonic solution to reduce cell adhesion, while maintaining suitable medium conductivity for electrokinetics-based cell separation. In short, we have demonstrated that OEK technology could be a promising tool for efficient and

  3. Technetium-99m labeled red blood cells for the detection and localization of cavernous hemangiomas of the bone

    SciTech Connect

    Lenane, P.

    1986-09-01

    Labeled red blood cells (RBCs) have already been proven useful in the detection and localization of many vascular abnormalities. One such abnormality is that of a cavernous hemangioma. Cavernous hemangiomas have a distinct circulation and have been found in many areas of the body. The ability to utilize this unique circulation is important to consider when choosing a diagnostic exam. This paper reports a case demonstrating the usefulness of labeled red blood cells for the detection and localization of cavernous hemangioma of the bone. A 31-yr-old female present with a history of persistent generalized headaches for many years. About 1 yr prior to the exam, she noticed that her headaches had become more localized to the right side of her head. Physical examination revealed a palpable lump developing on the right side of her head which was sensitive to the touch. The patient was then scheduled for a CT scan to be followed by both a bone scan and a /sup 99m/Tc blood-pool scan. A flow study using 15 mCi /sup 99m/Tc labeled RBCs was performed in the right lateral position at 1.5 sec/frame for 32 frames. Immediate blood-pool images 30-min, and 1-hr delayed images were recorded.

  4. A case of metastatic malignancy masquerading as a hepatic hemangioma on labeled red blood cell scintigraphy.

    PubMed

    Farlow, D C; Little, J M; Gruenewald, S M; Antico, V F; O'Neill, P

    1993-07-01

    A 36-yr-old woman with a past history of gastric neuro-endocrine carcinoma (carcinoid tumor) underwent 99mTc-red blood cell (RBC) scintigraphy for evaluation of a 2-cm echogenic liver mass demonstrated on ultrasound. Scan findings were typical of a cavernous hemangioma. On follow-up, however, there was progressive lesion enlargement; histopathology of the resected mass revealed neuro-endocrine carcinoma. This case report, one of the few examples of a false-positive 99mTc-RBC scan, highlights the need for cautious evaluation of focal liver masses, even when there are typical scintigraphic features of cavernous hemangioma. PMID:8315498

  5. Effect of exercise on erythrocyte count and blood activity concentration after technetium-99m in vivo red blood cell labeling

    SciTech Connect

    Konstom, M.A.; Tu'meh, S.; Wynne, J.; Beck, J.R.; Kozlowski, J.; Holman, B.L.

    1982-09-01

    The effects of exercise on blood radiotracer concentration after technetium-99m in vivo red blood cell labeling was studied. After red blood cell labeling, 13 subjects underwent maximal supine bicycle exercise. Radioactivity, analyzed with a well counter, was measured in heparinized venous blood samples drawn at rest and during peak exercise. Changes in activity were compared with changes in erythrocyte count. Activity and erythrocyte counts increased in erythrocyte count (r=0.78), but did not correlate with either duration of exercise or maximal heart rate. Twenty minutes after termination of exercise, activity and erythrocyte count had decreased from peak exercise values but remained higher than preexercise values. In nine nonexercised control subjects, samples drawn 20 minutes apart showed no change in activity or in erythrocyte count. It was concluded that exercise increases blood activity, primarily because of an increase in erythrocyte count. During radionuclide ventriculography, blood activity must be measured before and after any intervention, particularly exercise, before a change in left ventricular activity can be attributed to a change in left ventricular volume.

  6. Effect of Peumus boldus on the labeling of red blood cells and plasma proteins with technetium-99m.

    PubMed

    Reiniger, I W; de Oliveira, J F; Caldeira-de-Araújo, A; Bernardo-Filho, M

    1999-08-01

    Peumus boldus is used in popular medicine in Brazil. The influence of Peumus boldus on the labeling of red blood cells and plasma proteins with 99mTc was studied. Stannous chloride and 99mTc pertechnetate were incubated with blood and a tincture of Peumus boldus. Aliquots of plasma and blood cells were isolated from the mixture and treated with trichloroacetic acid (TCA). After separation, analysis of the soluble and insoluble fractions showed a rapid uptake of the radioactivity by blood cells in the presence of the drug, whereas there was a slight decrease in the amount of 99mTc radioactivity in the TCA-insoluble fraction of plasma. PMID:10376326

  7. Effect of Thuya occidentalis on the labeling of red blood cells and plasma proteins with technetium-99m.

    PubMed Central

    Oliveira, J. F.; Braga, A. C.; Avila, A. S.; Fonseca, L. M.; Gutfilen, B.; Bernardo-Filho, M.

    1996-01-01

    Thuya occidentalis is used in popular medicine in the treatment of condyloma and has antibacterial action. Red blood cells (RBC) labeled with technetium-99m (99mTc) are used for several evaluations in nuclear medicine. This labeling depends on a reducing agent, usually stannous ion. Any drug which alters the labeling of the tracer could be expected to modify the disposition of the radiopharmaceutical. We have evaluated the influence of T. occidentalis extract on the labeling of RBC and plasma proteins with 99mTc. Blood was withdrawn and incubated with T. occidentalis (0.25; 2.5; 20.5; and 34.1 percent v/v). Stannous chloride (1.2 micrograms/ml) was added and then 99mTc was added. Plasma (P) and blood cells (BC) were isolated, also precipitated with trichloroacetic acid and soluble (SF) and insoluble fractions (IF) separated. The analysis of the results shows that there is a decrease in radioactivity (from 97.64 to 75.89 percent) in BC with 34.1 percent of the drug. In the labeling process of RBC with 99mTc, the stannous and pertechnetate ions pass through the membrane, so we suggest that the T. occidentalis effect can be explained (i) by an inhibition of the transport of these ions, (ii) by damage in membrane, (iii) by competition with the cited ions for the same binding sites, or (iv) by possible generation of reactive oxygen species that could oxidize the stannous ion. PMID:9436292

  8. Computational design of a red fluorophore ligase for site-specific protein labeling in living cells

    DOE PAGESBeta

    Liu, Daniel S.; Nivon, Lucas G.; Richter, Florian; Goldman, Peter J.; Deerinck, Thomas J.; Yao, Jennifer Z.; Richardson, Douglas; Phipps, William S.; Ye, Anne Z.; Ellisman, Mark H.; et al

    2014-10-13

    In this study, chemical fluorophores offer tremendous size and photophysical advantages over fluorescent proteins but are much more challenging to target to specific cellular proteins. Here, we used Rosetta-based computation to design a fluorophore ligase that accepts the red dye resorufin, starting from Escherichia coli lipoic acid ligase. X-ray crystallography showed that the design closely matched the experimental structure. Resorufin ligase catalyzed the site-specific and covalent attachment of resorufin to various cellular proteins genetically fused to a 13-aa recognition peptide in multiple mammalian cell lines and in primary cultured neurons. We used resorufin ligase to perform superresolution imaging of themore » intermediate filament protein vimentin by stimulated emission depletion and electron microscopies. This work illustrates the power of Rosetta for major redesign of enzyme specificity and introduces a tool for minimally invasive, highly specific imaging of cellular proteins by both conventional and superresolution microscopies.« less

  9. Computational design of a red fluorophore ligase for site-specific protein labeling in living cells

    SciTech Connect

    Liu, Daniel S.; Nivon, Lucas G.; Richter, Florian; Goldman, Peter J.; Deerinck, Thomas J.; Yao, Jennifer Z.; Richardson, Douglas; Phipps, William S.; Ye, Anne Z.; Ellisman, Mark H.; Drennan, Catherine L.; Baker, David; Ting, Alice Y.

    2014-10-13

    In this study, chemical fluorophores offer tremendous size and photophysical advantages over fluorescent proteins but are much more challenging to target to specific cellular proteins. Here, we used Rosetta-based computation to design a fluorophore ligase that accepts the red dye resorufin, starting from Escherichia coli lipoic acid ligase. X-ray crystallography showed that the design closely matched the experimental structure. Resorufin ligase catalyzed the site-specific and covalent attachment of resorufin to various cellular proteins genetically fused to a 13-aa recognition peptide in multiple mammalian cell lines and in primary cultured neurons. We used resorufin ligase to perform superresolution imaging of the intermediate filament protein vimentin by stimulated emission depletion and electron microscopies. This work illustrates the power of Rosetta for major redesign of enzyme specificity and introduces a tool for minimally invasive, highly specific imaging of cellular proteins by both conventional and superresolution microscopies.

  10. Computational design of a red fluorophore ligase for site-specific protein labeling in living cells

    PubMed Central

    Liu, Daniel S.; Nivón, Lucas G.; Richter, Florian; Goldman, Peter J.; Deerinck, Thomas J.; Yao, Jennifer Z.; Richardson, Douglas; Phipps, William S.; Ye, Anne Z.; Ellisman, Mark H.; Drennan, Catherine L.; Baker, David; Ting, Alice Y.

    2014-01-01

    Chemical fluorophores offer tremendous size and photophysical advantages over fluorescent proteins but are much more challenging to target to specific cellular proteins. Here, we used Rosetta-based computation to design a fluorophore ligase that accepts the red dye resorufin, starting from Escherichia coli lipoic acid ligase. X-ray crystallography showed that the design closely matched the experimental structure. Resorufin ligase catalyzed the site-specific and covalent attachment of resorufin to various cellular proteins genetically fused to a 13-aa recognition peptide in multiple mammalian cell lines and in primary cultured neurons. We used resorufin ligase to perform superresolution imaging of the intermediate filament protein vimentin by stimulated emission depletion and electron microscopies. This work illustrates the power of Rosetta for major redesign of enzyme specificity and introduces a tool for minimally invasive, highly specific imaging of cellular proteins by both conventional and superresolution microscopies. PMID:25313043

  11. Technetium-99m-labeled red blood cells in the evaluation of hemangiomas of the liver in infants and children

    SciTech Connect

    Miller, J.H.

    1987-09-01

    The vascular origin lesions of the liver (capillary hemangioma/infantile hemangioendothelioma) that present in infancy or early childhood often have a typical clinical picture of hepatomegaly and congestive heart failure. These lesions rarely present as asymptomatic hepatomegaly, simulating a primary hepatic malignancy. These lesions may also simulate a primary or secondary hepatic malignancy on cross-sectional imaging or angiography. Scintigraphic evaluations with technetium-99m-labeled red blood cells offers an accurate method of identification of these lesions, and allows differentiation from other common primary or secondary hepatic masses in infancy or childhood. This scintigraphic method may also be used to follow these patients after medical, radiation, or embolization therapy. Experience with seven patients with these tumors is reported and compared with eight children with other primary or secondary liver tumors also evaluated by this method.

  12. Technetium-99m red blood cell labeling in patients treated with doxorubicin

    SciTech Connect

    Ballinger, J.R.; Gerson, B.; Gulenchyn, K.Y.; Ruddy, T.D.; Davies, R.A.

    1988-03-01

    Radionuclide angiography is useful in monitoring cardiotoxicity of doxorubicin, but in vivo RBC labeling in these patients is believed to be poorer than that in general patients. The left ventricle-to-background activity ratio (R) was not significantly lower in patients treated with doxorubicin (3.24 +/- 1.15, N = 13) than in control patients (3.89 +/- 1.60, N = 14). With both modified in vivo and in vitro labeling, R was significantly improved in patients treated with doxorubicin (4.37 +/- 0.91, N = 8, and 4.37 +/- 1.22, N = 13, respectively). However, with the modified in vivo method, labeling efficiency remained a function of hematocrit, whereas the in vitro method removed this dependency. Both modified in vivo and in vitro labeling result in improved image quality over in vivo labeling in patients treated with doxorubicin, and the choice of method can be based on other factors.

  13. Paramagnetic Gd(3+) labeled red blood cells for magnetic resonance angiography.

    PubMed

    Aryal, Santosh; Stigliano, Cinzia; Key, Jaehong; Ramirez, Maricela; Anderson, Jeff; Karmonik, Christof; Fung, Steve; Decuzzi, Paolo

    2016-08-01

    Despite significant advances in contrast enhanced-magnetic resonance angiography, the lack of truly blood-pool agents with long circulating property is limiting the clinical impact of this imaging technique. The terminal half-life for blood elimination of most small molecular weight gadolinium (Gd) based extracellular fluid agents is about 1.5 h when administered intravenously to subjects with normal renal function. The small size of these extracellular fluid agents does not prevent them from extravasating, especially from damaged vessels which are generally hyperpermeable. Therefore, the development of novel, clinically relevant blood pool contrast agents is critically needed to improve outcomes in the prevention, detection, and treatment of vascular diseases. We have demonstrated the fusion strategies in which the Gd-liposome without any stealth property radically fuses with red blood cells (RBCs) forming MR glowing Gd-RBC with the order of magnitude enhancements in circulation half-life (t1/2 = 50 h) and r1 relaxivity (r1 = 19.0 mM(-1) s(-1)) of Gd. The in vivo contrast enhancement of Gd-RBC was studied by using 3T clinical MR scanner for extended period of time, which clearly visualized the abdominal aorta. In summary, the vascular delivery of blood pool agents may benefit from carriage by RBCs because it naturally stays within the vascular lumen. PMID:27192419

  14. Accelerated removal of antibody-coated red blood cells from the circulation is accurately tracked by a biotin label

    PubMed Central

    Mock, Donald M.; Lankford, Gary L.; Matthews, Nell I.; Burmeister, Leon F.; Kahn, Daniel; Widness, John A.; Strauss, Ronald G.

    2013-01-01

    BACKGROUND Safe, accurate methods to reliably measure circulating red blood cell (RBC) kinetics are critical tools to investigate pathophysiology and therapy of anemia, including hemolytic anemias. This study documents the ability of a method using biotin-labeled RBCs (BioRBCs) to measure RBC survival (RCS) shortened by coating with a highly purified monomeric immunoglobulin G antibody to D antigen. STUDY DESIGN AND METHODS Autologous RBCs from 10 healthy D+ subjects were labeled with either biotin or 51Cr (reference method), coated (opsonized) either lightly (n = 4) or heavily (n = 6) with anti-D, and transfused. RCS was determined for BioRBCs and for 51Cr independently as assessed by three variables: 1) posttransfusion recovery at 24 hours (PTR24) for short-term RCS; 2) time to 50% decrease of the label (T50), and 3) mean potential life span (MPL) for long-term RCS. RESULTS BioRBCs tracked both normal and shortened RCS accurately relative to 51Cr. For lightly coated RBCs, mean PTR24, T50, and MPL results were not different between BioRBCs and 51Cr. For heavily coated RBCs, both short-term and long-term RCS were shortened by approximately 17 and 50%, respectively. Mean PTR24 by BioRBCs (84 ± 18%) was not different from 51Cr (81 ± 10%); mean T50 by BioRBCs (23 ± 17 days) was not different from 51Cr (22 ± 18 days). CONCLUSION RCS shortened by coating with anti-D can be accurately measured by BioRBCs. We speculate that BioRBCs will be useful for studying RCS in conditions involving accelerated removal of RBCs including allo- and autoimmune hemolytic anemias. PMID:22023312

  15. Placental localization in abdominal pregnancy using technetium-99m-labeled red blood cells

    SciTech Connect

    Martin, B.; Payan, J.M.; Jones, J.S.; Buse, M.G. )

    1990-06-01

    In a patient with third trimester abdominal pregnancy with fetal demise, technetium-99m-labeled erythrocytes ({sup 99m}Tc-RBCs) localized the placenta preoperatively, after nonvisualization by ultrasonography and arteriography. Extrauterine placental localization by blood-pool imaging may be useful when ultrasound fails.

  16. Preparation and radiochemical control of 99mTc labeled blood pool agent for in vivo labelling of the red blood cells.

    PubMed

    Ahmad, Israr; Amir, Noshad; Durr-E-Sabih; Bin Asad, Muhammad Hassham Hassan; Rahim, Muhammad Kashif; Hussain, Muhammad Shahzad; Murtaza, Ghulam; Shah, Syed Nisar Hussaian

    2014-01-01

    Our aim was to prepare cheap blood pool imaging kits by simplified method to overcome the burden on purchase department of MINAR, Nishtar Hospital, Multan, Pakistan. Secondarily, prompt supply of kits should save the time of patient during transportation. A total of 24 subjects selected for this study were equally divided into two groups. Mixture of stannous chloride and sodium pyrophosphate solution at pH 7 was injected to these subjects. Various concentrations (ranging from 200 to 800 microg) of stannous chloride dihydrate were injected to group one, followed by intravenous administration of technetium-99m (99mTc) pertechnetate at 30 min interval in 12 subjects. Labeling percentage of each sample was calculated afterwards followed by imaging under gamma camera. Each parameter was tested on three different patients and average of these three was calculated. In second set of experiments done on group two the same procedure was repeated in another 12 subjects, while keeping the concentration of Sn PYP constant at 400 microg. In this case, 99mTc was administered at different time intervals in different subjects ranging from 15 to 120 min (15, 30, 60 and 120 min) followed by calculation of labeling percentage and imaging under gamma camera. In group one, average percentage values of binding of red blood cells with 99mTc were 23.24, 84.88, 83.78 and 60.33% for concentrations of 200, 400, 600 and 800 microg, respectively. In group two, average percentage binging values of 22.26, 84.36. 55.54 and 28.67% were calculated at time intervals of 15, 30, 60 and 120 min, respectively. It is concluded from the results that the best blood pool imaging under gamma camera was observed for the concentration of 400 microg and the time interval of 30 min. The maximum percentage binding of red blood cells with 99mTc was calculated at concentration of 400 microg after 30 min interval that also correlated with imaging results. PMID:25272643

  17. Red blood cell production

    MedlinePlus

    ... cells are an important element of blood. Their job is to transport oxygen to the body’s tissues in exchange for carbon dioxide, which is carried to and eliminated by the lungs. Red blood cells are formed in the red bone marrow of bones. Stem cells in the red bone marrow called hemocytoblasts ...

  18. Effect of exercise on erythrocyte count and blood activity concentration after /sup 99m/Tc in vivo red blood cell labeling

    SciTech Connect

    Konstam, M.A.; Tu'meh, S.; Wynne, J.; Beck, J.R.; Kozlowski, J.; Holman, B.L.

    1982-09-01

    We studied the effect of exercise on blood radiotracer concentration after /sup 99m/Tc in vivo red blood cell labeling. After red blood cell labeling, 13 subjects underwent maximal supine bicycle exercise. Radioactivity, analyzed with a well counter, was measured in heparinized venous blood samples drawn at rest and during peak exercise. Changes in activity were compared with changes in erythrocyte count. Activity and erythrocyte counts increased during exercise in all 13 subjects. Percent increase in activity correlated with percent increase in erythrocyte count (r . -0.78), but did not correlate with either duration of exercise or maximal heart rate. Twenty minutes after termination of exercise, activity and erythrocyte count had decreased from peak exercise values but remained higher than preexercise values. In nine nonexercised control subjects, samples drawn 20 minutes apart showed no change in activity or in erythrocyte count. We conclude that exercise increases blood activity, primarily because of an increase in erythrocyte count. During radionuclide ventriculography, blood activity must be measured before and after any intervention, particularly exercise, before a change in left ventricular activity can be attributed to a change in left ventricular volume.

  19. Radiolabeled red blood cells: status, problems, and prospects

    SciTech Connect

    Srivastava, S.C.

    1983-01-01

    Radionuclidic labels for red cells can be divided into two main categories - cohort or pulse labels, and random labels. The random labels are incorporated into circulating cells of all ages and the labeling process is usually carried out in vitro. The red cell labels in predominant use involve random labeling and employ technetium-99m, chromium-51, indium-111, and gallium-68, roughly in that order. The extent of usefulness depends on the properties of the label such as the half-life, decay mode, and in-vivo stability, etc. Labeled cells can be used for red cell survival measurements when the half-life of the radionuclide is sufficiently long. The major portion of this article deals with random labels.

  20. Use of an oral stable isotope label to confirm variation in red blood cell mean age that influences HbA1c interpretation

    PubMed Central

    Lindsell, Christopher J.; Rogge, Mary Colleen; Haggerty, Shannon; Wagner, David A.; Palascak, Mary B.; Mehta, Shilpa; Hibbert, Jacqueline M.; Joiner, Clinton H.; Franco, Robert S.; Cohen, Robert M.

    2014-01-01

    HbA1c is commonly used to monitor glycemic control. However, there is growing evidence that the relationship between HbA1c and mean blood glucose (MBG) is influenced by variation in red blood cell (RBC) lifespan in hematologically normal individuals. Correction of HbA1c for mean RBC age (MRBC) requires a noninvasive, accurate, and affordable method to measure RBC survival. In this study, we evaluated whether a stable isotope approach would satisfy these requirements. RBC lifespan and MRBC were determined in a group of nine hematologically normal diabetic and nondiabetic subjects using oral 15N-glycine to label heme in an age cohort of RBC. The MRBC was 58.7 ± 9.1 (2SD) days and RBC lifespan was 106 ± 21 (2SD) days. This degree of variation (±15 - 20%) is consistent with previous studies using other techniques. In a subset of seven subjects, MRBC determined with the biotin label technique were available from approximately five years prior, and strongly correlated with the stable isotope values (R2 = 0.79). This study suggests that the MRBC is stable over time but varies substantially among individuals, and supports the importance of its variation in HbA1c interpretation. The characteristics of the stable isotope method support its suitability for studies to directly evaluate the impact of variation in MRBC on the interpretation of HbA1c. PMID:25293624

  1. Red blood cell production

    MedlinePlus Videos and Cool Tools

    ... or another. Red blood cells are an important element of blood. Their job is to transport oxygen ... hemocytoblasts give rise to all of the formed elements in blood. If a hemocytoblast commits to becoming ...

  2. Red blood cells, multiple sickle cells (image)

    MedlinePlus

    Sickle cell anemia is an inherited disorder in which abnormal hemoglobin (the red pigment inside red blood cells) is produced. The abnormal hemoglobin causes red blood cells to assume a sickle shape, like the ones seen in this photomicrograph.

  3. Recent developments in blood cell labeling research

    SciTech Connect

    Srivastava, S.C.; Straub, R.F.; Meinken, G.E.

    1988-09-07

    A number of recent developments in research on blood cell labeling techniques are presented. The discussion relates to three specific areas: (1) a new in vitro method for red blood cell labeling with /sup 99m/Tc; (2) a method for labeling leukocytes and platelets with /sup 99m/Tc; and (3) the use of monoclonal antibody technique for platelet labeling. The advantages and the pitfalls of these techniques are examined in the light of available mechanistic information. Problems that remain to be resolved are reviewed. An assessment is made of the progress as well as prospects in blood cell labeling methodology including that using the monoclonal antibody approach. 37 refs., 4 figs.

  4. Red blood cells, multiple sickle cells (image)

    MedlinePlus

    ... inherited disorder in which abnormal hemoglobin (the red pigment inside red blood cells) is produced. The abnormal hemoglobin causes red blood cells to assume a sickle shape, like the ones seen in this photomicrograph.

  5. Phosphatidylserine exposure and red cell viability in red cell aging and in hemolytic anemia

    PubMed Central

    Boas, Franz Edward; Forman, Linda; Beutler, Ernest

    1998-01-01

    Phosphatidylserine (PS) normally localizes to the inner leaflet of cell membranes but becomes exposed in abnormal or apoptotic cells, signaling macrophages to ingest them. Along similar lines, it seemed possible that the removal of red cells from circulation because of normal aging or in hemolytic anemias might be triggered by PS exposure. To investigate the role of PS exposure in normal red cell aging, we used N-hydroxysuccinimide-biotin to tag rabbit red cells in vivo, then used phycoerythrin-streptavidin to label the biotinylated cells, and annexin V-fluorescein isothiocyanate (FITC) to detect the exposed PS. Flow cytometric analysis of these cells drawn at 10-day intervals up to 70 days after biotinylation indicated that older, biotinylated cells expose more PS. Furthermore, our data match a simple model of red cell senescence that assumes both an age-dependent destruction of senescent red cells preceded by several hours of PS exposure and a random destruction of red cells without PS exposure. By using this model, we demonstrated that the exposure of PS parallels the rate at which biotinylated red cells are removed from circulation. On the other hand, using an annexin V-FITC label and flow cytometry demonstrates that exposed PS does not cause the reduced red cell life span of patients with hemolytic anemia, with the possible exception of those with unstable hemoglobins or sickle cell anemia. Thus, in some cases PS exposure on the cell surface may signal the removal of red cells from circulation, but in other cases some other signal must trigger the sequestration of cells. PMID:9501218

  6. Preoperative localization of intermittently bleeding small intestinal tumors using Tc-99m labeled red blood cell scanning. Report of two cases

    SciTech Connect

    Oliver, G.C.; Rubin, R.J.; Park, Y.H.; Ashton, J.K.

    1987-09-01

    Frequent tagged red blood cell scans offer an important diagnostic adjunct to help define a site of intermittent bleeding. Success is based upon scanning at two-to-four-hour intervals. Two patients are presented who experienced intermittent episodes of melena and hematochezia over prolonged periods of time. In each case an extensive diagnostic work-up had been performed on multiple occasions and failed to demonstrate the source. Utilizing a Technetium-99 macroaggregated albumin (Tc-99m) tagged red blood cell scan, an intermittently bleeding lesion within the small bowel was identified in each instance. In order to detect an intermittently bleeding lesion within the small bowel, more frequent scanning intervals are recommended. Due to rapid clearing of tagged red blood cells into the colon from the small-bowel bleeding point, the source may be obscured by longer, routine scanning intervals.

  7. Far-Red Fluorescent Lipid-Polymer Probes for an Efficient Labeling of Enveloped Viruses.

    PubMed

    Lacour, William; Adjili, Salim; Blaising, Julie; Favier, Arnaud; Monier, Karine; Mezhoud, Sarra; Ladavière, Catherine; Place, Christophe; Pécheur, Eve-Isabelle; Charreyre, Marie-Thérèse

    2016-08-01

    Far-red emitting fluorescent lipid probes are desirable to label enveloped viruses, for their efficient tracking by optical microscopy inside autofluorescent cells. Most used probes are rapidly released from membranes, leading to fluorescence signal decay and loss of contrast. Here, water-soluble lipid-polymer probes are synthesized harboring hydrophilic or hydrophobic far-red emitting dyes, and exhibiting enhanced brightness. They efficiently label Hepatitis C Virus pseudotyped particles (HCVpp), more stably and reproducibly than commercial probes, and a strong fluorescence signal is observed with a high contrast. Labeling with such probes do not alter virion morphology, integrity, nor infectivity. Finally, it is shown by fluorescence microscopy that these probes enable efficient tracking of labeled HCVpp inside hepatocarcinoma cells used as model hepatocytes, in spite of their autofluorescence up to 700 nm. These novel fluorescent lipid-polymer probes should therefore enable a better characterization of early stages of infection of autofluorescent cells by enveloped viruses. PMID:27113918

  8. Red blood cells, sickle cell (image)

    MedlinePlus

    Sickle cell anemia is an inherited blood disease in which the red blood cells produce abnormal pigment (hemoglobin). ... abnormal hemoglobin causes deformity of the red blood cells into crescent or sickle-shapes, as seen in this photomicrograph.

  9. Red blood cells, sickle cell (image)

    MedlinePlus

    Sickle cell anemia is an inherited blood disease in which the red blood cells produce abnormal pigment (hemoglobin). The abnormal hemoglobin causes deformity of the red blood cells into crescent or sickle-shapes, as seen in this photomicrograph.

  10. Red blood cells, sickle cells (image)

    MedlinePlus

    These crescent or sickle-shaped red blood cells (RBCs) are present with Sickle cell anemia, and stand out clearly against the normal round RBCs. These abnormally shaped cells may become entangled and ...

  11. Red Blood Cell Antibody Identification

    MedlinePlus

    ... be limited. Home Visit Global Sites Search Help? RBC Antibody Identification Share this page: Was this page helpful? Also known as: Alloantibody Identification; Antibody ID, RBC; RBC Ab ID Formal name: Red Blood Cell ...

  12. Comparison of technetium-99m IgG with technetium-99m red blood cells labeling in cardiac blood-pool scintigraphy: a preliminary study.

    PubMed

    Javadi, Hamid; Asli, Isa Neshandar; Semnani, Shahriar; Jallalat, Sara; Ansari, Mojtaba; Amini, Abdullatif; Barekat, Maryam; Assadi, Majid

    2011-01-01

    This first clinical prospective study was conducted to use of technetium-99m immunoglobulin G ((99m)Tc-IgG) as compared with autologous (99m)Tc-red blood cells (RBC) in gated blood pool ventriculography. We studied 12 patients who referred to us for a possible diagnosis of liver hemangioma or infection. Six patients underwent gated planar blood pool (GPBP) acquisition using (99m)Tc-RBC and 6 GPBP acquisition using (99m)Tc-IgG. The use of (99m)Tc-IgG in cardiac blood pool studies provided comparable images to (99m)Tc-RBC. In conclusion, (99m)Tc-IgG, which is readily available and needs only a single injection, may be an attractive alternative to (99m)Tc-RBC for the estimation of various cardiac function parameters like left ventricular function. PMID:21512662

  13. Estimation of anti-D IgG in red blood cell eluates using the specific radioactivity of 125I-labeled IgG: effect of unlabeled, cytophilic IgG

    SciTech Connect

    Masouredis, S.P.; Mahan, L.C.; Sudora, E.J.; Langley, J.W.; Victoria, E.J.

    1981-01-01

    The specific radioactivity of conventionally prepared 125I IgG anti-D eluates is significantly less (from 1/5 to 1/20) than that of the 125I IgG fraction used to prepare the eluate. This discrepancy is due to the release of unlabeled, cytophilic IgG from normal red blood cells during eluate preparation and does not represent an underestimation of the eluate anti-D IgG content. Cytophilic IgG content of eluates plays an important role in reducing the nonimmunologic binding of labeled antibody IgG. The results justify the assumption used in numerous studies that the specific radioactivity of 125I IgG fractions can be used to provide a valid estimate of the anti-D IgG content of eluates.

  14. Red cell metabolism studies on Skylab

    NASA Technical Reports Server (NTRS)

    Mengel, C. E.

    1977-01-01

    Blood samples from Spacelab crewmembers were studied for possible environment effects on red cell components. Analysis involved peroxidation of red cell lipids, enzymes of red cell metabolism, and levels of 2,3-diphosphoglyceric acid and adenosine triphosphate. Results show that there is no evidence of lipid peroxidation, that biochemical effect known to be associated with irreversible red cell damage. Changes observed in glycolytic intermediates and enzymes cannot be directly implicated as indicating evidence of red cell damage.

  15. Red cell DAMPs and inflammation.

    PubMed

    Mendonça, Rafaela; Silveira, Angélica A A; Conran, Nicola

    2016-09-01

    Intravascular hemolysis, or the destruction of red blood cells in the circulation, can occur in numerous diseases, including the acquired hemolytic anemias, sickle cell disease and β-thalassemia, as well as during some transfusion reactions, preeclampsia and infections, such as those caused by malaria or Clostridium perfringens. Hemolysis results in the release of large quantities of red cell damage-associated molecular patterns (DAMPs) into the circulation, which, if not neutralized by innate protective mechanisms, have the potential to activate multiple inflammatory pathways. One of the major red cell DAMPs, heme, is able to activate converging inflammatory pathways, such as toll-like receptor signaling, neutrophil extracellular trap formation and inflammasome formation, suggesting that this DAMP both activates and amplifies inflammation. Other potent DAMPs that may be released by the erythrocytes upon their rupture include heat shock proteins (Hsp), such as Hsp70, interleukin-33 and Adenosine 5' triphosphate. As such, hemolysis represents a major inflammatory mechanism that potentially contributes to the clinical manifestations that have been associated with the hemolytic diseases, such as pulmonary hypertension and leg ulcers, and likely plays a role in specific complications of sickle cell disease such as endothelial activation, vaso-occlusive processes and tissue injury. PMID:27251171

  16. Red cell distribution width and nonalcoholic steatohepatitis

    PubMed Central

    Gulcan Kurt, Yasemin; Cayci, Tuncer; Aydin, Fevzi Nuri; Agilli, Mehmet

    2014-01-01

    Red cell distribution width is a measure of deviation of the volume of red blood cells. It is a marker of anisocytosis and often used to evaluate the possible causes of anemia. Elevated red cell distribution width levels are also associated with acute and chronic inflammatory responses. In nonalcoholic steatohepatitis, inflammation is accompanied with steatosis. For assuming red cell distribution width as a marker of nonalcoholic steatohepatitis, intervening factors such as levels of inflammatory markers should also be evaluated. PMID:25473202

  17. Carbon dots as a fluorescent probe for label-free detection of physiological potassium level in human serum and red blood cells.

    PubMed

    Zhang, Lingyang; Chen, Shenna; Zhao, Qian; Huang, Haowen

    2015-06-23

    A unique photoluminescence carbon dots (CDs) with larger size were prepared by microwave-assisted method. Complex functional groups on the surface of the CDs facilitate the nanoparticles to form affinity with some metal ions. Taking advantage of the effective fluorescence quenching effect of K(+), a highly sensitive CD-based fluorescence analytical system for label-free detection of K(+) with limit of detection (LOD) 1.0×10(-12) M was established. The concentrations of potassium ion in biological samples such as human serum are usually found at millimolar levels or even higher. The proposed method begins with a substantial dilution of the sample to place the K(+) concentration in the dynamic range for quantification, which covers 3 orders of magnitude. This offers some advantages: the detection of K(+) only needs very small quantities of biological samples, and the dilution of samples such as serum may effectively eliminate the potential interferences that often originate from the background matrix. The determined potassium levels were satisfactory and closely comparable with the results given by the hospital, indicating that this fluorescent probe is applicable to detection of physiological potassium level with high accuracy. Compared with other relative biosensors requiring modified design, bio-molecular modification or/and sophisticated instruments, this CD-based sensor is very simple, cost-effective and easy detection, suggesting great potential applications for successively monitoring physiological potassium level and the change in biological system. PMID:26092345

  18. What is red cell deformability?

    PubMed

    Schmid-Schönbein, H; Gaehtgens, P

    1981-01-01

    Microscopic flow visualization of the process of red cell adaptation to flow shows that red cell deformation in flow is the consequence of a continuous viscous rather than an elastic deformation. This fluid drop-like adaptation primarily depends on: (a) the fluidity of the cytoplasm and (b) the favourable surface-area-to-volume ratio, with an excess of surface area allowing strong deformations without an increase in surface area (a real strain). (c) In contrast to previous notions, the modulus of shear elasticity of the membrane is probably less significant. After many attempts to differentiate the contribution of bending and shear stiffness to the elastic recovery of the normal biconcave cell shape have not produced equivocal results, we have changed the elastic shear modulus experimentally by cross-linking the spectrin using the membrane-permeant, bifunctional SH-reagent DIAMIDE, which allows to increase the elastic shear modulus in a dose-dependent manner. Despite a 25-fold decrease in compliance the DIAMIDE-treated cells have normal shape and show remarkably small changes in the rheological behaviour when tested in vitro and in vivo. PMID:6948373

  19. Red blood cell volume in preterm neonates

    SciTech Connect

    Quaife, M.A.; Dirksen, J.W.; Paxson, C.L. Jr.; McIntire, R.H. Jr.

    1981-10-01

    In the high-risk neonate, the direct determination of the red cell volume by radionuclide dilution technique appears to be the singularly definitive method of defining treatment efficacy, and is thus a useful evaluation and management tool for the pediatrician. For effective patient management, the red blood cell(RBC) volume of 69 preterm and term neonates was determined. The method utilized, Tc-99m-labeled RBCs, provided a fast and accurate answer with a large reduction in the absorbed radiation dose. In the population studied within a high-risk newborn ICU, the mean RBC volumes between the preterm and term neonates were without significant difference. Grouping and analysis of the RBC volume data with respect to birth weight, gestational ages, and 1- and 5-minute Apgar scores revealed on statistical difference. The mean value found in our population, 32.2 +/- 9.2 ml/kg, however, does differ from those previously reported in which the determinations were made using an indirect estimation from the plasma compartment.

  20. Chemical toxicity of red cells.

    PubMed Central

    Piomelli, S

    1981-01-01

    Exposure to toxic chemicals may result in alterations of red cell function. In certain cases, the toxic effect requires a genetic predisposition and thus affects only a restricted number of individuals; in other instances, the toxic effect is exerted on the hematopoietic system of every person. Glucose-6-phosphate dehydrogenase deficiency is probably the most widespread genetic disorder. It is observed at highest frequency in populations from subtropical countries as a result of its selective advantage vis à vis falciparum malaria. The gene controlling this enzyme is located on the X-chromosome; thus, the defect is sex-linked. Individuals with a genetic defect of this enzyme are extremely susceptible to hemolysis, when exposed to oxidant drugs (such as certain antimalarials and sulfonamides) because of the inability of their red cells to regenerate NADPH. Lead poisoning result in profound effects on the process of heme synthesis. Among the steps most sensitive to lead toxicity are the enzyme delta-aminolevulinic acid dehydratase and the intramitochondrial step that leads to the incorporation of iron into protoporphyrin. By these mechanisms, in severe lead intoxication there is an accumulation of large amounts of delta-aminolevulinic acid (a compound with inherent neurotoxicity), and there are abnormalities of mitochondrial function in all cells of the body. Individuals living in an industrialized society are unavoidably exposed to some environmental lead. Recent evidence indicates that, even at levels of exposure which do not increase the blood lead level above values presently considered normal, abnormalities of heme synthesis are clearly detectable. PMID:7016524

  1. Inflight Assay of Red Blood Cell Deformability

    NASA Technical Reports Server (NTRS)

    Ingram, M.; Paglia, D. E.; Eckstein, E. C.; Frazer, R. E.

    1985-01-01

    Studies on Soviet and American astronauts have demonstrated that red blood cell production is altered in response to low gravity (g) environment. This is associated with changes in individual red cells including increased mean cell volume and altered membrane deformability. During long orbital missions, there is a tendency for the red cell mass deficit to be at least partly corrected although the cell shape anomalies are not. Data currently available suggest that the observed decrease in red cell mass is the result of sudden suppression of erythropoieses and that the recovery trend observed during long missions reflects re-establishment of erythropoietic homeostasis at a "set point" for the red cell mass that is slightly below the normal level at 1 g.

  2. Stem cell labeling for magnetic resonance imaging.

    PubMed

    Himmelreich, Uwe; Hoehn, Mathias

    2008-01-01

    In vivo applications of cells for the monitoring of their cell dynamics increasingly use non-invasive magnetic resonance imaging. This imaging modality allows in particular to follow the migrational activity of stem cells intended for cell therapy strategies. All these approaches require the prior labeling of the cells under investigation for excellent contrast against the host tissue background in the imaging modality. The present review discusses the various routes of cell labeling and describes the potential to observe both cell localization and their cell-specific function in vivo. Possibilities for labeling strategies, pros and cons of various contrast agents are pointed out while potential ambiguities or problems of labeling strategies are emphasized. PMID:18465447

  3. Red blood cell membrane defects.

    PubMed

    Iolascon, Achille; Perrotta, Silverio; Stewart, Gordon W

    2003-03-01

    We present an overview of the currently known molecular basis of red cell membrane disorders. A detailed discussion of the structure of the red cell membrane and the pathophysiology and clinical aspects of its disorders is reported. Generally speaking, hereditary spherocytosis (HS) results from a loss of erythrocyte surface area. The mutations of most cases of HS are located in the following genes: ANK1, SPTB, SLC4A1, EPB42 and SPTA1, which encode for ankyrin, spectrin beta-chain, the anion exchanger 1 (band 3), protein 4.2 and spectrin alpha-chain, respectively. Hereditary elliptocytosis (HE) reflects a diminished elasticity of the skeleton. Its aggravated form, hereditary pyropoikilocytosis (HPP), implies that the skeleton undergoes further destabilization. The mutations responsible for HE and HPP, lie in the SPTA1 and SPTB gene, and in the EPB41 gene encoding protein 4.1. Allele alpha LELY is a common polymorphic allele, which plays the role of an aggravating factor when it occurs in trans of an elliptocytogenic allele of the SPTA1 gene. Southeast Asian ovalocytosis derives from a change in band 3. The genetic disorders of membrane permeability to monovalent cations required a positional cloning approach. In this respect, channelopathies represent a new frontier in the field. Dehydrated hereditary stomatocytosis (DHS) was shown to belong to a pleiotropic syndrome: DHS + fetal edema + pseudohyperkalemia, which maps 16q23-24. Splenectomy is strictly contraindicated in DHS and another disease of the same class, overhydrated hereditary stomatocytosis, because it increases the risk of thromboembolic accidents. PMID:14692233

  4. In vivo red cell destruction by anti-Lu6

    SciTech Connect

    Issitt, P.D.; Valinsky, J.E.; Marsh, W.L.; DiNapoli, J.; Gutgsell, N.S. )

    1990-03-01

    An example is presented of an IgG1, anti-Lu6, that reacted by indirect antiglobulin test and was capable of destroying antigen-positive red cells in vivo. Two methods for the measurement of red cell survival, {sup 51}Cr labeling and flow cytometry, gave the same result: 20 percent of the test dose of Lu:6 red cells was destroyed in the first hour after injection and 80 percent in the first 24 hours. The clinical relevance of the antibody was correctly predicted by an in vitro monocyte monolayer assay. The finding that this example of anti-Lu6 was clinically significant should not be taken to mean that all antibodies directed against high-incidence Lutheran and Lutheran system-related antigens will behave similarly. When such antibodies are encountered, in vivo and/or in vitro studies to assess their clinical significance are necessary before rare blood is used for transfusion.

  5. RED: a red-cell antibody identification expert module.

    PubMed

    Smith, J W; Svirbely, J R; Evans, C A; Strohm, P; Josephson, J R; Tanner, M

    1985-06-01

    We describe a software module in an expert system RED, which interprets data related to red cell antibody identification. There are three portions to this module: the problem-solving component, which incorporates the knowledge required for antibody identification as a hierarchy of programs. The programs in the hierarchy organize within themselves small pieces of knowledge represented in the form of production rules, which are capable of making judgments concerning a specific hypothesis; an intelligent data base for storage of patient data, red cell attributes, and test results; the "overview critic" portion, which combines the atomic hypotheses judged favorably by the antibody programs into a unified judgment concerning the case. Overview makes the decision to terminate processing with a conclusion about which antibodies are actually present and what specific further tests need to be performed to resolve any remaining ambiguities. PMID:3840517

  6. Red blood cell decreases of microgravity

    NASA Technical Reports Server (NTRS)

    Johnson, P. C.

    1985-01-01

    Postflight decreases in red blood cell mass (RBCM) have regularly been recorded after exposure to microgravity. These 5-25 percent decreases do not relate to the mission duration, workload, caloric intake or to the type of spacecraft used. The decrease is accompanied by normal red cell survivals, increased ferritin levels, normal radioactive iron studies, and increases in mean red blood cell volume. Comparable decreases in red blood cell mass are not found after bed rest, a commonly used simulation of the microgravity state. Inhibited bone marrow erythropoiesis has not been proven to date, although reticulocyte numbers in the peripheral circulation are decreased about 50 percent. To date, the cause of the microgravity induced decreases in RBCM is unknown. Increased splenic trapping of circulating red blood cells seem the most logical way to explain the results obtained.

  7. 21 CFR 640.10 - Red Blood Cells.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 7 2013-04-01 2013-04-01 false Red Blood Cells. 640.10 Section 640.10 Food and... ADDITIONAL STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Red Blood Cells § 640.10 Red Blood Cells. The proper name of this product shall be Red Blood Cells. The product is defined as red blood cells...

  8. 21 CFR 640.10 - Red Blood Cells.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 7 2010-04-01 2010-04-01 false Red Blood Cells. 640.10 Section 640.10 Food and... ADDITIONAL STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Red Blood Cells § 640.10 Red Blood Cells. The proper name of this product shall be Red Blood Cells. The product is defined as red blood cells...

  9. 21 CFR 640.10 - Red Blood Cells.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 7 2014-04-01 2014-04-01 false Red Blood Cells. 640.10 Section 640.10 Food and... ADDITIONAL STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Red Blood Cells § 640.10 Red Blood Cells. The proper name of this product shall be Red Blood Cells. The product is defined as red blood cells...

  10. 21 CFR 640.10 - Red Blood Cells.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 7 2011-04-01 2010-04-01 true Red Blood Cells. 640.10 Section 640.10 Food and... ADDITIONAL STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Red Blood Cells § 640.10 Red Blood Cells. The proper name of this product shall be Red Blood Cells. The product is defined as red blood cells...

  11. 21 CFR 640.10 - Red Blood Cells.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 7 2012-04-01 2012-04-01 false Red Blood Cells. 640.10 Section 640.10 Food and... ADDITIONAL STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Red Blood Cells § 640.10 Red Blood Cells. The proper name of this product shall be Red Blood Cells. The product is defined as red blood cells...

  12. The antibody approach of labeling blood cells

    SciTech Connect

    Srivastava, S.C.

    1991-12-31

    Although the science of blood cell labeling using monoclonal antibodies directed against specific cellular antigens is still in its early stages, considerable progress has recently been accomplished in this area. The monoclonal antibody approach offers the promise of greater selectivity and enhanced convenience since specific cell types can be labeled in vivo, thus eliminating the need for complex and damaging cell separation procedures. This article focuses on these developments with primary emphasis on antibody labeling of platelets and leukocytes. The advantages and the shortcomings of the recently reported techniques are criticality assessed and evaluated.

  13. The antibody approach of labeling blood cells

    SciTech Connect

    Srivastava, S.C.

    1991-01-01

    Although the science of blood cell labeling using monoclonal antibodies directed against specific cellular antigens is still in its early stages, considerable progress has recently been accomplished in this area. The monoclonal antibody approach offers the promise of greater selectivity and enhanced convenience since specific cell types can be labeled in vivo, thus eliminating the need for complex and damaging cell separation procedures. This article focuses on these developments with primary emphasis on antibody labeling of platelets and leukocytes. The advantages and the shortcomings of the recently reported techniques are criticality assessed and evaluated.

  14. The antibody approach of labeling blood cells

    SciTech Connect

    Srivastava, S.C.

    1992-12-31

    Although the science of blood cell labeling using monoclonal antibodies directed against specific cellular antigens is still in its early stages, considerable progress has recently been accomplished in this area. The monoclonal antibody approach offers the promise of greater selectivity and enhanced convenience since specific cell types can be labeled in vivo, thus eliminating the need for complex and damaging cell separation procedures. This article focuses on these developments with primary emphasis on antibody labeling of platelets and leukocytes. The advantages and the shortcomings of the recently reported techniques are critically assessed and evaluated.

  15. Red cell membrane: past, present, and future

    PubMed Central

    Gallagher, Patrick G.

    2008-01-01

    As a result of natural selection driven by severe forms of malaria, 1 in 6 humans in the world, more than 1 billion people, are affected by red cell abnormalities, making them the most common of the inherited disorders. The non-nucleated red cell is unique among human cell type in that the plasma membrane, its only structural component, accounts for all of its diverse antigenic, transport, and mechanical characteristics. Our current concept of the red cell membrane envisions it as a composite structure in which a membrane envelope composed of cholesterol and phospholipids is secured to an elastic network of skeletal proteins via transmembrane proteins. Structural and functional characterization of the many constituents of the red cell membrane, in conjunction with biophysical and physiologic studies, has led to detailed description of the way in which the remarkable mechanical properties and other important characteristics of the red cells arise, and of the manner in which they fail in disease states. Current studies in this very active and exciting field are continuing to produce new and unexpected revelations on the function of the red cell membrane and thus of the cell in health and disease, and shed new light on membrane function in other diverse cell types. PMID:18988878

  16. Colour and label evaluation of commercial pasteurised red juices and related drinks.

    PubMed

    Fallico, B; Arena, E; Chiappara, E; Ballistreri, G

    2010-01-01

    Despite growing demand by consumers for healthy beverages, artificial colours are still widely used. Levels of anthocyanins and artificial colours were determined by HPLC with UV-Vis detection in red orange juices and other red beverages (nectar, juice-based, health, carbonated and sports drinks). The contribution of pigments to the visible colour of the beverage was calculated. Red orange juice samples contained about 34 mg l(-1) of anthocyanins, which were responsible for about 92% of the visible colour. Red juice-based drinks, containing from 0% to 30% of red orange, berry, grape or pomegranate juices, had low levels of anthocyanins (about 7 mg l(-1)) and high levels of E129 (about 32 mg l(-1)), which were responsible for about 90.7% of the colour of these beverages. Red health drinks, enriched with vitamins and polyphenols, contained from 3% to 50% of red fruit juices. Also in this case the E129 levels were higher (about 22 mg l(-1)) than anthocyanins (about 9 mg l(-1)), and were responsible for the colour of the beverages (76.1%). High levels of artificial colours were found in red orange carbonated drinks, but in comparable amounts with those found in the other beverage samples, while anthocyanins were only present in trace amounts. Although all of the beverages claimed to contain red fruits on the labels, no correlation was found between the level of anthocyanins and the declared percentage of red fruits. These labels generally conformed with the requirements of the law, but food product labels can often be misleading to consumers about the real characteristics of the product. PMID:24779619

  17. Studies with nonradioisotopic sodium chromate. I. Development of a technique for measuring red cell volume

    SciTech Connect

    Heaton, W.A.; Hanbury, C.M.; Keegan, T.E.; Pleban, P.; Holme, S. )

    1989-10-01

    A nonradioisotopic method for measuring red cell volume that involves the use of 52Cr-sodium chromate as the red cell label and of graphite furnace atomic absorption analysis of chromium is described. The technique allows the labelling of 20 mL of packed red cells with 40 to 50 micrograms of sodium chromate (Na2CrO4) in 30 minutes at 22 degrees C with 94 +/- 6 percent uptake. Approximately 40 micrograms of Na2CrO4 was injected for in vivo studies. This results in posttransfusion in vivo red cell chromium levels after sample processing in the range of 1 to 7 micrograms per L, which could be quantitated accurately (coefficient of variation = 4.7%) by Zeeman electrothermal atomic absorption spectrophotometry. The labeling concentration of chromium did not cause increased hemolysis, and the labeled cells exhibited an osmotic fragility curve similar to that of unlabeled, fresh ACD red cells. Red cell glutathione peroxidase was unaffected by labeling, although glutathione reductase was reduced by approximately 13 percent (p less than 0.05). The 52Cr red cell volume-measuring method was evaluated by concurrent in vivo studies with the standard 51Cr and 125I-albumin methods for that procedure. Simultaneous measurement of red cell volumes in seven volunteers by the 51Cr, 52Cr, and 125I-albumin techniques correlated highly with each other (r greater than 0.76), with mean values of 2294 +/- 199, 2191 +/- 180, and 2243 +/- 291 mL, respectively. The standard deviations of the differences were small: 134 mL for 52Cr versus 51Cr and 183 mL for 52Cr versus 125I.

  18. Viscoelastic transient of confined red blood cells.

    PubMed

    Prado, Gaël; Farutin, Alexander; Misbah, Chaouqi; Bureau, Lionel

    2015-05-01

    The unique ability of a red blood cell to flow through extremely small microcapillaries depends on the viscoelastic properties of its membrane. Here, we study in vitro the response time upon flow startup exhibited by red blood cells confined into microchannels. We show that the characteristic transient time depends on the imposed flow strength, and that such a dependence gives access to both the effective viscosity and the elastic modulus controlling the temporal response of red cells. A simple theoretical analysis of our experimental data, validated by numerical simulations, further allows us to compute an estimate for the two-dimensional membrane viscosity of red blood cells, η(mem)(2D) ∼ 10(-7) N ⋅ s ⋅ m(-1). By comparing our results with those from previous studies, we discuss and clarify the origin of the discrepancies found in the literature regarding the determination of η(mem)(2D), and reconcile seemingly conflicting conclusions from previous works. PMID:25954871

  19. Avoiding Anemia: Boost Your Red Blood Cells

    MedlinePlus

    ... link, please review our exit disclaimer . Subscribe Avoiding Anemia Boost Your Red Blood Cells If you’re ... and sluggish, you might have a condition called anemia. Anemia is a common blood disorder that many ...

  20. Isotope Labeling in Mammalian Cells

    PubMed Central

    Dutta, Arpana; Saxena, Krishna; Klein-Seetharaman, Judith

    2011-01-01

    Isotope labeling of proteins represents an important and often required tool for the application of nuclear magnetic resonance (NMR) spectroscopy to investigate the structure and dynamics of proteins. Mammalian expression systems have conventionally been considered to be too weak and inefficient for protein expression. However, recent advances have significantly improved the expression levels of these systems. Here, we provide an overview of some of the recent developments in expression strategies for mammalian expression systems in view of NMR investigations. PMID:22167668

  1. Red Fluorescent Carbon Nanoparticle-Based Cell Imaging Probe.

    PubMed

    Ali, Haydar; Bhunia, Susanta Kumar; Dalal, Chumki; Jana, Nikhil R

    2016-04-13

    Fluorescent carbon nanoparticle-based probes with tunable visible emission are biocompatible, environment friendly and most suitable for various biomedical applications. However, synthesis of red fluorescent carbon nanoparticles and their transformation into functional nanoparticles are very challenging. Here we report red fluorescent carbon nanoparticle-based nanobioconjugates of <25 nm hydrodynamic size and their application as fluorescent cell labels. Hydrophobic carbon nanoparticles are synthesized via high temperature colloid-chemical approach and transformed into water-soluble functional nanoparticles via coating with amphiphilic polymer followed by covalent linking with desired biomolecules. Following this approach, carbon nanoparticles are functionalized with polyethylene glycol, primary amine, glucose, arginine, histidine, biotin and folic acid. These functional nanoparticles can be excited with blue/green light (i.e., 400-550 nm) to capture their emission spanning from 550 to 750 nm. Arginine and folic acid functionalized nanoparticles have been demonstrated as fluorescent cell labels where blue and green excitation has been used for imaging of labeled cells. The presented method can be extended for the development of carbon nanoparticle-based other bioimaging probes. PMID:27011336

  2. 21 CFR 864.8540 - Red cell lysing reagent.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Red cell lysing reagent. 864.8540 Section 864.8540...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Reagents § 864.8540 Red cell lysing reagent. (a) Identification. A red cell lysing reagent is a device used to lyse (destroy) red blood cells...

  3. 21 CFR 864.8540 - Red cell lysing reagent.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Red cell lysing reagent. 864.8540 Section 864.8540...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Reagents § 864.8540 Red cell lysing reagent. (a) Identification. A red cell lysing reagent is a device used to lyse (destroy) red blood cells...

  4. 21 CFR 864.8540 - Red cell lysing reagent.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Red cell lysing reagent. 864.8540 Section 864.8540...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Reagents § 864.8540 Red cell lysing reagent. (a) Identification. A red cell lysing reagent is a device used to lyse (destroy) red blood cells...

  5. 21 CFR 864.8540 - Red cell lysing reagent.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Red cell lysing reagent. 864.8540 Section 864.8540...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Reagents § 864.8540 Red cell lysing reagent. (a) Identification. A red cell lysing reagent is a device used to lyse (destroy) red blood cells...

  6. 21 CFR 864.8540 - Red cell lysing reagent.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Red cell lysing reagent. 864.8540 Section 864.8540...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Reagents § 864.8540 Red cell lysing reagent. (a) Identification. A red cell lysing reagent is a device used to lyse (destroy) red blood cells...

  7. 21 CFR 864.5300 - Red cell indices device.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Red cell indices device. 864.5300 Section 864.5300....5300 Red cell indices device. (a) Identification. A red cell indices device, usually part of a larger... corpuscular hemoglobin (MCH), and the mean corpuscular hemoglobin concentration (MCHC). The red cell...

  8. 21 CFR 864.5300 - Red cell indices device.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Red cell indices device. 864.5300 Section 864.5300....5300 Red cell indices device. (a) Identification. A red cell indices device, usually part of a larger... corpuscular hemoglobin (MCH), and the mean corpuscular hemoglobin concentration (MCHC). The red cell...

  9. 21 CFR 864.5300 - Red cell indices device.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Red cell indices device. 864.5300 Section 864.5300....5300 Red cell indices device. (a) Identification. A red cell indices device, usually part of a larger... corpuscular hemoglobin (MCH), and the mean corpuscular hemoglobin concentration (MCHC). The red cell...

  10. 21 CFR 864.5300 - Red cell indices device.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Red cell indices device. 864.5300 Section 864.5300....5300 Red cell indices device. (a) Identification. A red cell indices device, usually part of a larger... corpuscular hemoglobin (MCH), and the mean corpuscular hemoglobin concentration (MCHC). The red cell...

  11. 21 CFR 864.5300 - Red cell indices device.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Red cell indices device. 864.5300 Section 864.5300....5300 Red cell indices device. (a) Identification. A red cell indices device, usually part of a larger... corpuscular hemoglobin (MCH), and the mean corpuscular hemoglobin concentration (MCHC). The red cell...

  12. 99mTc-red blood cells SPECT and planar scintigraphy in the diagnosis of hepatic hemangiomas.

    PubMed

    Artiko, M V; Sobić-Saranović, P D; Perisić-Savić, S M; Stojković, V M; Radoman, B I; Knezević, S J; Petrović, S N; Obradović, B V; Milović, V

    2008-01-01

    The aim of the study is the assessment of the value of SPECT (single photon emission computerized tomography) using 99mTc-labeled red blood cells in the detection of liver hemangioma, in comparison to planar imaging. With planar red blood cell scintigraphy, sensitivity of the method was 76%, specificity 98%, positive predictive value 98% and negative predictive value 79%. With SPECT, sensitivity of the method was 95%, specificity 98%, positive predictive value 98% and negative predictive value 94%. The smallest lesion detected by planar red blood cell scintigraphy was 1.2 cm, and with SPECT red blood cell scintigraphy 0.8 cm. The use of 99mTc-labeled red blood cells SPECT improved the sensitivity much more in smaller lesions (0.8 to 2 cm), than in bigger ones (2-5 cm). SPECT with radiolabeled red blood cells significantlyy improves the results of scintigraphic findings, especially in the small lesions. PMID:19245136

  13. Density increment and decreased survival of rat red blood cells induced by cadmium

    SciTech Connect

    Kunimoto, M.; Miura, T.

    1986-01-01

    Male Wistar rats were injected with CdCl/sub 2/ subcutaneously to examine in vivo effects of Cd on density and survival of red blood cells. During the 7 days after administration of 1.0 mg Cd/kg, the following sequence of events occurred: (1) a progressive increase in the amount of more dense red blood cells concomitant with a decrease in that of light red blood cells from the first to the third day; (2) an increase in the spleen weight at the third day; (3) a decrease in the hematocrit value and an increase in the amount of light red blood cells at the fifth day; and (4) a recovery of the hematocrit value at the seventh day. Five days after administration, the hematocrit value decreased in a dose-dependent mode and the decrease was significant at the 1% level at 1.0 and 1.5 mg Cd/kg. A highly significant splenomegaly was also observed at 0.5 to 1.5 mg Cd/kg. In order to label red blood cells in vivo, (/sup 3/H) diisopropylfluorophosphate ((/sup 3/H)DFP) was injected into rats. At Day 11, Cd at either 0.5 or 1.0 mg/kg was administered to (/sup 3/H)DFP-prelabeled animals. Cd administration accelerated /sup 3/H-labeled red cell clearance from the blood. Six days after Cd administration, the radioactivity of red blood cells was 76 and 68% of the control at 0.5 and 1.0 mg Cd/kg, respectively. In vitro treatment of rat red density and accelerated in vivo clearance of red blood cells from the recipient circulation. These results show that Cd at low dose can cause anemia by increasing red cell density and by accelerating red cell sequestration, presumably in the spleen.

  14. Red cell metabolism studies on Skylab

    NASA Technical Reports Server (NTRS)

    Mengel, C. E.

    1974-01-01

    On the basis of these background data, metabolic studies were performed on humans involved in space flight. These studies included the Skylab experiences. The primary purpose of the investigations was to study red cells for: (1) evidences of lipid peroxidation, or (2) changes at various points in the glycolytic pathway. The Skylab missions were an opportunity to study blood samples before, during, and after flight and to compare results with simultaneous controls. No direct evidence that lipid peroxidation had occurred in the red blood cells was apparent in the studies.

  15. Models for the red blood cell lifespan.

    PubMed

    Shrestha, Rajiv P; Horowitz, Joseph; Hollot, Christopher V; Germain, Michael J; Widness, John A; Mock, Donald M; Veng-Pedersen, Peter; Chait, Yossi

    2016-06-01

    The lifespan of red blood cells (RBCs) plays an important role in the study and interpretation of various clinical conditions. Yet, confusion about the meanings of fundamental terms related to cell survival and their quantification still exists in the literature. To address these issues, we started from a compartmental model of RBC populations based on an arbitrary full lifespan distribution, carefully defined the residual lifespan, current age, and excess lifespan of the RBC population, and then derived the distributions of these parameters. For a set of residual survival data from biotin-labeled RBCs, we fit models based on Weibull, gamma, and lognormal distributions, using nonlinear mixed effects modeling and parametric bootstrapping. From the estimated Weibull, gamma, and lognormal parameters we computed the respective population mean full lifespans (95 % confidence interval): 115.60 (109.17-121.66), 116.71 (110.81-122.51), and 116.79 (111.23-122.75) days together with the standard deviations of the full lifespans: 24.77 (20.82-28.81), 24.30 (20.53-28.33), and 24.19 (20.43-27.73). We then estimated the 95th percentiles of the lifespan distributions (a surrogate for the maximum lifespan): 153.95 (150.02-158.36), 159.51 (155.09-164.00), and 160.40 (156.00-165.58) days, the mean current ages (or the mean residual lifespans): 60.45 (58.18-62.85), 60.82 (58.77-63.33), and 57.26 (54.33-60.61) days, and the residual half-lives: 57.97 (54.96-60.90), 58.36 (55.45-61.26), and 58.40 (55.62-61.37) days, for the Weibull, gamma, and lognormal models respectively. Corresponding estimates were obtained for the individual subjects. The three models provide equally excellent goodness-of-fit, reliable estimation, and physiologically plausible values of the directly interpretable RBC survival parameters. PMID:27039311

  16. Viscoelastic Transient of Confined Red Blood Cells

    PubMed Central

    Prado, Gaël; Farutin, Alexander; Misbah, Chaouqi; Bureau, Lionel

    2015-01-01

    The unique ability of a red blood cell to flow through extremely small microcapillaries depends on the viscoelastic properties of its membrane. Here, we study in vitro the response time upon flow startup exhibited by red blood cells confined into microchannels. We show that the characteristic transient time depends on the imposed flow strength, and that such a dependence gives access to both the effective viscosity and the elastic modulus controlling the temporal response of red cells. A simple theoretical analysis of our experimental data, validated by numerical simulations, further allows us to compute an estimate for the two-dimensional membrane viscosity of red blood cells, ηmem2D ∼ 10−7 N⋅s⋅m−1. By comparing our results with those from previous studies, we discuss and clarify the origin of the discrepancies found in the literature regarding the determination of ηmem2D, and reconcile seemingly conflicting conclusions from previous works. PMID:25954871

  17. Red Blood Cells Play a Role in Reverse Cholesterol Transport

    PubMed Central

    Hung, Kimberly T.; Berisha, Stela Z.; Ritchey, Brian M.; Santore, Jennifer; Smith, Jonathan D.

    2012-01-01

    Objective Reverse cholesterol transport (RCT) involves the removal of cholesterol from peripheral tissue for excretion in the feces. Here, we determined whether red blood cells (RBCs) can contribute to RCT. Methods and Results We performed a series of studies in apoAI-deficient mice where the HDL-mediated pathway of RCT is greatly diminished. RBCs carried a higher fraction of whole blood cholesterol than plasma in apoAI-deficient mice, and as least as much of the labeled cholesterol derived from injected foam cells appeared in RBCs compared to plasma. To determine if RBCs mediate RCT to the fecal compartment, we measured RCT in anemic and control apoAI-deficient mice and found that anemia decreased RCT to the feces by over 35% after correcting for fecal mass. Transfusion of [3H]cholesterol labeled RBCs led to robust delivery of the labeled cholesterol to the feces in apoAI-deficient hosts. In wild type mice, the majority of the blood cholesterol mass, as well as [3H]cholesterol derived from the injected foam cells, was found in plasma, and anemia did not significantly alter RCT to the feces after correction for fecal mass. Conclusion The RBC cholesterol pool is dynamic and facilitates RCT of peripheral cholesterol to the feces, particularly in the low HDL state. PMID:22499994

  18. 21 CFR 864.8185 - Calibrator for red cell and white cell counting.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Calibrator for red cell and white cell counting... Calibrator for red cell and white cell counting. (a) Identification. A calibrator for red cell and white cell counting is a device that resembles red or white blood cells and that is used to set instruments...

  19. 21 CFR 864.8185 - Calibrator for red cell and white cell counting.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Calibrator for red cell and white cell counting... Calibrator for red cell and white cell counting. (a) Identification. A calibrator for red cell and white cell counting is a device that resembles red or white blood cells and that is used to set instruments...

  20. 21 CFR 864.8185 - Calibrator for red cell and white cell counting.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Calibrator for red cell and white cell counting... Calibrator for red cell and white cell counting. (a) Identification. A calibrator for red cell and white cell counting is a device that resembles red or white blood cells and that is used to set instruments...

  1. 21 CFR 864.8185 - Calibrator for red cell and white cell counting.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Calibrator for red cell and white cell counting... Calibrator for red cell and white cell counting. (a) Identification. A calibrator for red cell and white cell counting is a device that resembles red or white blood cells and that is used to set instruments...

  2. 21 CFR 864.8185 - Calibrator for red cell and white cell counting.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Calibrator for red cell and white cell counting... Calibrator for red cell and white cell counting. (a) Identification. A calibrator for red cell and white cell counting is a device that resembles red or white blood cells and that is used to set instruments...

  3. Determination of Fc function with frozen red blood cells.

    PubMed

    Gurevich, V; Bertolini, J; Lyons, K

    2006-09-01

    The Fc function of immunoglobulins is commonly determined by an assay based on monitoring immunoglobulin induced, complement mediated red cell lysis. This assay requires a continuous source of fresh red cells. We have shown that the assay can be successfully performed with frozen red cells. The possibility of access to a stored standard stock of red cells will improve the convenience of performing the assay and could contribute to improved assay reproducibility. PMID:16500112

  4. Chloride transport in human red cells.

    PubMed Central

    Dalmark, M

    1975-01-01

    1. The chloride equilibrium flux (chloride self-exchange) was determined by measuring the rate of 36Cl efflux from radioactively labelled human red cells. The cellular chloride concentration was varied between 5 and 700 mM by the nystatin technique (Cass & Dalmark, 1973). The chloride transport capacity was not affected by the nystatin technique. 2. The chloride equilibrium flux showed saturation kinetics in the pH range between 6-2 and 9-2 (0 degrees C). The chloride transport decreased at chloride concentrations higher than those which gave the maximum transport. 3. The apparent half-saturation constant, (K1/2), depended on the pH and whether the chloride transport was perceived as a function of the chloride concentration in the medium or in the cell water. The (K1/2)m increased and the (K1/2)c decreased with increasing pH. The dependence of the chloride transport on the chloride concentration was described by Michaelis-Menten kinetics at pH 7-2, but at values of pH outside pH 7-8 S-shaped or steeper graphs were observed. 4. The chloride equilibrium flux varied with the pH at constant chloride concentration in the medium (pH 5-7-9-5). The transport had a bell-shaped pH dependence at chloride concentrations below 200 mM. At chloride concentrations between 300 and 600 mM the chloride transport increased with increasing pH to reach a plateau around pH 8. The position of the acidic branches of the pH graphs was independent of the chloride concentration (25-600 mM), but the position of the alkaline branches moved towards higher values of pH with increasing chloride concentration (5-150 mM). Thus, the position of the pH optimum increased with increasing chloride concentration. The chloride transport at low pH values was a function of the inverse second power of the hydrogen ion concentration. The pK of the groups which caused the inhibition was approximately 6 and independent of the temperature (0-18 degrees C). 5. The chloride equilibrium flux as a function of

  5. Survival of Er(a+) red cells in a patient with allo-anti-Era

    SciTech Connect

    Thompson, H.W.; Skradski, K.J.; Thoreson, J.R.; Polesky, H.F.

    1985-03-01

    /sup 51/Chromium-labeled Er(a+) red cells survived nearly normally (T1/2 of 21 days) in a patient with allo-anti-Era. Transfusion of Er(a+) blood was without significant reaction and did not affect the anti-Era titer.

  6. 21 CFR 864.7100 - Red blood cell enzyme assay.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Red blood cell enzyme assay. 864.7100 Section 864... enzyme assay. (a) Identification. Red blood cell enzyme assay is a device used to measure the activity in... kinase or 2,3-diphosphoglycerate. A red blood cell enzyme assay is used to determine the enzyme...

  7. 21 CFR 864.7100 - Red blood cell enzyme assay.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Red blood cell enzyme assay. 864.7100 Section 864... enzyme assay. (a) Identification. Red blood cell enzyme assay is a device used to measure the activity in... kinase or 2,3-diphosphoglycerate. A red blood cell enzyme assay is used to determine the enzyme...

  8. 21 CFR 864.7100 - Red blood cell enzyme assay.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Red blood cell enzyme assay. 864.7100 Section 864... enzyme assay. (a) Identification. Red blood cell enzyme assay is a device used to measure the activity in... kinase or 2,3-diphosphoglycerate. A red blood cell enzyme assay is used to determine the enzyme...

  9. 21 CFR 864.7100 - Red blood cell enzyme assay.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Red blood cell enzyme assay. 864.7100 Section 864... enzyme assay. (a) Identification. Red blood cell enzyme assay is a device used to measure the activity in... kinase or 2,3-diphosphoglycerate. A red blood cell enzyme assay is used to determine the enzyme...

  10. 21 CFR 864.7100 - Red blood cell enzyme assay.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Red blood cell enzyme assay. 864.7100 Section 864... enzyme assay. (a) Identification. Red blood cell enzyme assay is a device used to measure the activity in... kinase or 2,3-diphosphoglycerate. A red blood cell enzyme assay is used to determine the enzyme...

  11. 21 CFR 660.30 - Reagent Red Blood Cells.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... identify human blood-group antibodies. (b) Source. Reagent Red Blood Cells shall be prepared from human... 21 Food and Drugs 7 2010-04-01 2010-04-01 false Reagent Red Blood Cells. 660.30 Section 660.30...) BIOLOGICS ADDITIONAL STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Reagent Red Blood Cells §...

  12. 21 CFR 660.30 - Reagent Red Blood Cells.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 7 2013-04-01 2013-04-01 false Reagent Red Blood Cells. 660.30 Section 660.30...) BIOLOGICS ADDITIONAL STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Reagent Red Blood Cells § 660.30 Reagent Red Blood Cells. (a) Proper name and definition. The proper name of the product shall...

  13. 21 CFR 660.30 - Reagent Red Blood Cells.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 7 2014-04-01 2014-04-01 false Reagent Red Blood Cells. 660.30 Section 660.30...) BIOLOGICS ADDITIONAL STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Reagent Red Blood Cells § 660.30 Reagent Red Blood Cells. (a) Proper name and definition. The proper name of the product shall...

  14. 21 CFR 660.30 - Reagent Red Blood Cells.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 7 2012-04-01 2012-04-01 false Reagent Red Blood Cells. 660.30 Section 660.30...) BIOLOGICS ADDITIONAL STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Reagent Red Blood Cells § 660.30 Reagent Red Blood Cells. (a) Proper name and definition. The proper name of the product shall...

  15. 21 CFR 660.30 - Reagent Red Blood Cells.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 7 2011-04-01 2010-04-01 true Reagent Red Blood Cells. 660.30 Section 660.30 Food... ADDITIONAL STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Reagent Red Blood Cells § 660.30 Reagent Red Blood Cells. (a) Proper name and definition. The proper name of the product shall be Reagent...

  16. Responder individuality in red blood cell alloimmunization.

    PubMed

    Körmöczi, Günther F; Mayr, Wolfgang R

    2014-11-01

    Many different factors influence the propensity of transfusion recipients and pregnant women to form red blood cell alloantibodies (RBCA). RBCA may cause hemolytic transfusion reactions, hemolytic disease of the fetus and newborn and may be a complication in transplantation medicine. Antigenic differences between responder and foreign erythrocytes may lead to such an immune answer, in part with suspected specific HLA class II associations. Biochemical and conformational characteristics of red blood cell (RBC) antigens, their dose (number of transfusions and pregnancies, absolute number of antigens per RBC) and the mode of exposure impact on RBCA rates. In addition, individual circumstances determine the risk to form RBCA. Responder individuality in terms of age, sex, severity of underlying disease, disease- or therapy-induced immunosuppression and inflammation are discussed with respect to influencing RBC alloimmunization. For particular high-risk patients, extended phenotype matching of transfusion and recipient efficiently decreases RBCA induction and associated clinical risks. PMID:25670932

  17. Multiscale simulation of red blood cell aggregation

    NASA Astrophysics Data System (ADS)

    Bagchi, P.; Popel, A. S.

    2004-11-01

    In humans and other mammals, aggregation of red blood cells (RBC) is a major determinant to blood viscosity in microcirculation under physiological and pathological conditions. Elevated levels of aggregation are often related to cardiovascular diseases, bacterial infection, diabetes, and obesity. Aggregation is a multiscale phenomenon that is governed by the molecular bond formation between adjacent cells, morphological and rheological properties of the cells, and the motion of the extra-cellular fluid in which the cells circulate. We have developed a simulation technique using front tracking methods for multiple fluids that includes the multiscale characteristics of aggregation. We will report the first-ever direct computer simulation of aggregation of deformable cells in shear flows. We will present results on the effect of shear rate, strength of the cross-bridging bonds, and the cell rheological properties on the rolling motion, deformation and subsequent breakage of an aggregate.

  18. Reversibility of red blood cell deformation

    NASA Astrophysics Data System (ADS)

    Zeitz, Maria; Sens, P.

    2012-05-01

    The ability of cells to undergo reversible shape changes is often crucial to their survival. For red blood cells (RBCs), irreversible alteration of the cell shape and flexibility often causes anemia. Here we show theoretically that RBCs may react irreversibly to mechanical perturbations because of tensile stress in their cytoskeleton. The transient polymerization of protein fibers inside the cell seen in sickle cell anemia or a transient external force can trigger the formation of a cytoskeleton-free membrane protrusion of μm dimensions. The complex relaxation kinetics of the cell shape is shown to be responsible for selecting the final state once the perturbation is removed, thereby controlling the reversibility of the deformation. In some case, tubular protrusion are expected to relax via a peculiar “pearling instability.”

  19. Red blood cell transfusion in newborn infants.

    PubMed

    Whyte, Robin K; Jefferies, Ann L

    2014-04-01

    Red blood cell transfusion is an important and frequent component of neonatal intensive care. The present position statement addresses the methods and indications for red blood cell transfusion of the newborn, based on a review of the current literature. The most frequent indications for blood transfusion in the newborn are the acute treatment of perinatal hemorrhagic shock and the recurrent correction of anemia of prematurity. Perinatal hemorrhagic shock requires immediate treatment with large quantities of red blood cells; the effects of massive transfusion on other blood components must be considered. Some guidelines are now available from clinical trials investigating transfusion in anemia of prematurity; however, considerable uncertainty remains. There is weak evidence that cognitive impairment may be more severe at follow-up in extremely low birth weight infants transfused at lower hemoglobin thresholds; therefore, these thresholds should be maintained by transfusion therapy. Although the risks of transfusion have declined considerably in recent years, they can be minimized further by carefully restricting neonatal blood sampling. PMID:24855419

  20. From Red Cells to Soft Porous Lubrication

    NASA Astrophysics Data System (ADS)

    Wu, Qianhong; Gacka, Thomas; Nathan, Rungun; Crawford, Robert; Vucbmss Team

    2014-11-01

    Biological scientists have wondered, since the motion of red cells was first observed in capillaries, how the highly flexible red cell can move with so little friction in tightly fitting microvessels without being damaged or undergoing hemolysis. Theoretical studies (Feng and Weinbaum, 2000, JFM; Wu et al., 2004, PRL) attributed this frictionless motion to the dramatically enhanced hydrodynamic lifting force generated inside the soft, porous, endothelial surface layer (ESL) covering the inner surfaces of our capillaries, as a red blood cell glides over it. Herein we report the first experimental examination of this concept. The results conclusively demonstrate that significant fraction of the overall lifting force generated in a soft porous layer as a planing surface glides over it, is contributed by the pore fluid pressure, and thus frictional loss is reduced significantly. Moreover, the experimental predictions showed excellent agreement with the experimental data. This finding has the potential of dramatically changing existing lubrication approaches, and can result in substantial savings in energy consumption and thus reduction in greenhouse gas emissions.

  1. Single-cell label-free photoacoustic flowoxigraphy in vivo

    PubMed Central

    Wang, Lidai; Maslov, Konstantin; Wang, Lihong V.

    2013-01-01

    Label-free functional imaging of single red blood cells (RBCs) in vivo holds the key to uncovering the fundamental mechanism of oxygen metabolism in cells. To this end, we developed single-RBC photoacoustic flowoxigraphy (FOG), which can image oxygen delivery from single flowing RBCs in vivo with millisecond-scale temporal resolution and micrometer-scale spatial resolution. Using intrinsic optical absorption contrast from oxyhemoglobin (HbO2) and deoxyhemoglobin (HbR), FOG allows label-free imaging. Multiple single-RBC functional parameters, including total hemoglobin concentration (CHb), oxygen saturation (sO2), sO2 gradient (), flow speed (vf), and oxygen release rate (rO2), have been quantified simultaneously in real time. Working in reflection instead of transmission mode, the system allows minimally invasive imaging at more anatomical sites. We showed the capability to measure relationships among sO2, , vf, and rO2 in a living mouse brain. We also demonstrated that single-RBC oxygen delivery was modulated by changing either the inhalation gas or blood glucose. Furthermore, we showed that the coupling between neural activity and oxygen delivery could be imaged at the single-RBC level in the brain. The single-RBC functional imaging capability of FOG enables numerous biomedical studies and clinical applications. PMID:23536296

  2. Design of polymeric immunomicrospheres for cell labelling and cell separation

    NASA Technical Reports Server (NTRS)

    Rembaum, A.; Margel, S.

    1978-01-01

    Synthesis of several classes of hydrophylic microspheres applied to cell labeling and cell separation is described. Five classes of cross-linked microspheres with functional groups such as carboxyl, hydroxyl, amide and/or pyridine groups were synthesized. These functional groups were used to bind covalently antibodies and other proteins to the surface of the microspheres. To optimize the derivatisation technique, polyglutaraldehyde immunomicrospheres were prepared and utilized. Specific populations of human and murine lymphocytes were labelled with microspheres synthesized by the emulsion of the ionizing radiation technique. The labelling of the cells by means of microspheres containing an iron core produced successful separation of B from T lymphocytes by means of a magnetic field.

  3. Red blood cell (RBC) transfusion rates among US chronic dialysis patients during changes to Medicare end-stage renal disease (ESRD) reimbursement systems and erythropoiesis stimulating agent (ESA) labels

    PubMed Central

    2014-01-01

    Background Several major ESRD-related regulatory and reimbursement changes were introduced in the United States in 2011. In several large, national datasets, these changes have been associated with decreases in erythropoiesis stimulating agent (ESA) utilization and hemoglobin concentrations in the ESRD population, as well as an increase in the use of red blood cell (RBC) transfusions in this population. Our objective was to examine the use of RBC transfusion before and after the regulatory and reimbursement changes implemented in 2011 in a prevalent population of chronic dialysis patients in a large national claims database. Methods Patients in the Truven Health MarketScan Commercial and Medicare Databases with evidence of chronic dialysis were selected for the study. The proportion of chronic dialysis patients who received any RBC transfusion and RBC transfusion event rates per 100 patient-months were calculated in each month from January 1, 2007 to March 31, 2012. The results were analyzed overall and stratified by primary health insurance payer (commercial payer or Medicare). Results Overall, the percent of chronic dialysis patients with RBC transfusion and RBC transfusion event rates per 100 patient-months increased between January 2007 and March 2012. When stratified by primary health insurance payer, it appears that the increase was driven by the primary Medicare insurance population. While the percent of patients with RBC transfusion and RBC transfusion event rates did not increase in the commercially insured population between 2007 and 2012 they did increase in the primary Medicare insurance population; the majority of the increase occurred in 2011 during the same time frame as the ESRD-related regulatory and reimbursement changes. Conclusions The regulatory and reimbursement changes implemented in 2011 may have contributed to an increase in the use of RBC transfusions in chronic dialysis patients in the MarketScan dataset who were covered by Medicare plus

  4. Neocytolysis: physiological down-regulator of red-cell mass

    NASA Technical Reports Server (NTRS)

    Alfrey, C. P.; Rice, L.; Udden, M. M.; Driscoll, T. B.

    1997-01-01

    It is usually considered that red-cell mass is controlled by erythropoietin-driven bone marrow red-cell production, and no physiological mechanisms can shorten survival of circulating red cells. In adapting to acute plethora in microgravity, astronauts' red-cell mass falls too rapidly to be explained by diminished red-cell production. Ferrokinetics show no early decline in erythropolesis, but red cells radiolabelled 12 days before launch survive normally. Selective destruction of the youngest circulating red cells-a process we call neocytolysis-is the only plausible explanation. A fall in erythropoietin below a threshold is likely to initiate neocytolysis, probably by influencing surface-adhesion molecules. Recognition of neocytolysis will require re-examination of the pathophysiology and treatment of several blood disorders, including the anaemia of renal disease.

  5. ELECTRON MICROSCOPE ANALYSIS OF YOUNG AND OLD RED BLOOD CELLS STAINED WITH COLLOIDAL IRON FOR SURFACE CHARGE EVALUATION

    PubMed Central

    Marikovsky, Y.; Danon, D.

    1969-01-01

    Human and rabbit red blood cells, separated into "young" and "old" age groups by differential flotation on phthalate esters, were fixed with glutaraldehyde and labeled with colloidal ferric oxide. Electron micrographs of thin sections of young cells showed a uniform and dense depostion of positive iron particles. Old cells showed particles deposited irregularly, leaving unlabeled gaps on the membrane surface. Red cells incubated with 10 units/ml receptor-destroying enzyme (RDE) demonstrate a reduced labeling, similar to that of old cells. After neuraminic acid had been removed from red cells by 20 units/ml RDE, no iron particles were found on membrane surfaces. The different labeling of young, old, and RDE-treated human and rabbit red cells was correlated with their electric mobility and agglutinability by poly-L-lysine. The contradiction between the apparent similarity in charge density of human and rabbit red cells as estimated by density of iron particles and the markedly lower electric mobility of rabbit red cells is discussed. PMID:4186411

  6. Electron microscope analysis of young and old red blood cells stained with colloidal iron for surface charge evaluation.

    PubMed

    Marikovsky, Y; Danon, D

    1969-10-01

    Human and rabbit red blood cells, separated into "young" and "old" age groups by differential flotation on phthalate esters, were fixed with glutaraldehyde and labeled with colloidal ferric oxide. Electron micrographs of thin sections of young cells showed a uniform and dense depostion of positive iron particles. Old cells showed particles deposited irregularly, leaving unlabeled gaps on the membrane surface. Red cells incubated with 10 units/ml receptor-destroying enzyme (RDE) demonstrate a reduced labeling, similar to that of old cells. After neuraminic acid had been removed from red cells by 20 units/ml RDE, no iron particles were found on membrane surfaces. The different labeling of young, old, and RDE-treated human and rabbit red cells was correlated with their electric mobility and agglutinability by poly-L-lysine. The contradiction between the apparent similarity in charge density of human and rabbit red cells as estimated by density of iron particles and the markedly lower electric mobility of rabbit red cells is discussed. PMID:4186411

  7. Red Rain Cells Recovered from Interior of the Polonnaruwa Meteorite

    NASA Astrophysics Data System (ADS)

    Wickramarathne, K.; Wickramasinghe, N. C.

    2013-03-01

    Red rain cells were discovered in extracts from the Polonnaruwa (Aralaganwila) meteorite that fell nearly ten days before a red rain event in the same location in Sri Lanka. A causal connection is speculated.

  8. Anesthetics and red blood cell rheology

    NASA Astrophysics Data System (ADS)

    Aydogan, Burcu; Aydogan, Sami

    2014-05-01

    There are many conditions where it is useful for anesthetists to have a knowledge of blood rheology. Blood rheology plays an important role in numerous clinical situations. Hemorheologic changes may significantly affect the induction and recovery times with anesthetic agents. But also, hemorheologic factors are directly or indirectly affected by many anesthetic agents or their metabolites. In this review, the blood rheology with special emphasis on its application in anesthesiology, the importance hemorheological parameters in anesthesiology and also the effect of some anesthetic substances on red blood cell rheology were presented.

  9. The effect of osmotic pressure of aqueous PEG solutions on red blood cells.

    PubMed

    Herrmann, A; Arnold, K; Pratsch, L

    1985-08-01

    A drastic increase of the intracellular microviscosity of red blood cells in the presence of polyethylene glycol (PEG) was established by electron spin resonance using the small spin label molecule 2,2,6,6-tetramethyl-piperidine-N-oxyl-4-one (TEMPONE). The effective osmotic pressure of PEG solutions stressing the cells was estimated by comparison with those cytoplasmic rotational correlation times of TEMPONE measured in NaCl or sucrose containing media of known osmotic pressure. PMID:2998502

  10. Mechanosensing Dynamics of Red blood Cells

    NASA Astrophysics Data System (ADS)

    Wan, Jiandi

    2015-11-01

    Mechanical stress-induced deformation of human red blood cells (RBCs) plays important physiopathological roles in oxygen delivery, blood rheology, transfusion, and malaria. Recent studies demonstrate that, in response to mechanical deformation, RBCs release adenosine-5'-triphosphate (ATP), suggesting the existence of mechanotransductive pathways in RBCs. Most importantly, the released ATP from RBCs regulates vascular tone and impaired release of ATP from RBCs has been linked to diseases such as type II diabetes and cystic fibrosis. To date, however, the mechanisms of mechanotransductive release of ATP from RBCs remain unclear. Given that RBCs experience shear stresses continuously during the circulation cycle and the released ATP plays a central role in vascular physiopathology, understanding the mechanotransductive release of ATP from RBCs will provide not only fundamental insights to the role of RBCs in vascular homeostasis but also novel therapeutic strategies for red cell dysfunction and vascular disease. This talk describes the main research in my group on integrating microfluidic-based approaches to study the mechanosensing dynamics of RBCs. Specifically, I will introduce a micro?uidic approach that can probe the dynamics of shear-induced ATP release from RBCs with millisecond resolution and provide quantitative understandings of the mechanosensitive ATP release processes in RBCs. Furthermore, I will also describe our recent findings about the roles of the Piezo1 channel, a newly discovered mechanosensitive cation channel in the mechanotransductive ATP release in RBCs. Last, possible functions of RBCs in the regulation of cerebral blood flow will be discussed.

  11. Classification of blood cells and tumor cells using label-free ultrasound and photoacoustics.

    PubMed

    Strohm, Eric M; Kolios, Michael C

    2015-08-01

    A label-free method that can identify cells in a blood sample using high frequency photoacoustic and ultrasound signals is demonstrated. When the wavelength of the ultrasound or photoacoustic wave is similar to the size of a single cell (frequencies of 100-500 MHz), unique periodic features occur within the ultrasound and photoacoustic power spectrum that depend on the cell size, structure, and morphology. These spectral features can be used to identify different cell types present in blood, such as red blood cells (RBCs), white blood cells (WBCs), and circulating tumor cells. Circulating melanoma cells are ideal for photoacoustic detection due to their endogenous optical absorption properties. Using a 532 nm pulsed laser and a 375 MHz transducer, the ultrasound and photoacoustic signals from RBCs, WBCs, and melanoma cells were individually measured in an acoustic microscope to examine how the signals change between cell types. A photoacoustic and ultrasound signal was detected from RBCs and melanoma cells; only an ultrasound signal was detected from WBCs. The different cell types were distinctly separated using the ultrasound and photoacoustic signal amplitude and power spectral periodicity. The size of each cell was also estimated from the spectral periodicity. For the first time, sound waves generated using pulse-echo ultrasound and photoacoustics have been used to identify and size single cells, with applications toward counting and identifying cells, including circulating melanoma cells. PMID:26079610

  12. Microconfined flow behavior of red blood cells.

    PubMed

    Tomaiuolo, Giovanna; Lanotte, Luca; D'Apolito, Rosa; Cassinese, Antonio; Guido, Stefano

    2016-01-01

    Red blood cells (RBCs) perform essential functions in human body, such as gas exchange between blood and tissues, thanks to their ability to deform and flow in the microvascular network. The high RBC deformability is mainly due to the viscoelastic properties of the cell membrane. Since an impaired RBC deformability could be found in some diseases, such as malaria, sickle cell anemia, diabetes and hereditary disorders, there is the need to provide further insight into measurement of RBC deformability in a physiologically relevant flow field. Here, RBCs deformability has been studied in terms of the minimum apparent plasma-layer thickness by using high-speed video microscopy of RBCs flowing in cylindrical glass capillaries. An in vitro systematic microfluidic investigation of RBCs in micro-confined conditions has been performed, resulting in the determination of the RBCs time recovery constant, RBC volume and surface area and RBC membrane shear elastic modulus and surface viscosity. It has been noticed that the deformability of RBCs induces cells aggregation during flow in microcapillaries, allowing the formation of clusters of cells. Overall, our results provide a novel technique to estimate RBC deformability and also RBCs collective behavior, which can be used for the analysis of pathological RBCs, for which reliable quantitative methods are still lacking. PMID:26071649

  13. Label-Free and Continuous-Flow Ferrohydrodynamic Separation of HeLa Cells and Blood Cells in Biocompatible Ferrofluids

    PubMed Central

    Zhao, Wujun; Zhu, Taotao; Cheng, Rui; Liu, Yufei; He, Jian; Qiu, Hong; Wang, Lianchun; Nagy, Tamas; Querec, Troy D.; Unger, Elizabeth R.

    2016-01-01

    In this study, a label-free, low-cost, and fast ferrohydrodynamic cell separation scheme is demonstrated using HeLa cells (an epithelial cell line) and red blood cells. The separation is based on cell size difference, and conducted in a custom-made biocompatible ferrofluid that retains the viability of cells during and after the assay for downstream analysis. The scheme offers moderate-throughput (≈106 cells h−1 for a single channel device) and extremely high recovery rate (>99%) without the use of any label. It is envisioned that this separation scheme will have clinical applications in settings where rapid cell enrichment and removal of contaminating blood will improve efficiency of screening and diagnosis such as cervical cancer screening based on mixed populations in exfoliated samples. PMID:27478429

  14. Cells labeled with multiple fluorophores bound to a nucleic acid carrier

    SciTech Connect

    Dattagupta, N.; Kamarch, M.E.

    1989-04-25

    In passing labeled cells through a cell sorter, the improvement which comprises employing a labeled cell comprising a cell, an antibody specific to and bound to such cell, a nucleic acid fragment joined to the antibody, and a plurality of labels on the nucleic acid fragment. Because of the presence of multiple labels, the sensitivity of the separation of labeled cells in increased.

  15. Fluorometric assay for red blood cell antibodies

    SciTech Connect

    Schreiber, A.B.; Lambermont, M.; Strosberg, A.D.; Wybran, J.

    1981-03-01

    A fluorometric assay is described for the detection of red blood cell antibodies. The assay reveals as little as 600 molecules of bound, fluoroesceinated rabbit anti-human IgG antibodies per erythrocyte. Eleven patients with possible autoimmune erythrocyte disorder and negative direct antiglobulin test were studied by the fluorometric assay. The outcome of the fluorometric assay was compared with that of the human allogeneic rosette test. Results obtained by the two methods were in complete agreement. Five of the patients were shown to possess unexpectedly high levels of erythrocyte-bound IgG in spite of a negative, direct antiglobulin test. These findings and the validity of the fluorometric assay are discussed.

  16. Optical analysis of red blood cell suspension

    NASA Astrophysics Data System (ADS)

    Szołna, Alicja A.; Grzegorzewski, Bronisław

    2008-12-01

    The optical properties of suspensions of red blood cells (RBCs) were studied. Fresh human venues blood was obtained from adult healthy donors. RBCs were suspended in isotonic salt solution, and in autologous plasma. Suspensions with haematocrit 0.25 - 3% were investigated. Novel technique was proposed to determine the scattering coefficient μs for the suspensions. The intensity of He-Ne laser light transmitted through a wedge-shape container filled with a suspension was recorded. To find the dependence of the intensity on the thickness of the sample the container was moved horizontally. The dependence of μs on the haematocrit was determined for RBCs suspended in the isotonic salt solution. RBCs suspended in plasma tend to form rouleaux. For the RBCs suspended in plasma, the scattering coefficient as a function of time was obtained. It is shown that this technique can be useful in the study of rouleaux formation.

  17. State of the science of blood cell labeling

    SciTech Connect

    Srivastava, S.C.; Straub, R.F.

    1989-01-01

    Blood cell labeling can be considered a science in as far as it is based on precise knowledge and can be readily reproduced. This benchmark criterion is applied to all current cell labeling modalities and their relative merits and deficiencies are discussed. Mechanisms are given where they are known as well as labeling yields, label stability, and cell functionality. The focus is on the methodology and its suitability to the clinical setting rather than on clinical applications per se. Clinical results are cited only as proof of efficacy of the various methods. The emphasis is on technetium as the cell label, although comparisons are made between technetium and indium, and all blood cells are covered. 52 refs., 6 figs., 7 tabs.

  18. Tissue engineering red blood cells: a therapeutic.

    PubMed

    van Veen, Theun; Hunt, John A

    2015-07-01

    The use of red blood cells (RBCs) in transfusion is widespread in modern medicine. Limitations in blood transfusion have made an urgent argument for the focus on alternatives, as particular medical treatments heavily rely on the supply of donated blood. Stem cells have been successfully used in vitro to produce RBCs and researchers are currently challenged with developing larger-scale culture methods to meet the requirements for clinically relevant cell numbers. The ultimate conditions that will be beneficial for this type of research are trivial. A successful human clinical trial has shown that tremendous progress has already been made in this field. Other alternatives are based on the oxygen carrier protein that RBCs contain, i.e. haemoglobin (Hb). Chemically defined molecules and crosslinked proteins, which are able to bind and transport oxygen, have been found to be functional in vivo. Major progress has been achieved, but developing highly suitable products for the transfusion market still remains an enormous challenge for these acellular blood substitutes. We provide a review about developing alternatives for blood transfusion, with the emphasis on tissue-engineering approaches. PMID:24753354

  19. Growth and replication of red rain cells at 121°C and their red fluorescence

    NASA Astrophysics Data System (ADS)

    Gangappa, Rajkumar; Wickramasinghe, Chandra; Wainwright, Milton; Kumar, A. Santhosh; Louis, Godfrey

    2010-09-01

    We have shown that the red cells found in the Red Rain (which fell on Kerala, India, in 2001) survive and grow after incubation for periods of up to two hours at 121°C . Under these conditions daughter cells appear within the original mother cells and the number of cells in the samples increases with length of exposure to 121°C. No such increase in cells occurs at room temperature, suggesting that the increase in daughter cells is brought about by exposure of the Red Rain cells to high temperatures. This is an independent confirmation of results reported earlier by two of the present authors, claiming that the cells can replicate under high pressure at temperatures upto 300°C. The flourescence behaviour of the red cells is shown to be in remarkable correspondence with the extended red emission observed in the Red Rectagle planetary nebula and other galactic and extragalactic dust clouds, suggesting, though not proving an extraterrestrial origin.

  20. High efficiency labeling of glycoproteins on living cells

    PubMed Central

    Zeng, Ying; Ramya, T. N. C.; Dirksen, Anouk; Dawson, Philip E.; Paulson, James C.

    2010-01-01

    We describe a simple method for efficiently labeling cell surface glycans on virtually any living animal cell. The method employs mild Periodate oxidation to generate an aldehyde on sialic acids, followed by Aniline-catalyzed oxime Ligation with a suitable tag (PAL). Aniline catalysis dramatically accelerates oxime ligation, allowing use of low concentrations of aminooxy-biotin at neutral pH to label the majority of cell surface glycoproteins while maintaining high cell viability. PMID:19234450

  1. Distribution of chloride permeabilities in normal human red cells.

    PubMed Central

    Raftos, J E; Bookchin, R M; Lew, V L

    1996-01-01

    1. The rate of dehydration of K+ permeabilized red cells is influenced by their Cl- permeability (PCl). In instances of pathological K+ permeabilization, cell-to-cell differences in PCl may determine which red cells dehydrate most. The present study was designed to investigate whether PCl differed significantly among red cells from a single blood sample. 2. Previously available methods measure only the mean PCl of red cell populations. We describe a 'profile migration' method in which dilute red cell suspensions in low-K+ media were permeabilized to K+ with a high concentration of valinomycin, rendering PCl the main rate-limiting factor for cell dehydration. As the cells dehydrated, samples were processed to obtain full haemolysis curves at precise times. Variations in PCl among cells would have appeared as progressive changes in the profile of their haemolysis curves, as the curves migrated towards lower tonicities. 3. Red cells from five normal volunteers showed no change in profile of the migrating haemolysis curves, suggesting that their PCl distributions were fairly uniform. Quantitative analysis demonstrated that intercell variation in PCl was less than 7.5%. 4. Results obtained with this technique were analysed using the Lew-Bookchin red cell model. The calculated PCl was within the normal range described in earlier studies. PMID:8815210

  2. Destruction of newly released red blood cells in space flight

    NASA Technical Reports Server (NTRS)

    Alfrey, C. P.; Udden, M. M.; Huntoon, C. L.; Driscoll, T.

    1996-01-01

    Space flight results in a rapid change in total blood volume, plasma volume, and red blood cell mass because the space to contain blood is decreased. The plasma volume and total blood volume decreases during the first hours in space and remain at a decreased level for the remainder of the flight. During the first several hours following return to earth, plasma volume and total blood volume increase to preflight levels. During the first few days in space recently produced red blood cells disappear from the blood resulting in a decrease in red blood cell mass of 10-15%. Red cells 12 d old or older survive normally and production of new cells continues at near preflight levels. After the first few days in space, the red cell mass is stable at the decreased level. Following return to earth the hemoglobin and red blood cell mass concentrations decrease reflecting the increase in plasma volume. The erythropoietin levels increase responding to "postflight anemia"; red cell production increases, and the red cell mass is restored to preflight levels after several weeks.

  3. Kit for the rapid preparation of .sup.99m Tc red blood cells

    DOEpatents

    Richards, Powell; Smith, Terry D.

    1976-01-01

    A method and sample kit for the preparation of .sup.99m Tc-labeled red blood cells in a closed, sterile system. A partially evacuated tube, containing a freeze-dried stannous citrate formulation with heparin as an anticoagulant, allows whole blood to be automatically drawn from the patient. The radioisotope is added at the end of the labeling sequence to minimize operator exposure. Consistent 97% yields in 20 minutes are obtained with small blood samples. Freeze-dried kits have remained stable after five months.

  4. Control of red blood cell mass during spaceflight

    NASA Technical Reports Server (NTRS)

    Lane, H. W.; Alfrey, C. P.; Driscoll, T. B.; Smith, S. M.; Nyquist, L. E.

    1996-01-01

    Data are reviewed from twenty-two astronauts from seven space missions in a study of red blood cell mass. The data show that decreased red cell mass in all astronauts exposed to space for more than nine days, although the actual dynamics of mass changes varies with flight duration. Possible mechanisms for these changes, including alterations in erythropoietin levels, are discussed.

  5. Instant magnetic labeling of tumor cells by ultrasound in vitro

    NASA Astrophysics Data System (ADS)

    Mo, Runyang; Yang, Jian; Wu, Ed X.; Lin, Shuyu

    2011-09-01

    Magnetic labeling of living cells creates opportunities for numerous biomedical applications. Here we describe an instantly cell magnetic labeling method based on ultrasound. We present a detailed study on the ultrasound performance of a simple and efficient labeling protocol for H-22 cells in vitro. High frequency focus ultrasound was investigated as an alternative method to achieve instant cell labeling with the magnetic particles without the need for adjunct agents or initiating cell cultures. Mean diameter of 168 nm dextran-T40 coated superparamagnetic iron oxide (SPIO) nanoparticles were prepared by means of classical coprecipitation in solution in our laboratory. H-22 tumor cells suspended in phosphate-buffered saline (PBS, pH=7.2) were exposed to ultrasound at 1.37 MHz for up to 120 s in the presence of SPIOs. The cellular uptake of iron oxide nanoparticles was detected by prussion blue staining. The viability of cells was determined by a trypan blue exclusion test. At 2 W power and 60 s ultrasound exposure in presence of 410 μg/ml SPIOs, H-22 cell labeling efficiency reached 69.4±6.3% and the labeled cells exhibited an iron content of 10.38±2.43 pg per cell. Furthermore, 95.2±3.2% cells remained viable. The results indicated that the ultrasound protocol could be potentially applied to label cells with large-sized magnetic particles. We also calculated the shear stress at the 2 W power and 1.37 MHz used in experiments. The results showed that the shear stress threshold for ultrasonically induced H-22 cell reparable sonoporation was 697 Pa. These findings provide a quantitative guidance in designing ultrasound protocols for cell labeling.

  6. A novel cell permeant and far red-fluorescing DNA probe, DRAQ5, for blood cell discrimination by flow cytometry.

    PubMed

    Smith, P J; Wiltshire, M; Davies, S; Patterson, L H; Hoy, T

    1999-10-29

    The deep red fluorescing agent (DRAQ5) is a synthetic anthraquinone with a high affinity for DNA and a high capacity to rapidly enter living cells or stain fixed cells. DRAQ5 is optimally excited by red-light emitting sources and yields a deep red emission spectrum which extends into the low infra-red. DRAQ5 shows excitation at sub-optimal wavelengths including the 488 nm line and the multi-line UV wavelengths emitted by argon-ion lasers. Single beam (488 nm) flow cytometry has been used to demonstrate the utility of DRAQ5-nuclear DNA fluorescence as a discriminating parameter for human leucocytes and lymphoma cells, in combination with fluorochrome-labelled antibodies for the detection of surface antigens and subpopulation recognition. DRAQ5 fluorescence was found to reflect cellular DNA content as evidenced by cell cycle distribution profiles for asynchronous and cell cycle-perturbed populations. Importantly, DRAQ5 can be used in combination with FITC and RPE-labelled antibodies, without the need for fluorescence compensation. PMID:10556697

  7. Red blood cells in sports: effects of exercise and training on oxygen supply by red blood cells.

    PubMed

    Mairbäurl, Heimo

    2013-01-01

    During exercise the cardiovascular system has to warrant substrate supply to working muscle. The main function of red blood cells in exercise is the transport of O2 from the lungs to the tissues and the delivery of metabolically produced CO2 to the lungs for expiration. Hemoglobin also contributes to the blood's buffering capacity, and ATP and NO release from red blood cells contributes to vasodilation and improved blood flow to working muscle. These functions require adequate amounts of red blood cells in circulation. Trained athletes, particularly in endurance sports, have a decreased hematocrit, which is sometimes called "sports anemia." This is not anemia in a clinical sense, because athletes have in fact an increased total mass of red blood cells and hemoglobin in circulation relative to sedentary individuals. The slight decrease in hematocrit by training is brought about by an increased plasma volume (PV). The mechanisms that increase total red blood cell mass by training are not understood fully. Despite stimulated erythropoiesis, exercise can decrease the red blood cell mass by intravascular hemolysis mainly of senescent red blood cells, which is caused by mechanical rupture when red blood cells pass through capillaries in contracting muscles, and by compression of red cells e.g., in foot soles during running or in hand palms in weightlifters. Together, these adjustments cause a decrease in the average age of the population of circulating red blood cells in trained athletes. These younger red cells are characterized by improved oxygen release and deformability, both of which also improve tissue oxygen supply during exercise. PMID:24273518

  8. Red blood cell vesiculation in hereditary hemolytic anemia

    PubMed Central

    Alaarg, Amr; Schiffelers, Raymond M.; van Solinge, Wouter W.; van Wijk, Richard

    2013-01-01

    Hereditary hemolytic anemia encompasses a heterogeneous group of anemias characterized by decreased red blood cell survival because of inherited membrane, enzyme, or hemoglobin disorders. Affected red blood cells are more fragile, less deformable, and more susceptible to shear stress and oxidative damage, and show increased vesiculation. Red blood cells, as essentially all cells, constitutively release phospholipid extracellular vesicles in vivo and in vitro in a process known as vesiculation. These extracellular vesicles comprise a heterogeneous group of vesicles of different sizes and intracellular origins. They are described in literature as exosomes if they originate from multi-vesicular bodies, or as microvesicles when formed by a one-step budding process directly from the plasma membrane. Extracellular vesicles contain a multitude of bioactive molecules that are implicated in intercellular communication and in different biological and pathophysiological processes. Mature red blood cells release in principle only microvesicles. In hereditary hemolytic anemias, the underlying molecular defect affects and determines red blood cell vesiculation, resulting in shedding microvesicles of different compositions and concentrations. Despite extensive research into red blood cell biochemistry and physiology, little is known about red cell deformability and vesiculation in hereditary hemolytic anemias, and the associated pathophysiological role is incompletely assessed. In this review, we discuss recent progress in understanding extracellular vesicles biology, with focus on red blood cell vesiculation. Also, we review recent scientific findings on the molecular defects of hereditary hemolytic anemias, and their correlation with red blood cell deformability and vesiculation. Integrating bio-analytical findings on abnormalities of red blood cells and their microvesicles will be critical for a better understanding of the pathophysiology of hereditary hemolytic anemias. PMID

  9. Developmental Plasticity of Red Blood Cell Homeostasis

    PubMed Central

    Golub, Mari S.; Hogrefe, Casey E.; Malka, Roy; Higgins, John M.

    2014-01-01

    Most human physiologic set points like body temperature are tightly regulated and show little variation between healthy individuals. Red blood cell (RBC) characteristics such as hematocrit (HCT) and mean cell volume (MCV) are stable within individuals but can vary by 20% from one healthy person to the next. The mechanisms for the majority of this inter-individual variation are unknown and do not appear to involve common genetic variation. Here we show that environmental conditions present during development, namely in utero iron availability, can exert long-term influence on a set point related to the RBC life cycle. In a controlled study of rhesus monkeys and a retrospective study of humans, we use a mathematical model of in vivo RBC population dynamics to show that in utero iron deficiency is associated with a lowered threshold for RBC clearance and turnover. This in utero effect is plastic, persisting at least two years after birth and after the cessation of iron deficiency. Our study reports a rare instance of developmental plasticity in the human hematologic systems and also shows how mathematical modeling can be used to identify cellular mechanisms involved in the adaptive control of homeostatic set points. PMID:24415575

  10. Red blood cell in simple shear flow

    NASA Astrophysics Data System (ADS)

    Chien, Wei; Hew, Yayu; Chen, Yeng-Long

    2013-03-01

    The dynamics of red blood cells (RBC) in blood flow is critical for oxygen transport, and it also influences inflammation (white blood cells), thrombosis (platelets), and circulatory tumor migration. The physical properties of a RBC can be captured by modeling RBC as lipid membrane linked to a cytoskeletal spectrin network that encapsulates cytoplasm rich in hemoglobin, with bi-concave equilibrium shape. Depending on the shear force, RBC elasticity, membrane viscosity, and cytoplasm viscosity, RBC can undergo tumbling, tank-treading, or oscillatory motion. We investigate the dynamic state diagram of RBC in shear and pressure-driven flow using a combined immersed boundary-lattice Boltzmann method with a multi-scale RBC model that accurately captures the experimentally established RBC force-deformation relation. It is found that the tumbling (TU) to tank-treading (TT) transition occurs as shear rate increases for cytoplasm/outer fluid viscosity ratio smaller than 0.67. The TU frequency is found to be half of the TT frequency, in agreement with experiment observations. Larger viscosity ratios lead to the disappearance of stable TT phase and unstable complex dynamics, including the oscillation of the symmetry axis of the bi-concave shape perpendicular to the flow direction. The dependence on RBC bending rigidity, shear modulus, the order of membrane spectrin network and fluid field in the unstable region will also be discussed.

  11. Cell labeling with magnetic nanoparticles: Opportunity for magnetic cell imaging and cell manipulation

    PubMed Central

    2013-01-01

    This tutorial describes a method of controlled cell labeling with citrate-coated ultra small superparamagnetic iron oxide nanoparticles. This method may provide basically all kinds of cells with sufficient magnetization to allow cell detection by high-resolution magnetic resonance imaging (MRI) and to enable potential magnetic manipulation. In order to efficiently exploit labeled cells, quantify the magnetic load and deliver or follow-up magnetic cells, we herein describe the main requirements that should be applied during the labeling procedure. Moreover we present some recommendations for cell detection and quantification by MRI and detail magnetic guiding on some real-case studies in vitro and in vivo. PMID:24564857

  12. The aging of the red blood cell. A multifactor process.

    PubMed

    Danon, D; Marikovsky, Y

    1988-01-01

    Red blood cell (rbc) senescence is associated with loss of surface sialic acid, which is the principal carrier of surface negative charge and determines the electrokinetic behavior of old rbcs. Loss of sialic acid in an old rbc is demonstrated in its decreased electric mobility and lower negative charge density, determined topographically with cationic particle labeling. Surface sialic acid determines also the mutual attraction--repulsion forces, as demonstrated in enhanced aggluinability with cationic molecules, lectins, and blood group antibodies. Loss of sialic acid accompanies ATP-depletion in vitro; thus, a T-antigen site is unmasked. Macrophages have specific receptors to the site as to newly exposed galactose and N-acetyl galactosamine sugars. Furthermore, the involvement of complement molecules in the recognition of old RBCs by macrophages has been shown. This is possibly due to loss of sialic acid or at least a regrouping--relocation of surface anionic sites due to cell shape changes from discocytes to crenated forms, which accompany both in vivo and in vitro rbc aging. In turn, shape changes are apparently controlled by the cytoskeletal network underlying the rbc membrane, which undergoes structural alteration with physiologic aging in changing the dimensions of oligomeric spectrin and the thickness of the spectrin-actin cytoskeletal assembly. PMID:3052636

  13. Synthesis and assembly of membrane skeletal proteins in mammalian red cell precursors

    SciTech Connect

    Hanspal, M.; Palek, J.

    1987-09-01

    The synthesis of membrane skeletal proteins in avian nucleated red cells has been the subject of extensive investigation, whereas little is known about skeletal protein synthesis in bone marrow erythroblasts and peripheral blood reticulocytes in mammals. To address this question, we have isolated nucleated red cell precursors and reticulocytes from spleens and from the peripheral blood, respectively, of rats with phenylhydrazine-induced hemolytic anemia and pulse-labeled them with (/sup 35/S)methionine. Pulse-labeling of nucleated red cell precursors shows that the newly synthesized alpha- and beta-spectrins are present in the cytosol, with a severalfold excess of alpha-spectrin over beta-spectrin. However, in the membrane-skeletal fraction, newly synthesized alpha- and beta-spectrins are assembled in stoichiometric amounts, suggesting that the association of alpha-spectrin with the membrane skeleton may- be rate-limited by the amount of beta-spectrin synthesized, as has been shown recently in avian erythroid cells. Pulse-chase experiments in the rat nucleated red cell precursors show that the newly synthesized alpha- and beta-spectrin of the cytosol turn over coordinately and extremely rapidly. In contrast, in the membrane-skeletal fraction, the newly synthesized polypeptides of spectrin are stable. In contrast to nucleated erythroid cells, in reticulocytes the synthesis of alpha- and beta-spectrins is markedly diminished compared with the synthesis and assembly of proteins comigrating with bands 2.1 and 4.1 on SDS gels. Thus, in nucleated red cell precursors, the newly synthesized spectrin may be attached to the plasma membrane before proteins 2.1 and 4.1 are completely synthesized and incorporated in the membrane.

  14. Identification and behavior of label-retaining cells in epithelia

    SciTech Connect

    Bickenbach, J.R.

    1982-01-01

    A subpopulation of stem cells has been demonstrated in several renewing tissues. Such cells have a slow cell cycle and provide differentiating cells during normal turnover and during regeneration of the tissue following damage. The presence of slowly-cycling cells in epithelia from regions of skin and oral mucosa was examined by labeling 10-day-old mice and 5-day-old hamsters with tritiated thymidine (/sup 3/H-TdR) and observing the rate at which label was diluted from the basal cells. Label was rapidly diluted by cell division in most cells but a small percentage of basal cells (label-retaining cells, LRCS) was found to retain label for up to ninety days. Electron microscopic autoradiography and ..beta..-glucuronidase histochemistry with autoradiography were used to distinguish slowly-cycling keratinocytes from Langerhans cells. Such findings of slowly-cycling keratinocytes in epithelia with the ability to proliferate in culture and with a direct relationship to patterns of tissue architecture suggest that LRCs in epithelia correspond to stem cells described in other continuously renewing tissues.

  15. Multiexcitation Fluorogenic Labeling of Surface, Intracellular, and Total Protein Pools in Living Cells.

    PubMed

    Naganbabu, Matharishwan; Perkins, Lydia A; Wang, Yi; Kurish, Jeffery; Schmidt, Brigitte F; Bruchez, Marcel P

    2016-06-15

    Malachite green (MG) is a fluorogenic dye that shows fluorescence enhancement upon binding to its engineered cognate protein, a fluorogen activating protein (FAP). Energy transfer donors such as cyanine and rhodamine dyes have been conjugated with MG to modify the spectral properties of the fluorescent complexes, where the donor dyes transfer energy through Förster resonance energy transfer to the MG complex resulting in binding-conditional fluorescence emission in the far-red region. In this article, we use a violet-excitable dye as a donor to sensitize the far-red emission of the MG-FAP complex. Two blue emitting fluorescent coumarin dyes were coupled to MG and evaluated for energy transfer to the MG-FAP complex via its secondary excitation band. 6,8-Difluoro-7-hydroxycoumarin-3-carboxylic acid (Pacific blue, PB) showed the most efficient energy transfer and maximum brightness in the far-red region upon violet (405 nm) excitation. These blue-red (BluR) tandem dyes are spectrally varied from other tandem dyes and are able to produce fluorescence images of the MG-FAP complex with a large Stokes shift (>250 nm). These dyes are cell-permeable and are used to label intracellular proteins. Used together with a cell-impermeable hexa-Cy3-MG (HCM) dye that labels extracellular proteins, we are able to visualize extracellular, intracellular, and total pools of cellular protein using one fluorogenic tag that combines with distinct dyes to effect different spectral characteristics. PMID:27159569

  16. Light scattering by aggregated red blood cells.

    PubMed

    Tsinopoulos, Stephanos V; Sellountos, Euripides J; Polyzos, Demosthenes

    2002-03-01

    In low flow rates, red blood cells (RBCs) fasten together along their axis of symmetry and form a so-called rouleaux. The scattering of He-Ne laser light by a rouleau consisting of n (2 < or = n < or = 8) average-sized RBCs is investigated. The interaction problem is treated numerically by means of an advanced axisymmetric boundary element--fast Fourier transform methodology. The scattering problem of one RBC was solved first, and the results showed that the influence of the RBC's membrane on the scattering patterns is negligible. Thus the rouleau is modeled as an axisymmetric, homogeneous, low-contrast dielectric cylinder, on the surface of which appears, owing to aggregated RBCs, a periodic roughness along the direction of symmetry. The direction of the incident laser light is considered to be perpendicular to the scatterer's axis of symmetry. The differential scattering cross sections in both perpendicular and parallel scattering planes and for all the scattering angles are calculated and presented in detail. PMID:11900021

  17. Light scattering by aggregated red blood cells

    NASA Astrophysics Data System (ADS)

    Tsinopoulos, Stephanos V.; Sellountos, Euripides J.; Polyzos, Demosthenes

    2002-03-01

    In low flow rates, red blood cells (RBCs) fasten together along their axis of symmetry and form a so-called rouleaux. The scattering of He-Ne laser light by a rouleau consisting of n (2 less-than-or-equal n less-than-or-equal 8) average-sized RBCs is investigated. The interaction problem is treated numerically by means of an advanced axisymmetric boundary element--fast Fourier transform methodology. The scattering problem of one RBC was solved first, and the results showed that the influence of the RBC's membrane on the scattering patterns is negligible. Thus the rouleau is modeled as an axisymmetric, homogeneous, low-contrast dielectric cylinder, on the surface of which appears, owing to aggregated RBCs, a periodic roughness along the direction of symmetry. The direction of the incident laser light is considered to be perpendicular to the scatterer's axis of symmetry. The differential scattering cross sections in both perpendicular and parallel scattering planes and for all the scattering angles are calculated and presented in detail.

  18. Electrochemical Red Blood Cell Counting: One at a Time.

    PubMed

    Sepunaru, Lior; Sokolov, Stanislav V; Holter, Jennifer; Young, Neil P; Compton, Richard G

    2016-08-01

    We demonstrate that the concentration of a red blood cell solution under physiological conditions can be determined by electrochemical voltammetry. The magnitude of the oxygen reduction currents produced at an edge-plane pyrolytic graphite electrode was diagnosed analytically at concentrations suitable for a point-of-care test device. The currents could be further enhanced when the solution of red blood cells was exposed to hydrogen peroxide. We show that the enhanced signal can be used to detect red blood cells at a single entity level. The method presented relies on the catalytic activity of red blood cells towards hydrogen peroxide and on surface-induced haemolysis. Each single cell activity is expressed as current spikes decaying within a few seconds back to the background current. The frequency of such current spikes is proportional to the concentration of cells in solution. PMID:27355839

  19. Cell-to-Cell Trafficking of Macromolecules through Plasmodesmata Potentiated by the Red Clover Necrotic Mosaic Virus Movement Protein.

    PubMed

    Fujiwara, T.; Giesman-Cookmeyer, D.; Ding, B.; Lommel, S. A.; Lucas, W. J.

    1993-12-01

    Direct evidence is presented for cell-to-cell trafficking of macromolecules via plasmodesmata in higher plants. The fluorescently labeled 35-kD movement protein of red clover necrotic mosaic virus (RCNMV) trafficked rapidly from cell to cell when microinjected into cowpea leaf mesophyll cells. Furthermore, this protein potentiated rapid cell-to-cell trafficking of RCNMV RNA, but not DNA. Electron microscopic studies demonstrated that the 35-kD movement protein does not unfold the RCNMV RNA molecules. Thus, if unfolding of RNA is necessary for cell-to-cell trafficking, it may well involve participation of endogenous cellular factors. These findings support the hypothesis that trafficking of macromolecules is a normal plasmodesmal function, which has been usurped by plant viruses for their cell-to-cell spread. PMID:12271056

  20. Effects of helicopter transport on red blood cell components

    PubMed Central

    Otani, Taiichi; Oki, Ken-ichi; Akino, Mitsuaki; Tamura, Satoru; Naito, Yuki; Homma, Chihiro; Ikeda, Hisami; Sumita, Shinzou

    2012-01-01

    Background There are no reported studies on whether a helicopter flight affects the quality and shelf-life of red blood cells stored in mannitol-adenine-phosphate. Materials and methods Seven days after donation, five aliquots of red blood cells from five donors were packed into an SS-BOX-110 container which can maintain the temperature inside the container between 2 °C and 6 °C with two frozen coolants. The temperature of an included dummy blood bag was monitored. After the box had been transported in a helicopter for 4 hours, the red blood cells were stored again and their quality evaluated at day 7 (just after the flight), 14, 21 and 42 after donation. Red blood cell quality was evaluated by measuring adenosine triphosphate, 2,3-diphosphoglycerate, and supernatant potassium, as well as haematocrit, intracellular pH, glucose, supernatant haemoglobin, and haemolysis rate at the various time points. Results During the experiment the recorded temperature remained between 2 and 6 °C. All data from the red blood cells that had undergone helicopter transportation were the same as those from a control group of red blood cell samples 7 (just after the flight), 14, 21, and 42 days after the donation. Only supernatant Hb and haemolysis rate 42 days after the donation were slightly increased in the helicopter-transported group of red blood cell samples. All other parameters at 42 days after donation were the same in the two groups of red blood cells. Discussion These results suggest that red blood cells stored in mannitol-adenine-phosphate are not significantly affected by helicopter transportation. The differences in haemolysis by the end of storage were small and probably not of clinical significance. PMID:22153688

  1. Label-free high-throughput cell screening in flow

    PubMed Central

    Mahjoubfar, Ata; Chen, Claire; Niazi, Kayvan R.; Rabizadeh, Shahrooz; Jalali, Bahram

    2013-01-01

    Flow cytometry is a powerful tool for cell counting and biomarker detection in biotechnology and medicine especially with regards to blood analysis. Standard flow cytometers perform cell type classification both by estimating size and granularity of cells using forward- and side-scattered light signals and through the collection of emission spectra of fluorescently-labeled cells. However, cell surface labeling as a means of marking cells is often undesirable as many reagents negatively impact cellular viability or provide activating/inhibitory signals, which can alter the behavior of the desired cellular subtypes for downstream applications or analysis. To eliminate the need for labeling, we introduce a label-free imaging-based flow cytometer that measures size and cell protein concentration simultaneously either as a stand-alone instrument or as an add-on to conventional flow cytometers. Cell protein concentration adds a parameter to cell classification, which improves the specificity and sensitivity of flow cytometers without the requirement of cell labeling. This system uses coherent dispersive Fourier transform to perform phase imaging at flow speeds as high as a few meters per second. PMID:24049682

  2. Red Blood Cells Preconditioned with Hemin Are Less Permissive to Plasmodium Invasion In Vivo and In Vitro

    PubMed Central

    Gaudreault, Véronique; Wirbel, Jakob; Jardim, Armando; Rohrbach, Petra; Scorza, Tatiana

    2015-01-01

    Malaria is a parasitic disease that causes severe hemolytic anemia in Plasmodium-infected hosts, which results in the release and accumulation of oxidized heme (hemin). Although hemin impairs the establishment of Plasmodium immunity in vitro and in vivo, mice preconditioned with hemin develop lower parasitemia when challenged with Plasmodium chabaudi adami blood stage parasites. In order to understand the mechanism accounting for this resistance as well as the impact of hemin on eryptosis and plasma levels of scavenging hemopexin, red blood cells were labeled with biotin prior to hemin treatment and P. c. adami infection. This strategy allowed discriminating hemin-treated from de novo generated red blood cells and to follow the infection within these two populations of cells. Fluorescence microscopy analysis of biotinylated-red blood cells revealed increased P. c. adami red blood cells selectivity and a decreased permissibility of hemin-conditioned red blood cells for parasite invasion. These effects were also apparent in in vitro P. falciparum cultures using hemin-preconditioned human red blood cells. Interestingly, hemin did not alter the turnover of red blood cells nor their replenishment during in vivo infection. Our results assign a function for hemin as a protective agent against high parasitemia, and suggest that the hemolytic nature of blood stage human malaria may be beneficial for the infected host. PMID:26465787

  3. Live cell immunogold labelling of RNA polymerase II

    PubMed Central

    Orlov, Igor; Schertel, Andreas; Zuber, Guy; Klaholz, Bruno; Drillien, Robert; Weiss, Etienne; Schultz, Patrick; Spehner, Danièle

    2015-01-01

    Labeling nuclear proteins with electron dense probes in living cells has been a major challenge due to their inability to penetrate into nuclei. We developed a lipid-based approach for delivering antibodies coupled to 0.8 nm ultrasmall gold particles into the nucleus to label RNA polymerase II. Focussed Ion Beam slicing coupled to Scanning Electron Microscopy (FIB/SEM) enabled visualization of entire cells with probe localization accuracy in the 10 nm range. PMID:25662860

  4. Red Blood Cell Dysfunction Induced by High-Fat Diet

    PubMed Central

    Unruh, Dusten; Srinivasan, Ramprasad; Benson, Tyler; Haigh, Stephen; Coyle, Danielle; Batra, Neil; Keil, Ryan; Sturm, Robert; Blanco, Victor; Palascak, Mary; Franco, Robert S.; Tong, Wilson; Chatterjee, Tapan; Hui, David Y.; Davidson, W. Sean; Aronow, Bruce J.; Kalfa, Theodosia; Manka, David; Peairs, Abigail; Blomkalns, Andra; Fulton, David J.; Brittain, Julia E.; Weintraub, Neal L.; Bogdanov, Vladimir Y.

    2015-01-01

    Background High-fat diet (HFD) promotes endothelial dysfunction and proinflammatory monocyte activation, which contribute to atherosclerosis in obesity. We investigated whether HFD also induces the dysfunction of red blood cells (RBCs), which serve as a reservoir for chemokines via binding to Duffy antigen receptor for chemokines (DARC). Methods and Results A 60% HFD for 12 weeks, which produced only minor changes in lipid profile in C57/BL6 mice, markedly augmented the levels of monocyte chemoattractant protein-1 bound to RBCs, which in turn stimulated macrophage migration through an endothelial monolayer. Levels of RBC-bound KC were also increased by HFD. These effects of HFD were abolished in DARC−/− mice. In RBCs from HFD-fed wild-type and DARC−/− mice, levels of membrane cholesterol and phosphatidylserine externalization were increased, fostering RBC-macrophage inflammatory interactions and promoting macrophage phagocytosis in vitro. When labeled ex vivo and injected into wild-type mice, RBCs from HFD-fed mice exhibited ≈3-fold increase in splenic uptake. Finally, RBCs from HFD-fed mice induced increased macrophage adhesion to the endothelium when they were incubated with isolated aortic segments, indicating endothelial activation. Conclusions RBC dysfunction, analogous to endothelial dysfunction, occurs early during diet-induced obesity and may serve as a mediator of atherosclerosis. These findings may have implications for the pathogenesis of atherosclerosis in obesity, a worldwide epidemic. PMID:26467254

  5. Labeling of lectin receptors during the cell cycle.

    PubMed

    Garrido, J

    1976-12-01

    Labeling of lectin receptors during the cell cycle. (Localizabión de receptores para lectinas durante el ciclo celular). Arch. Biol. Med. Exper. 10: 100-104, 1976. The topographic distribution of specific cell surface receptors for concanavalin A and wheat germ agglutinin was studied by ultrastructural labeling in the course of the cell cycle. C12TSV5 cells were synchronized by double thymidine block or mechanical selection (shakeoff). They were labeled by means of lectin-peroxidase techniques while in G1 S, G2 and M phases of the cycle. The results obtained were similar for both lectins employed. Interphase cells (G1 S, G2) present a stlihtly discontinous labeling pattern that is similar to the one observed on unsynchronized cells of the same line. Cells in mitosis, on the contrary, present a highly discontinous distribution of reaction product. This pattern disappears after the cells enters G1 and is not present on mitotic cells fixed in aldehyde prior to labeling. PMID:1030938

  6. 21 CFR 660.35 - Labeling.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Reagent Red Blood Cells § 660.35 Labeling. In... or end of the label, oustide of the main panel. (2) If washing the cells is required by the manufacturer, the container label shall include appropriate instructions; if the cells should not be...

  7. 21 CFR 660.35 - Labeling.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Reagent Red Blood Cells § 660.35 Labeling. In... or end of the label, oustide of the main panel. (2) If washing the cells is required by the manufacturer, the container label shall include appropriate instructions; if the cells should not be...

  8. 21 CFR 660.35 - Labeling.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Reagent Red Blood Cells § 660.35 Labeling. In... or end of the label, oustide of the main panel. (2) If washing the cells is required by the manufacturer, the container label shall include appropriate instructions; if the cells should not be...

  9. Dysferlin and Other Non-Red Cell Proteins Accumulate in the Red Cell Membrane of Diamond-Blackfan Anemia Patients

    PubMed Central

    Pesciotta, Esther N.; Sriswasdi, Sira; Tang, Hsin-Yao; Speicher, David W.; Mason, Philip J.; Bessler, Monica

    2014-01-01

    Diamond Blackfan Anemia (DBA) is a congenital anemia usually caused by diverse mutations in ribosomal proteins. Although the genetics of DBA are well characterized, the mechanisms that lead to macrocytic anemia remain unclear. We systematically analyzed the proteomes of red blood cell membranes from multiple DBA patients to determine whether abnormalities in protein translation or erythropoiesis contribute to the observed macrocytosis or alterations in the mature red blood cell membrane. In depth proteome analysis of red cell membranes enabled highly reproducible identification and quantitative comparisons of 1100 or more proteins. These comparisons revealed clear differences between red cell membrane proteomes in DBA patients and healthy controls that were consistent across DBA patients with different ribosomal gene mutations. Proteins exhibiting changes in abundance included those known to be increased in DBA such as fetal hemoglobin and a number of proteins not normally found in mature red cell membranes, including proteins involved in the major histocompatibility complex class I pathway. Most striking was the presence of dysferlin in the red blood cell membranes of DBA patients but absent in healthy controls. Immunoblot validation using red cell membranes isolated from additional DBA patients and healthy controls confirmed a distinct membrane protein signature specific to patients with DBA. PMID:24454878

  10. Molecular basis of red cell membrane disorders.

    PubMed

    Delaunay, Jean

    2002-01-01

    We will consider an array of genetic disorders of the red cell membrane. Some affect well-known genes. The mutations of most cases of hereditary spherocytosis (HS) are located in the following genes: ANK1, SPTB, SLC4A1, EPB42 and SPTA1, which encode ankyrin, spectrin beta-chain, the anion exchanger 1 (band 3), protein 4.2 and spectrin alpha-chain, respectively. A dominant form of distal renal tubular acidosis also stems from distinct mutations in the SLC4A1 gene. The mutations responsible for hereditary elliptocytosis (HE) and its aggravated form, poikilocytosis (HP), lie in the SPTA1 and SPTB gene, already mentioned, and in the EPB41 gene encoding protein 4.1. Whereas in HS, the SPTA1 and SPTB gene mutations tend to abolish the synthesis of the corresponding chains, in HE/HP, they hinder spectrin tetramerization. Allele alpha(LELY) is a common polymorphic allele which plays the role of an aggravating factor when it occurs in trans of an elliptocytogenic allele of the SPTA1 gene. Southeast Asian ovalocytosis results from a 27- nucleotide deletion in the SLC4A1 gene. Besides these conditions in which the mutations were reached from known alterations in the proteins, other conditions required a positional cloning approach. Such are the genetic disorders of membrane permeability to monovalent cations. Knowledge is the most advanced as regards dehydrated hereditary stomatocytois (DHS). DHS was shown to belong to a pleiotropic syndrome: DHS + fetal edema + pseudohyperkalemia, which maps to 16q23-24. Concerning DHS and another disease of the same class, overhydrated hereditary stomatocytosis, splenectomy almost certainly appears to elicit thromboembolic accidents. PMID:12432217

  11. Molecular characterization of the human red cell Rho(D) antigen.

    PubMed Central

    Gahmberg, C G

    1983-01-01

    Human red cells of Rh blood groups -D-/-D- ('super-D'), -/- (Rhnull) and normal Rho(D)+ cells were radioactively surface-labeled using the lactoperoxidase 125I method. Polyacrylamide gel electrophoresis in the presence of SDS followed by fluorography showed a strong enrichment of a polypeptide with an apparent mol. wt. of 28,0000-33,000 in the 125I-labeled -D-/-D- membranes. This polypeptide was specifically immune precipitated with anti-Rho(D) antiserum. Treatment of intact cells with trypsin or Pronase did not digest the protein. The Rho polypeptide migrated identically on polyacrylamide gel electrophoresis under reducing and non-reducing conditions. It was not phosphorylated after in vitro incubation of red cells with 32P. When whole labeled membranes were solubilized in neutral detergent and applied to lectin-Sepharose columns the Rho(D) polypeptide adsorbed to Ricinus communis lectin but not to wheat germ lectin or Lens culinaris lectin. The purified molecule did not adsorb to R. communis lectin-Sepharose. Treatment of the Rho(D) antigen with endo-N-acetyl glucosaminidase H, endo-beta-galactosidase or mild alkali did not lower its apparent mol. wt. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 7. PMID:11894930

  12. Single-cell measurement of red blood cell oxygen affinity

    PubMed Central

    Di Caprio, Giuseppe; Stokes, Chris; Higgins, John M.; Schonbrun, Ethan

    2015-01-01

    Oxygen is transported throughout the body by hemoglobin (Hb) in red blood cells (RBCs). Although the oxygen affinity of blood is well-understood and routinely assessed in patients by pulse oximetry, variability at the single-cell level has not been previously measured. In contrast, single-cell measurements of RBC volume and Hb concentration are taken millions of times per day by clinical hematology analyzers, and they are important factors in determining the health of the hematologic system. To better understand the variability and determinants of oxygen affinity on a cellular level, we have developed a system that quantifies the oxygen saturation, cell volume, and Hb concentration for individual RBCs in high throughput. We find that the variability in single-cell saturation peaks at an oxygen partial pressure of 2.9%, which corresponds to the maximum slope of the oxygen–Hb dissociation curve. In addition, single-cell oxygen affinity is positively correlated with Hb concentration but independent of osmolarity, which suggests variation in the Hb to 2,3-diphosphoglycerate (2–3 DPG) ratio on a cellular level. By quantifying the functional behavior of a cellular population, our system adds a dimension to blood cell analysis and other measurements of single-cell variability. PMID:26216973

  13. IgG red blood cell autoantibodies in autoimmune hemolytic anemia bind to epitopes on red blood cell membrane band 3 glycoprotein

    SciTech Connect

    Victoria, E.J.; Pierce, S.W.; Branks, M.J.; Masouredis, S.P. )

    1990-01-01

    Red blood cell (RBC) autoantibodies from patients with IgG warm-type autoimmune hemolytic anemia were labeled with iodine 125 and their RBC binding behavior characterized. Epitope-bearing RBC membrane polypeptides were identified after autoantibody immunoprecipitation of labeled membranes and immunoblotting. Immunoaffinity isolation of labeled membrane proteins with 12 different IgG hemolytic autoantibodies with protein A-agarose revealed a major polypeptide at Mr 95 to 110 kd, which coelectrophoresed on sodium dodecylsulfate-polyacrylamide gel electrophoresis with a membrane component isolated with sheep IgG anti-band 3. Immunoprecipitation studies with chymotrypsinized RBCs resulted in the recovery of two labeled membrane polypeptides with molecular weights characteristically resulting from the chymotryptic fragmentation of band 3. Immunoblotting with sheep IgG anti-band 3 of the immunoprecipitated polypeptides confirmed that hemolytic autoantibody binding led to recovery of band 3 or its fragments. Two 125I-labeled IgG hemolytic autoantibodies showed binding behavior consistent with epitope localization on band 3. The labeled RBC autoantibodies bound immunospecifically to all types of human RBC tested, including those of rare Rh type (Rh-null, D--) at a site density of approximately 10(6) per RBC. The 125I-IgG in two labeled autoantibodies was 84% and 92% adsorbable by human and higher nonhuman primate RBCs. Antigen-negative animal RBC bound less than 10%, consistent with immunospecific RBC binding. IgG-1 was the major subclass in five autoantibodies tested; one of six fixed complement; and autoantibody IgG appeared polyclonal by isoelectric focusing. We conclude that IgG eluted from RBCs of patients with autoimmune hemolytic anemia consists predominantly of a single totally RBC-adsorbable antibody population that binds to antigenic determinants on band 3.

  14. Delayed positive gastrointestinal bleeding studies with technetium-99m-red blood cells: Utility of a second injection

    SciTech Connect

    Jacobson, A.F. )

    1991-02-01

    Two patients studied with technetium-99m-labeled red blood cells (RBCs) for gastrointestinal bleeding had positive findings only on 24-hr delayed images, at which time the site of bleeding could not be ascertained. In each instance, when additional delayed images suggested that active bleeding was occurring, a second aliquot of RBCs was labeled and injected. Sites of active hemorrhage were identified following further imaging in both patients. When delayed GI bleeding images are positive, further views should be obtained to ascertain if the pattern of intraluminal activity changes. If renewed active hemorrhage is suspected, reinjection with a second dose of labeled RBCs may identify the bleeding site.

  15. Formation of dimethylthioarsenicals in red blood cells

    SciTech Connect

    Naranmandura, Hua; Suzuki, Kazuo T.

    2008-03-15

    The bladder and skin are the primary targets for arsenic-induced carcinogenicity in mammals. Thioarsenicals dimethylmonothioarsinic (DMMTA{sup V}) and dimethyldithioarsinic (DMDTA{sup V}) acids are common urinary metabolites, the former being much more toxic than non-thiolated dimethylarsinic acid (DMA{sup V}) and comparable to dimethylarsinous acid (DMA{sup III}) in epidermoid cells, suggesting that the metabolic production of thioarsenicals may be a risk factor for the development of cancer in these organs. To reveal their production sites (tissues/body fluids), we examined the uptake and transformation of the four dimethylated arsenicals by incubation with rat and human red blood cells (RBCs). Although DMA{sup V} and DMDTA{sup V} were not taken up by either type of RBCs, DMA{sup III} and DMMTA{sup V} were taken up by both (more efficiently by rat ones), though DMMTA{sup V} was taken up slowly, and then the arsenic transformed into DMDTA{sup V} was excreted from both types of animal RBCs. On the other hand, although DMA{sup III} taken up rapidly by rat RBCs was retained in the RBCs, that taken up by human RBCs was immediately transformed into DMMTA{sup V} and then excreted into the incubation medium without being retained in the RBCs. In a separate experiment, arsenic remaining in primary rat hepatocytes after incubation with 1.5 {mu}M DMA{sup III} was recovered from the incubation medium in the forms of DMA{sup V} and DMMTA{sup V} in the presence of human RBCs, but not in the presence of rat RBCs (in which the arsenic was bound to hemoglobin). Thus, DMMTA{sup V} was detected in the medium only in the presence of human RBCs and increased with incubation time. It was proposed that arsenic is excreted from hepatocytes into the bloodstream in the form of DMA{sup III} and then taken up by RBCs in humans, where it is transformed into DMMTA{sup V} and then excreted again into the bloodstream.

  16. Attachment of antibody to biotinylated red blood cells: immuno-red blood cells display high affinity to immobilized antigen and normal biodistribution in rats.

    PubMed

    Muzykantov, V R; Murciano, J C

    1996-08-01

    Streptavidin-mediated attachment of biotinylated antibodies (b-Ab) to biotinylated red blood cells (b-RBC) is useful for preparation of immuno-red blood cells, a prospective vehicle for drug targeting. However, streptavidin (SA) induces lysis of extensively biotinylated RBC by complement due to cross-linking and inactivation of RBC complement regulators. To reduce cross-linking of RBC membrane proteins, we utilized mild biotinylation of RBC with 20 microM biotin ester (b20-RBC). SA effectively binds to rat b20-RBC (10(5) SA molecules/cell) and provides for following attachment of 5 x 10(4) molecules of b-IgG/SA per b20-RBC. By in vitro assay, b-Ab/SA/b20-RBC were stable in fresh rat serum. Serum-stable immuno-red blood cells (b-Ab/SA/b20-RBC) specifically bound to antigen-coated surfaces, but not to BSA-coated surfaces. Biodistribution of 51Cr-labelled b-Ab/SA/b20-RBC in rats was similar to that of control RBC, with no indication of lysis in vivo. These results suggest b-Ab/SA/b20-RBC may be explored as a vehicle for drug targeting. PMID:8756393

  17. Dynamic quantitative photothermal monitoring of cell death of individual human red blood cells upon glucose depletion

    NASA Astrophysics Data System (ADS)

    Vasudevan, Srivathsan; Chen, George Chung Kit; Andika, Marta; Agarwal, Shuchi; Chen, Peng; Olivo, Malini

    2010-09-01

    Red blood cells (RBCs) have been found to undergo ``programmed cell death,'' or eryptosis, and understanding this process can provide more information about apoptosis of nucleated cells. Photothermal (PT) response, a label-free photothermal noninvasive technique, is proposed as a tool to monitor the cell death process of living human RBCs upon glucose depletion. Since the physiological status of the dying cells is highly sensitive to photothermal parameters (e.g., thermal diffusivity, absorption, etc.), we applied linear PT response to continuously monitor the death mechanism of RBC when depleted of glucose. The kinetics of the assay where the cell's PT response transforms from linear to nonlinear regime is reported. In addition, quantitative monitoring was performed by extracting the relevant photothermal parameters from the PT response. Twofold increases in thermal diffusivity and size reduction were found in the linear PT response during cell death. Our results reveal that photothermal parameters change earlier than phosphatidylserine externalization (used for fluorescent studies), allowing us to detect the initial stage of eryptosis in a quantitative manner. Hence, the proposed tool, in addition to detection of eryptosis earlier than fluorescence, could also reveal physiological status of the cells through quantitative photothermal parameter extraction.

  18. Reflectance confocal microscopy of red blood cells: simulation and experiment

    PubMed Central

    Zeidan, Adel; Yelin, Dvir

    2015-01-01

    Measuring the morphology of red blood cells is important for clinical diagnosis, providing valuable indications on a patient’s health. In this work, we have simulated the appearance of normal red blood cells under a reflectance confocal microscope and discovered unique relations between the morphological parameters and the resulting characteristic interference patterns of the cell. The simulation results showed good agreement with in vitro reflectance confocal images of red blood cells, acquired using spectrally encoded flow cytometry that imaged the cells in a linear flow without artificial staining. By matching the simulated patterns to confocal images of the cells, this method could be used for measuring cell morphology in three dimensions and for studying their physiology. PMID:26600999

  19. Red blood cell-derived microparticles: An overview.

    PubMed

    Westerman, Maxwell; Porter, John B

    2016-07-01

    The red blood cell (RBC) is historically the original parent cell of microparticles (MPs). In this overview, we describe the discovery and the early history of red cell-derived microparticles (RMPs) and present an overview of the evolution of RMP. We report the formation, characteristics, effects of RMP and factors which may affect RMP evaluation. The review examines RMP derived from both normal and pathologic RBC. The pathologic RBC studies include sickle cell anemia (SCA), sickle cell trait (STr), thalassemia intermedia (TI), hereditary spherocytosis (HS), hereditary elliptocytosis (HE), hereditary stomatocytosis (HSt) and glucose-6-phosphate dehydrogenase deficiency (G6PD). PMID:27282583

  20. Method for determining properties of red blood cells

    DOEpatents

    Gourley, Paul L.

    2001-01-01

    A method for quantifying the concentration of hemoglobin in a cell, and indicia of anemia, comprises determining the wavelength of the longitudinal mode of a liquid in a laser microcavity; determining the wavelength of the fundamental transverse mode of a red blood cell in the liquid in the laser microcavity; and determining if the cell is anemic from the difference between the wavelength of the longitudinal mode and the fundamental transverse mode. In addition to measuring hemoglobin, the invention includes a method using intracavity laser spectroscopy to measure the change in spectra as a function of time for measuring the influx of water into a red blood cell and the cell's subsequent rupture.

  1. The Effect of Shape Memory on Red Blood Cell Motions

    NASA Astrophysics Data System (ADS)

    Niu, Xiting; Shi, Lingling; Pan, Tsorng-Whay; Glowinski, Roland

    2013-11-01

    An elastic spring model is applied to study the effect of the shape memory on the motion of red blood cell in flows. In shear flow, shape memory also plays an important role to obtain all three motions: tumbling, swinging, and tank-treading. In Poiseuille flow, cell has an equilibrium shape as a slipper or parachute depending on capillary number. To ensure the tank-treading motion while in slippery shape, a modified model is proposed by introducing a shape memory coefficient which describes the degree of shape memory in cells. The effect of the coefficient on the cell motion of red blood cell will be presented.

  2. Polyelectrolyte coating of ferumoxytol nanoparticles for labeling of dendritic cells

    NASA Astrophysics Data System (ADS)

    Celikkin, Nehar; Jakubcová, Lucie; Zenke, Martin; Hoss, Mareike; Wong, John Erik; Hieronymus, Thomas

    2015-04-01

    Engineered magnetic nanoparticles (MNPs) are emerging to be used as cell tracers, drug delivery vehicles, and contrast agents for magnetic resonance imaging (MRI) for enhanced theragnostic applications in biomedicine. In vitro labeling of target cell populations with MNPs and their implantation into animal models and patients shows promising outcomes in monitoring successful cell engraftment, differentiation and migration by using MRI. Dendritic cells (DCs) are professional antigen-presenting cells that initiate adaptive immune responses. Thus, DCs have been the focus of cellular immunotherapy and are increasingly applied in clinical trials. Here, we addressed the coating of different polyelectrolytes (PE) around ferumoxytol particles using the layer-by-layer technique. The impact of PE-coated ferumoxytol particles for labeling of DCs and Flt3+ DC progenitors was then investigated. The results from our studies revealed that PE-coated ferumoxytol particles can be readily employed for labeling of DC and DC progenitors and thus are potentially suitable as contrast agents for MRI tracking.

  3. Effect of an electrical left ventricular assist device on red blood cell and platelet survival in the cow. Technical report

    SciTech Connect

    Melaragno, A.J.; Vecchione, J.J.; Katchis, R.J.; Abdu, W.A.; Ouellet, R.P.

    1982-04-23

    Blood volume measurements were made in cows after infusion of human 125 iodine albumin and autologous 51 chromium-labeled red blood cells. Repeated intravenous infusions of iodinated human albumin did not appear to isosensitize the cows. When the cow red blood cells were incubated at 37 C after labeling with 51 chromium, there was elution of the 51 chromium, and the 51 chromium T 50 values were 45 hours in both healthy cows and cows with LVAD's. Measurements also were made in the cow platelets labeled with 51 chromium or 111 Indium-oxine. The platelets labeled with 51 chromium had T 50 values of 4 days, and platelets labeled with 111 Indium-oxine had T 50 values of 0.9 to 2.7 days. 51 chromium-labeled platelets had similar T 50 values in healthy cows and cows with LVAD's. Bovine platelets isolated from units of blood using serial differential centrifugation were labeled with 51 chromium or with 111 Indium-oxine, and after infusion in healthy cows and cows with LVAD's measurements were made of platelet circulation and distribution. The disappearance of platelet radioactivity from the blood was linear with time, and the platelet lifespan was 6-10 days. The presence of an LVAD did not affect initial recovery or lifespan of cow platelets.

  4. Separation of a single cell by red-laser manipulation

    NASA Astrophysics Data System (ADS)

    Shikano, Shuji; Horio, Koji; Ohtsuka, Yoshihiro; Eto, Yuzuro

    1999-10-01

    A single cell of yeast was separated from a bulk sample of yeast without causing damage to the cell. A focused red-laser light beam was used for trapping and transporting the cell. A specially designed microchannel separator played an essential role in the success of the separation.

  5. Acetylcholinesterase: an enzymatic marker of human red blood cell aging.

    PubMed

    Prall, Y G; Gambhir, K K; Ampy, F R

    1998-01-01

    The purpose of this investigation was to determine whether acetylcholinesterase (AChE) can be used as a marker of cell aging in human red blood cells (RBCs). This study used consented subjects; both males and females in an age range of 21-42 years. The blood samples (8-9 mL) were drawn in tubes containing sodium heparin or EDTA as anticoagulants. To avoid contamination with other cells, (lymphocytes, monocytes and reticulocytes), RBCs were purified (PRBC) by Hypaque-Ficoll gradient technique. The PRBCs were subfractionated into young (y) (1.08-1.09), mid (m) (1.09-1.11) and old (o) (1.11-1.12) percoll density (g/mL) fractions using a discontinuous percoll gradient. The mean +/- 1 SD AChE per gram hemoglobin (U/g Hgb) activities in whole blood (WB) purified human red blood cells (PRBCs), young human red blood cells (y-RBCs), mid age human red blood cells (m-RBCs) and old human red blood cells (o-RBCs) were 27.4 +/- 2.98, 26.0 +/- 2.33, 25.5 +/- 1.64, 20.3 +/- 3.84, 14.6 +/- 3.42 in males and 26.3 +/- 4.44, 24.8 /- 4.83, 26.4 +/- 4.59, 24.0 +/- 5.50 and 12.4 +/- 7.09 in females respectively. Although there was variation in the data, the results indicated that old human red blood cells showed significantly (p<.05) lower AChE activity compared to young human red blood cells of both sexes. These preliminary but novel observations suggest that AChE can be an excellent enzymatic marker for RBC aging in man. PMID:9698047

  6. Stable isotope labeling of oligosaccharide cell surface antigens

    SciTech Connect

    Unkefer, C.J.; Silks, L.A. III; Martinez, R.A.

    1998-12-31

    The overall goal of this Laboratory Directed Research and Development (LDRD) project was to develop new methods for synthesis of {sup 13}C-labeled oligosaccharides that are required for nuclear magnetic resonance (NMR) studies of their solution conformation. Oligosaccharides are components of the cell`s outer surface and are involved in important processes such as cell-cell recognition and adhesion. Recently, Danishefsky and coworkers at Slone-Kettering Cancer Center developed a method for the solid-phase chemical synthesis of oligosaccharides. The specific goal of this LDRD project was to prepare uniform {sup 13}C-labeled aldohexose precursors required for the solid-phase synthesis of the Lewis blood-group antigenic determinants. We report the synthesis of {sup 13}C-labeled D-glucal, D-galactal and Fucosyl precursors. We have been collaborating with the Danishefsky group on the synthesis of the Lewis oligosaccharides and the NMR analysis of their solution conformation.

  7. Transplantation of Adrenal Cortical Progenitor Cells Enriched by Nile Red

    PubMed Central

    Dunn, James C.Y.; Chu, Yinting; Qin, Harry H.; Zupekan, Tatiana

    2009-01-01

    Background The adrenal cortex may contain progenitor cells useful for tissue regeneration. Currently there are no established methods to isolate these cells. Material and Methods Murine adrenal cells were sorted into a Nile-Red-bright (NRbright) and a Nile-Red-dim (NRdim) population of cells according to their degree of cholesterol content revealed by Nile Red fluorescence. The cells were transplanted under the renal capsule to determine their ability for regeneration. Results The NRbright cells contained an abundance of lipid droplets, whereas the NRdim cells contained little. The NRbright cells expressed Sf1 and the more differentiated adrenal cortical genes including Cyp11a1, Cyp11b1, and Cyp11b2, whereas the NRdim cells expressed Sf1 but not the more differentiated adrenal cortical genes. After 56 days of implantation in unilateral adrenalectomized mice, the NRdim cells expressed Sf1 and the more differentiated adrenal cortical genes, whereas the NRbright cells ceased to express Sf1 as well as the more differentiated adrenal cortical genes. NRdim cells also proliferated in the presence of basic fibroblast growth factor. Conclusions The population of NRdim cells contained adrenal cortical progenitor cells that can proliferate and give rise to differentiated daughter cells. These cells may be useful for adrenal cortical regeneration. PMID:19592014

  8. Theory of the sphering of red blood cells.

    PubMed

    Fung, Y C; Tong, P

    1968-02-01

    A rigorous mathematical solution of the sphering of a red blood cell is obtained under the assumptions that the red cells is a fluid-filled shell and that it can swell into a perfect sphere in an appropriate hypotonic medium. The solution is valid for finite strain of the cell membrane provided that the membrane is isotropic, elastic and incompressible. The most general nonlinear elastic stress-strain law for the membrane in a state of generalized plane stress is used. A necessary condition for a red cell to be able to sphere is that its extensional stiffness follow a specific distribution over the membrane. This distribution is strongly influenced by the surface tension in the cell membrane. A unique relation exists between the extensional stiffness, pressure differential, surface tension, and the ratio of the radius of the sphere to that of the undeformed red cell. The functional dependence of this stiffness distribution on various physical parameters is presented. A critique of some current literature on red cell mechanics is presented. PMID:5639934

  9. Quantification of Depletion-Induced Adhesion of Red Blood Cells

    NASA Astrophysics Data System (ADS)

    Steffen, P.; Verdier, C.; Wagner, C.

    2013-01-01

    Red blood cells (RBCs) are known to form aggregates in the form of rouleaux due to the presence of plasma proteins under physiological conditions. The formation of rouleaux can also be induced in vitro by the addition of macromolecules to the RBC suspension. Current data on the adhesion strength between red blood cells in their natural discocyte shapes mostly originate from indirect measurements such as flow chamber experiments, but data is lacking at the single cell level. Here, we present measurements on the dextran-induced aggregation of red blood cells using atomic force microscopy-based single cell force spectroscopy. The effects of dextran concentration and molecular weight on the interaction energy of adhering RBCs were determined. The results on adhesion energy are in excellent agreement with a model based on the depletion effect and previous experimental studies. Furthermore, our method allowed to determine the adhesion force, a quantity that is needed in theoretical investigations on blood flow.

  10. Quantification of depletion-induced adhesion of red blood cells.

    PubMed

    Steffen, P; Verdier, C; Wagner, C

    2013-01-01

    Red blood cells (RBCs) are known to form aggregates in the form of rouleaux due to the presence of plasma proteins under physiological conditions. The formation of rouleaux can also be induced in vitro by the addition of macromolecules to the RBC suspension. Current data on the adhesion strength between red blood cells in their natural discocyte shapes mostly originate from indirect measurements such as flow chamber experiments, but data is lacking at the single cell level. Here, we present measurements on the dextran-induced aggregation of red blood cells using atomic force microscopy-based single cell force spectroscopy. The effects of dextran concentration and molecular weight on the interaction energy of adhering RBCs were determined. The results on adhesion energy are in excellent agreement with a model based on the depletion effect and previous experimental studies. Furthermore, our method allowed to determine the adhesion force, a quantity that is needed in theoretical investigations on blood flow. PMID:23383842

  11. Fluorescent Labeling of Yeast Cell Wall Components.

    PubMed

    Okada, Hiroki; Ohya, Yoshikazu

    2016-01-01

    Yeast cells stained with a fluorescent dye that specifically binds to one of the cell wall components can be observed under a fluorescent microscope. Visualization of the components 1,3-β-glucan, mannoproteins, and/or chitin not only provides information concerning the cell wall, but also reveals clues about various cellular activities such as cell polarity, vesicular transport, establishment of budding pattern, apical and isotropic bud growth, and replicative cell age. This protocol describes a standard method for visualizing different components of the yeast cell wall. PMID:27480714

  12. 21 CFR 660.35 - Labeling.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... matrix. (g) The package label or package insert shall state the blood group antigens that have been... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Reagent Red Blood Cells § 660.35 Labeling. In... container label of Group O cells shall state: “FOR USE IN DETECTION OF UNEXPECTED ANTIBODIES” or “FOR USE...

  13. X-ray microscopic studies of labeled nuclear cell structures

    NASA Astrophysics Data System (ADS)

    Vogt, S.; Schneider, G.; Steuernagel, A.; Lucchesi, J.; Schulze, E.; Rudolph, D.; Schmahl, G.

    2000-05-01

    In X-ray microscopy different proteins are not readily distinguishable. However, in cell biology it is often desirable to localize single proteins, e.g., inside the cell nucleus. This can be achieved by immunogold labeling. Colloidal gold conjugated antibodies are used to mark the protein specifically. With silver solution these are enlarged so as to heighten their contrast. The strong absorption of silver allows easy visualization of the label in the nuclei. In this study male specific lethal 1 protein in male Drosophila melanogaster cells was labeled. This protein forms, together with four other proteins, a complex that is associated with the male X chromosome. It regulates dosage compensation by enhancing X-linked gene transcription in males. Room temperature and cyro transmission X-ray microscopic images (taken with the Göttingen TXM at BESSY) of these labeled cells are shown. Confocal laser scan microscopy ascertains the correct identification of the label in the X-ray micrographs, and allows comparison of the structural information available from both instruments.

  14. 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. PMID:25536421

  15. 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

  16. Labeling proteins on live mammalian cells using click chemistry.

    PubMed

    Nikić, Ivana; Kang, Jun Hee; Girona, Gemma Estrada; Aramburu, Iker Valle; Lemke, Edward A

    2015-05-01

    We describe a protocol for the rapid labeling of cell-surface proteins in living mammalian cells using click chemistry. The labeling method is based on strain-promoted alkyne-azide cycloaddition (SPAAC) and strain-promoted inverse-electron-demand Diels-Alder cycloaddition (SPIEDAC) reactions, in which noncanonical amino acids (ncAAs) bearing ring-strained alkynes or alkenes react, respectively, with dyes containing azide or tetrazine groups. To introduce ncAAs site specifically into a protein of interest (POI), we use genetic code expansion technology. The protocol can be described as comprising two steps. In the first step, an Amber stop codon is introduced--by site-directed mutagenesis--at the desired site on the gene encoding the POI. This plasmid is then transfected into mammalian cells, along with another plasmid that encodes an aminoacyl-tRNA synthetase/tRNA (RS/tRNA) pair that is orthogonal to the host's translational machinery. In the presence of the ncAA, the orthogonal RS/tRNA pair specifically suppresses the Amber codon by incorporating the ncAA into the polypeptide chain of the POI. In the second step, the expressed POI is labeled with a suitably reactive dye derivative that is directly supplied to the growth medium. We provide a detailed protocol for using commercially available ncAAs and dyes for labeling the insulin receptor, and we discuss the optimal surface-labeling conditions and the limitations of labeling living mammalian cells. The protocol involves an initial cloning step that can take 4-7 d, followed by the described transfections and labeling reaction steps, which can take 3-4 d. PMID:25906116

  17. Imaging of inflammatory processes with labeled cells

    SciTech Connect

    Froelich, J.W.; Swanson, D.

    1984-04-01

    Radionuclide techniques for localizing inflammatory processes had relied heavily upon /sup 67/Ga-citrate until McAfee and Thakur described the technique for the radiolabeling of leukocytes with /sup 111/In-oxine. Since their initial description in 1976 there has been continued development of the radiopharmaceutical, as well as clinical efficacy. At present /sup 111/In-labeled leukocytes continue to be handled as an investigational new drug but this has not greatly limited its clinical availability. Indium-/sup 111/ leukocytes are the agent of choice for evaluation of patients with fever of unknown origin, osteomyelitis, and prosthetic graft infections; and preliminary data shows great promise in the area of detecting reoccurrence of inflammatory bowel disease. This article attempts to review currently accepted uses of 111In leukocytes as well as potential areas of application.

  18. Pure Red Cell Aplasia Following Interleukin-2 Therapy

    PubMed Central

    Dutcher, Janice P.; Fan, Wen; Wiernik, Peter H.

    2016-01-01

    A 61-year-old woman with metastatic renal cell carcinoma underwent systemic treatment with high-dose interleukin-2 (IL-2). Anemia requiring transfusion of 1 unit of packed red blood cells (PRBCs) was required during the second week of IL-2 therapy. One month following completion of high-dose IL-2 treatment, she was hospitalized for severe, symptomatic anemia and received 5 units of PRBCs. She was referred back for evaluation. A complete hematologic evaluation was performed including antiviral serology, evaluation for hemolysis, complete iron studies, and finally bone marrow aspiration and biopsy. The diagnosis was pure red cell aplasia, and no inciting viral cause could be ascertained. She required PRBCs for 5 months following IL-2 therapy. It was concluded that IL-2 was the cause of her red cell aplasia. This subsequently resolved spontaneously, and she had normal hemoglobin and hematocrit, respectively, 1 and 2 years after treatment. PMID:27144182

  19. A statistical model for red blood cell survival.

    PubMed

    Korell, Julia; Coulter, Carolyn V; Duffull, Stephen B

    2011-01-01

    A statistical model for the survival time of red blood cells (RBCs) with a continuous distribution of cell lifespans is presented. The underlying distribution of RBC lifespans is derived from a probability density function with a bathtub-shaped hazard curve, and accounts for death of RBCs due to senescence (age-dependent increasing hazard rate) and random destruction (constant hazard), as well as for death due to initial or delayed failures and neocytolysis (equivalent to early red cell mortality). The model yields survival times similar to those of previously published studies of RBC survival and is easily amenable to inclusion of drug effects and haemolytic disorders. PMID:20950630

  20. The fate of phenylhydroxylamine in human red cells.

    PubMed

    Kiese, M; Taeger, K

    1976-01-01

    Phenylhydroxylamine added to human red cells under aerobic conditions and in the presence of glucose was partly reduced to aniline. About half the hydroxylamine was recovered as amine after a 2-hr incubation. The aniline, after acetylation, was identified as acetanilide by melting point, Rf-value in TCL as well as UV, IR, and NMR spectroscopy. The fate of the remaining phenylhydroxylamine was followed by use of 14C-labeled phenylhydroxylamine. About 30% of the total radioactivity was bound to hemoglobin or other proteins and about 20% was found in highly polar low-molecular substances which were insoluble in organic solvents. The elucidation of the sites at which phenylhydroxylamine was bound to hemoglobin was complicated by the lability of the bonds. When purified human hemoglobin had reacted with radioactive phenylhydroxylamine, large proportions of the radioactivity bound to hemoglobin were removed by treatment with acid or with PMB for separation of alpha- and beta-chains. The radioactive compound liberated from hemoglobin by acid was found to be aniline. After reaction with phenylhydroxylamine the number of SH groups titrable with PMB was found to be diminished. Pretreatment of hemoglobin with N-ethylmaleimide or PMB decreased the amount of phenylhydroxylamine bound to hemoglobin but did not fully prevent the reaction. Tryptic digestion of hemoglobin after reaction with radioactive phenylhydroxylamine yielded tryptic peptides with lower specific activity than that of hemoglobin. Chymotryptic digestion of the tryptic core yielded a core with specific activity much higher than that of hemoglobin. Fingerprinting of the tryptic or chymotryptic hydrolyzates showed the presence of peptides with high and other ones with low or no radioactivity and of radioactive compounds which did not react with ninhydrin. In the covalent binding of phenylhydroxylamine to globin the SH group beta93 plays an important role, but other yet unknown sites are also reactive. PMID:934354

  1. Water soluble fluorescence quantum dot probe labeling liver cancer cells.

    PubMed

    Chang, Baoxing; Yang, Xianjun; Wang, Fang; Wang, Yinsong; Yang, Rui; Zhang, Ning; Wang, Baiqi

    2013-11-01

    Water soluble quantum dots (QDs) have been prepared by hydrothermal method and characterized by ultraviolet irradiation, XRD, TEM, UV-Vis absorption spectrometer and fluorescence spectrometer. Then the QD-antibody-AFP probes (QD-Ab-AFP) were synthesized by chemical process and specifically labeled AFP antigen in PLC/PRF/5 liver cancer cells. The results showed that the QDs were cubic structure and had excellent optical properties. Moreover, the QD-Ab-AFP with good stability could specifically label liver cancer cells. This work provides strong foundation for further studying and developing new approach to detect liver cancer at early stage. PMID:23888351

  2. Regulation of red cell membrane protein interactions: implications for red cell function.

    PubMed

    Takakuwa, Y

    2001-03-01

    This article presents new insights into the molecular mechanism for regulating red cell membrane protein interactions that are responsible for erythrocyte membrane mechanical properties. For various skeletal proteins, structure-function correlations of protein 4.1R have been studied in detail. Kinetic analysis with the resonant mirror detection method has determined the nature of 4.1R interactions with various binding partners such as band 3, glycophorin C, and p55, and their binding sites. More importantly, calmodulin (CaM) binds to 4.1R in a Ca2+-independent manner to modulate the 4.1R interactions in the presence of Ca2+ at microM. Crystal structure of the 30-kD domain of 4.1R has a cloverleaf-like architecture with three lobes, each of which contains a binding region specific for binding partners. CaM binds to the grooves situated in two regions between the three lobes, possibly leading to conformational changes of the three lobes with a consequent alteration in the capacity of 4.1R to bind to its partners. The present findings on erythrocyte 4.1R should provide a basis for better understanding the membrane functions of nonerythroid cells. PMID:11224681

  3. Deep Learning in Label-free Cell Classification

    DOE PAGESBeta

    Chen, Claire Lifan; Mahjoubfar, Ata; Tai, Li-Chia; Blaby, Ian K.; Huang, Allen; Niazi, Kayvan Reza; Jalali, Bahram

    2016-03-15

    Label-free cell analysis is essential to personalized genomics, cancer diagnostics, and drug development as it avoids adverse effects of staining reagents on cellular viability and cell signaling. However, currently available label-free cell assays mostly rely only on a single feature and lack sufficient differentiation. Also, the sample size analyzed by these assays is limited due to their low throughput. Here, we integrate feature extraction and deep learning with high-throughput quantitative imaging enabled by photonic time stretch, achieving record high accuracy in label-free cell classification. Our system captures quantitative optical phase and intensity images and extracts multiple biophysical features of individualmore » cells. These biophysical measurements form a hyperdimensional feature space in which supervised learning is performed for cell classification. We compare various learning algorithms including artificial neural network, support vector machine, logistic regression, and a novel deep learning pipeline, which adopts global optimization of receiver operating characteristics. As a validation of the enhanced sensitivity and specificity of our system, we show classification of white blood T-cells against colon cancer cells, as well as lipid accumulating algal strains for biofuel production. In conclusion, this system opens up a new path to data-driven phenotypic diagnosis and better understanding of the heterogeneous gene expressions in cells.« less

  4. Deep Learning in Label-free Cell Classification.

    PubMed

    Chen, Claire Lifan; Mahjoubfar, Ata; Tai, Li-Chia; Blaby, Ian K; Huang, Allen; Niazi, Kayvan Reza; Jalali, Bahram

    2016-01-01

    Label-free cell analysis is essential to personalized genomics, cancer diagnostics, and drug development as it avoids adverse effects of staining reagents on cellular viability and cell signaling. However, currently available label-free cell assays mostly rely only on a single feature and lack sufficient differentiation. Also, the sample size analyzed by these assays is limited due to their low throughput. Here, we integrate feature extraction and deep learning with high-throughput quantitative imaging enabled by photonic time stretch, achieving record high accuracy in label-free cell classification. Our system captures quantitative optical phase and intensity images and extracts multiple biophysical features of individual cells. These biophysical measurements form a hyperdimensional feature space in which supervised learning is performed for cell classification. We compare various learning algorithms including artificial neural network, support vector machine, logistic regression, and a novel deep learning pipeline, which adopts global optimization of receiver operating characteristics. As a validation of the enhanced sensitivity and specificity of our system, we show classification of white blood T-cells against colon cancer cells, as well as lipid accumulating algal strains for biofuel production. This system opens up a new path to data-driven phenotypic diagnosis and better understanding of the heterogeneous gene expressions in cells. PMID:26975219

  5. Deep Learning in Label-free Cell Classification

    NASA Astrophysics Data System (ADS)

    Chen, Claire Lifan; Mahjoubfar, Ata; Tai, Li-Chia; Blaby, Ian K.; Huang, Allen; Niazi, Kayvan Reza; Jalali, Bahram

    2016-03-01

    Label-free cell analysis is essential to personalized genomics, cancer diagnostics, and drug development as it avoids adverse effects of staining reagents on cellular viability and cell signaling. However, currently available label-free cell assays mostly rely only on a single feature and lack sufficient differentiation. Also, the sample size analyzed by these assays is limited due to their low throughput. Here, we integrate feature extraction and deep learning with high-throughput quantitative imaging enabled by photonic time stretch, achieving record high accuracy in label-free cell classification. Our system captures quantitative optical phase and intensity images and extracts multiple biophysical features of individual cells. These biophysical measurements form a hyperdimensional feature space in which supervised learning is performed for cell classification. We compare various learning algorithms including artificial neural network, support vector machine, logistic regression, and a novel deep learning pipeline, which adopts global optimization of receiver operating characteristics. As a validation of the enhanced sensitivity and specificity of our system, we show classification of white blood T-cells against colon cancer cells, as well as lipid accumulating algal strains for biofuel production. This system opens up a new path to data-driven phenotypic diagnosis and better understanding of the heterogeneous gene expressions in cells.

  6. Deep Learning in Label-free Cell Classification

    PubMed Central

    Chen, Claire Lifan; Mahjoubfar, Ata; Tai, Li-Chia; Blaby, Ian K.; Huang, Allen; Niazi, Kayvan Reza; Jalali, Bahram

    2016-01-01

    Label-free cell analysis is essential to personalized genomics, cancer diagnostics, and drug development as it avoids adverse effects of staining reagents on cellular viability and cell signaling. However, currently available label-free cell assays mostly rely only on a single feature and lack sufficient differentiation. Also, the sample size analyzed by these assays is limited due to their low throughput. Here, we integrate feature extraction and deep learning with high-throughput quantitative imaging enabled by photonic time stretch, achieving record high accuracy in label-free cell classification. Our system captures quantitative optical phase and intensity images and extracts multiple biophysical features of individual cells. These biophysical measurements form a hyperdimensional feature space in which supervised learning is performed for cell classification. We compare various learning algorithms including artificial neural network, support vector machine, logistic regression, and a novel deep learning pipeline, which adopts global optimization of receiver operating characteristics. As a validation of the enhanced sensitivity and specificity of our system, we show classification of white blood T-cells against colon cancer cells, as well as lipid accumulating algal strains for biofuel production. This system opens up a new path to data-driven phenotypic diagnosis and better understanding of the heterogeneous gene expressions in cells. PMID:26975219

  7. Isotopic Labeling of Red Cabbage Anthocyanins with Atmospheric 13-CO2

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Isotopic labeling of plants provides a unique opportunity for understanding metabolic processes. A significant challenge of isotopic labeling during plant growth is that isotopes must be administered without disrupting plant development and at sufficient levels for mass spectral analysis. We describ...

  8. Separating Magnetically Labeled and Unlabeled Biological Cells within Microfluidic Channels

    NASA Astrophysics Data System (ADS)

    Byvank, Tom; Vieira, Greg; Miller, Brandon; Yu, Bo; Chalmers, Jeffrey; Lee, L. James; Sooryakumar, R.

    2011-03-01

    The transport of microscopic objects that rely on magnetic forces have numerous advantages including flexibility of controlling many design parameters and the long range magnetic interactions generally do not adversely affect biological or chemical interactions. We present results on the use of magnetic micro-arrays imprinted within polydimethylsiloxane (PDMS) microfluidic channels that benefit from these features and the ability to rapidly reprogram the magnetic energy landscape for cell manipulation and sorting applications. A central enabling feature is the very large, tunable, magnetic field gradients (> 10 4) that can be designed within the microfluidic architecture. Through use of antibody-conjugated magnetic microspheres to label biological cells, results on the transport and sorting of heterogeneous cell populations are presented. The effects of micro-array and fluid channel design parameters, competition between magnetic forces and hydrodynamic drag forces, and cell-labeling efficiency on cell separation are discussed.

  9. Multiplex cell and lineage tracking with combinatorial labels.

    PubMed

    Loulier, Karine; Barry, Raphaëlle; Mahou, Pierre; Le Franc, Yann; Supatto, Willy; Matho, Katherine S; Ieng, Siohoi; Fouquet, Stéphane; Dupin, Elisabeth; Benosman, Ryad; Chédotal, Alain; Beaurepaire, Emmanuel; Morin, Xavier; Livet, Jean

    2014-02-01

    We present a method to label and trace the lineage of multiple neural progenitors simultaneously in vertebrate animals via multiaddressable genome-integrative color (MAGIC) markers. We achieve permanent expression of combinatorial labels from new Brainbow transgenes introduced in embryonic neural progenitors with electroporation of transposon vectors. In the mouse forebrain and chicken spinal cord, this approach allows us to track neural progenitor's descent during pre- and postnatal neurogenesis or perinatal gliogenesis in long-term experiments. Color labels delineate cytoarchitecture, resolve spatially intermixed clones, and specify the lineage of astroglial subtypes and adult neural stem cells. Combining colors and subcellular locations provides an expanded marker palette to individualize clones. We show that this approach is also applicable to modulate specific signaling pathways in a mosaic manner while color-coding the status of individual cells regarding induced molecular perturbations. This method opens new avenues for clonal and functional analysis in varied experimental models and contexts. PMID:24507188

  10. Optical painting and fluorescence activated sorting of single adherent cells labelled with photoswitchable Pdots.

    PubMed

    Kuo, Chun-Ting; Thompson, Alison M; Gallina, Maria Elena; Ye, Fangmao; Johnson, Eleanor S; Sun, Wei; Zhao, Mengxia; Yu, Jiangbo; Wu, I-Che; Fujimoto, Bryant; DuFort, Christopher C; Carlson, Markus A; Hingorani, Sunil R; Paguirigan, Amy L; Radich, Jerald P; Chiu, Daniel T

    2016-01-01

    The efficient selection and isolation of individual cells of interest from a mixed population is desired in many biomedical and clinical applications. Here we show the concept of using photoswitchable semiconducting polymer dots (Pdots) as an optical 'painting' tool, which enables the selection of certain adherent cells based on their fluorescence, and their spatial and morphological features, under a microscope. We first develop a Pdot that can switch between the bright (ON) and dark (OFF) states reversibly with a 150-fold contrast ratio on irradiation with ultraviolet or red light. With a focused 633-nm laser beam that acts as a 'paintbrush' and the photoswitchable Pdots as the 'paint', we select and 'paint' individual Pdot-labelled adherent cells by turning on their fluorescence, then proceed to sort and recover the optically marked cells (with 90% recovery and near 100% purity), followed by genetic analysis. PMID:27118210

  11. Optical painting and fluorescence activated sorting of single adherent cells labelled with photoswitchable Pdots

    PubMed Central

    Kuo, Chun-Ting; Thompson, Alison M.; Gallina, Maria Elena; Ye, Fangmao; Johnson, Eleanor S.; Sun, Wei; Zhao, Mengxia; Yu, Jiangbo; Wu, I-Che; Fujimoto, Bryant; DuFort, Christopher C.; Carlson, Markus A.; Hingorani, Sunil R.; Paguirigan, Amy L.; Radich, Jerald P.; Chiu, Daniel T.

    2016-01-01

    The efficient selection and isolation of individual cells of interest from a mixed population is desired in many biomedical and clinical applications. Here we show the concept of using photoswitchable semiconducting polymer dots (Pdots) as an optical ‘painting' tool, which enables the selection of certain adherent cells based on their fluorescence, and their spatial and morphological features, under a microscope. We first develop a Pdot that can switch between the bright (ON) and dark (OFF) states reversibly with a 150-fold contrast ratio on irradiation with ultraviolet or red light. With a focused 633-nm laser beam that acts as a ‘paintbrush' and the photoswitchable Pdots as the ‘paint', we select and ‘paint' individual Pdot-labelled adherent cells by turning on their fluorescence, then proceed to sort and recover the optically marked cells (with 90% recovery and near 100% purity), followed by genetic analysis. PMID:27118210

  12. Variation in Growth, Colonization of Maize, and Metabolic Parameters of GFP- and DsRed-Labeled Fusarium verticillioides Strains.

    PubMed

    Wu, Lei; Conner, R L; Wang, Xiaoming; Xu, Rongqi; Li, Hongjie

    2016-08-01

    Autofluorescent proteins are frequently applied as visual markers in the labeling of filamentous fungi. Genes gfp and DsRed were transformed into the genome of Fusarium verticillioides via the Agrobacterium tumefaciens-mediated transformation method. The selected transformants displayed a bright green or red fluorescence in all the organelles of the growing fungal mycelia and spores (except for the vacuoles) both in cultures and in the maize (Zea mays) roots they colonized. The results of gene-specific polymerase chain reaction (PCR) analysis and the thermal asymmetrical interlaced (TAIL)-PCR analysis demonstrated that gfp and DsRed were integrated on different chromosomes of the fungus. Reductions in the colony growth on the plates at pH 4.0 and 5.5 was observed for the green fluorescent protein (GFP)-transformant G3 and the DsRed-transformant R4, but transformants G4 and R1 grew as well as the wild-type strain at pH 4.0. The speed of growth of all the transformants was similar to the wild-type strain at pH ≥ 7. The insertion of gfp and DsRed did not alter the production of extracellular enzymes and fumonisin B by F. verticillioides. The transformants expressing GFP and DsRed proteins were able to colonize maize roots. However, the four transformants examined produced fewer CFU in the root samples than the wild-type strain during a sampling period of 7 to 28 days after inoculation. PMID:27088391

  13. [Effects of superparamagnetic iron-oxide particles-labeling on the multi-diffentiation of rabbit marrow mesenchymal stem cell in vitro].

    PubMed

    Jin, Xuhong; Yang, Liu; Zhang, Shou; Dun, Xiaojun; Wang, Fuyou; Tan, Hongbo

    2012-02-01

    The aim of this study was to label rabbit bone derived mesenchymal stem cells (BMSCs) with superparamagnetic iron oxide particles (SPIO) and to study the effects of magnetic labeling on the multi-differentiation of BMSCs. Rabbit BMSCs were isolated, purified, expanded, then coincubated with SPIO(25 microg/ml) complexed to protamine sulfate (Pro) transfection agents overnight. Prussian blue staining and transmission electron microscopy were performed to show intracellular iron. Cell differentiation was evaluated. Both labeled and unlabeled BMSCs were subjected to osteogenic, adipogenic and chondrogenic differentiation to assess their differentiation capacity for 21 d. Osteogenic cells were stained with alizarin red to reveal calcium deposition, adipogenic cells were stained with oil redO' respectively. Chondrogenic cells stained with Safranin-O, glycosamino glycans, and type II collagen production was assessed by standard immunohistochemistry. Cell with immunohistochemistry staining were detected by polarized light microscopy and analysed by Image-Pro Plus software. The results showed that intracytoplasmic nanoparticles were stained with Prussian blue and observed by transmission electron microscopy clearly except the unlabeled control. As compared with the nonlabeled cells, it showed no statistically significant difference on the differentiation of the labeled BMSCs. And the differentiation of the labeled cells were unaffected by the endosomal incorporation of SPIO. In summary, BMSCs can be labeled with SPIO without significant change in cell multi-differentiation capacity. PMID:22404022

  14. Specific cell surface labeling of GPCRs using split GFP.

    PubMed

    Jiang, Wen-Xue; Dong, Xu; Jiang, Jing; Yang, Yu-Hong; Yang, Ju; Lu, Yun-Bi; Fang, San-Hua; Wei, Er-Qing; Tang, Chun; Zhang, Wei-Ping

    2016-01-01

    Specific cell surface labeling is essential for visualizing the internalization processes of G-protein coupled receptors (GPCRs) and for gaining mechanistic insight of GPCR functions. Here we present a rapid, specific, and versatile labeling scheme for GPCRs at living-cell membrane with the use of a split green fluorescent protein (GFP). Demonstrated with two GPCRs, GPR17 and CysLT2R, we show that two β-stands (β-stands 10 and 11) derived from a superfolder GFP (sfGFP) can be engineered to one of the three extracellular loop of a GPCR. The complementary fragment of sfGFP has nine β-strands (β-stands 1-9) that carries the mature fluorophore, and can be proteolytically derived from the full-length sfGFP. Separately the GFP fragments are non-fluorescent, but become fluorescent upon assembly, thus allowing specific labeling of the target proteins. The two GFP fragments rapidly assemble and the resulting complex is extremely tight under non-denaturing conditions, which allows real-time and quantitative assessment of the internalized GPCRs. We envision that this labeling scheme will be of great use for labeling other membrane proteins in various biological and pharmacological applications. PMID:26857153

  15. Dual network model for red blood cell membranes

    NASA Astrophysics Data System (ADS)

    Boal, David H.; Seifert, Udo; Zilker, Andreas

    1992-12-01

    A two-component network is studied by Monte Carlo simulation to model the lipid/spectrin membrane of red blood cells. The model predicts that the shear modulus decreases rapidly with the maximum length of the model spectrin and should be in the 10-7 J/m2 range for human red blood cells. A simplified model for the isolated spectrin network shows a negative Lamé coefficient λ. Transverse fluctuations of the dual membrane are found to be fluidlike over the range of wavelengths investigated.

  16. Photoacoustic response of suspended and hemolyzed red blood cells

    NASA Astrophysics Data System (ADS)

    Saha, Ratan K.; Karmakar, Subhajit; Roy, Madhusudan

    2013-07-01

    The effect of confinement of hemoglobin molecules on photoacoustic (PA) signal is studied experimentally. The PA amplitudes for samples with suspended red blood cells (SRBCs) and hemolyzed red blood cells (HRBCs) were found to be comparable at each hematocrit for 532 nm illumination. The difference between the corresponding amplitudes increased with increasing hematocrit for 1064 nm irradiation. For example, the PA amplitude for the SRBCs was about 260% higher than that of the HRBCs at 40% hematocrit. This observation may help to develop a PA method detecting hemolysis noninvasively.

  17. Immunomicrospheres - Reagents for cell labeling and separation

    NASA Technical Reports Server (NTRS)

    Rembaum, A.; Dreyer, W. J.

    1980-01-01

    Immunomicrospheres are specially designed microscopic particles that have antibodies or similar molecules chemically bound to their surfaces. The antibody-coated microspheres react in a highly specific way with target cells, viruses, or other antigenic agents. Immunomicrospheres may be synthesized so that they incorporate compounds that are highly radioactive, intensely fluorescent, magnetic, electron opaque, highly colored, or pharmacologically active. These various types of microspheres may be coated with pure, highly specific monoclonal antibodies obtained by the new hybridoma cell cloning techniques or with conventional antibody preparations. Some of the many present and potential applications for these new reagents are (1) new types of radioimmune or immunofluorescent assays, (2) improved fluorescence microscopy, (3) separation of cells on the basis of the fluorescent, electrophoretic, or magnetic properties of bound immunomicrospheres, (4) markers for use in several types of electron or standard light microscopy, and (5) delivery of lethal compouds to specific undesirable living cells. The combination of the various new types of synthetic microspheres and the newly available homogeneous antibodies offers new opportunities in research, diagnosis, and therapy.

  18. Evaluation of the red cell storage lesion after irradiation in filtered packed red cell units

    SciTech Connect

    Hillyer, C.D.; Tiegerman, K.O.; Berkman, E.M. )

    1991-07-01

    Packed red cell units (n = 10) were filtered and divided equally. One-half unit from each donor was irradiated (x) (3500 cGy). On Days 0, 14, 28, and 42, ATP, K+, Na+, lactate dehydrogenase (LDH), plasma-free hemoglobin (PFH), and pH were determined. The reduction in ATP was greater in the irradiated than the nonirradiated (y) units by Day 42 (mean x-y: -70, p = 0.0005). The increase in K+ was greater in the irradiated than nonirradiated units on Days 14, 28, and 42 (mean x-y: 17-20, p = 0.0001). Decrease in pH and increases in LDH and PFH were significant (p less than 0.05) on Day 42 only. K+ increases added only 1.7 to 2.0 mmol per unit, a difference felt to be clinically insignificant. The changes noted in ATP, pH, LDH, and PFH are significant but minimal on Day 42 and imply that viability changes would also be minimal. These biochemical data support the storage of irradiated units for at least 28 days.

  19. Anatomy of the red cell membrane skeleton: unanswered questions.

    PubMed

    Lux, Samuel E

    2016-01-14

    The red cell membrane skeleton is a pseudohexagonal meshwork of spectrin, actin, protein 4.1R, ankyrin, and actin-associated proteins that laminates the inner membrane surface and attaches to the overlying lipid bilayer via band 3-containing multiprotein complexes at the ankyrin- and actin-binding ends of spectrin. The membrane skeleton strengthens the lipid bilayer and endows the membrane with the durability and flexibility to survive in the circulation. In the 36 years since the first primitive model of the red cell skeleton was proposed, many additional proteins have been discovered, and their structures and interactions have been defined. However, almost nothing is known of the skeleton's physiology, and myriad questions about its structure remain, including questions concerning the structure of spectrin in situ, the way spectrin and other proteins bind to actin, how the membrane is assembled, the dynamics of the skeleton when the membrane is deformed or perturbed by parasites, the role lipids play, and variations in membrane structure in unique regions like lipid rafts. This knowledge is important because the red cell membrane skeleton is the model for spectrin-based membrane skeletons in all cells, and because defects in the red cell membrane skeleton underlie multiple hemolytic anemias. PMID:26537302

  20. Effect of misoprostol and cimetidine on gastric cell labeling index

    SciTech Connect

    Fich, A.; Arber, N.; Sestieri, M.; Zajicek, G.; Rachmilewitz, D.

    1985-07-01

    The effect of misoprostol and cimetidine on gastric cell turnover was studied. Endoscopic biopsy specimens of fundic and antral mucosa were obtained from duodenal ulcer patients before and after 4 wk of therapy with cimetidine 1.2 g/day or misoprostol 800 micrograms/day. Biopsy specimens were incubated with (/sup 3/H)thymidine. Glandular column length and number of labeled cells were determined after autoradiography. There was no significant difference in column length of antral or fundic glands before or after therapy with cimetidine and misoprostol. The number of antral and fundic labeled cells was significantly decreased after misoprostol treatment (3.6 +/- 0.3 and 4.6 +/- 0.4, mean +/- SE), as opposed to their respective number before therapy (6.9 +/- 0.5 and 8.3 +/- 0.8) (p less than 0.01). On the other hand, after treatment with cimetidine, the number of antral and fundic labeled cells was significantly higher (11.8 +/- 0.9 and 7.5 +/- 1.0, respectively) as compared with their number before therapy (5.7 +/- 0.5 and 5.6 +/- 0.6, respectively). The decreased gastric cell turnover induced by misoprostol indicates that the trophic effect of prostanoids on gastric mucosa is not due to an increase in cellular kinetics. The increased gastric cell turnover induced by cimetidine may contribute to its therapeutic effect in peptic ulcer disease.

  1. Fermented red ginseng extract inhibits cancer cell proliferation and viability.

    PubMed

    Oh, Jisun; Jeon, Seong Bin; Lee, Yuri; Lee, Hyeji; Kim, Ju; Kwon, Bo Ra; Yu, Kang-Yeol; Cha, Jeong-Dan; Hwang, Seung-Mi; Choi, Kyung-Min; Jeong, Yong-Seob

    2015-04-01

    Red ginseng (Panax ginseng C.A. Meyer) is the most widely recognized medicinal herb due to its remedial effects in various disorders, such as cancers, diabetes, and heart problems. In this study, we investigated the anticancer effect of fermented red ginseng extract (f-RGE; provided by Jeonju Biomaterials Institute, Jeonju, South Korea) in a parallel comparison with the effect of nonfermented red ginseng extract (nf-RGE; control) on several cancer cell lines--MCF-7 breast cancer cells, HepG2 hepatocellular carcinoma cells, and reprogrammed MCF-7 cells (mimicking cancer stem cells). Cells were cultured at various concentrations of RGE (from 0.5 up to 5 mg/mL) and their viabilities and proliferative properties were examined. Our data demonstrate the following: (1) nf-RGE inhibited cell viability at ≥1 mg/mL for MCF-7 cells and ≥2 mg/mL for HepG2 cells, (2) in the presence of a carcinogenic agent, 12-O-tetradecanoylphorbol-13-acetate (TPA), nf-RGE treatment in combination with paclitaxel synergistically decreased MCF-7 as well as HepG2 cell viability, (3) f-RGE (which contained a greater level of Rg3 content) more effectively decreased the viability of MCF-7 and HepG2 cells compared to nf-RGE, and (4) f-RGE appeared more potent for inhibiting cancerous differentiation of reprogrammed MCF-7 cells in a synergistic fashion with paclitaxel, especially in the presence of TPA, compared to nf-RGE. These findings suggest that f-RGE treatment may be more effective for decreasing cancer cell survival by inducing apoptotic cell death and also presumably for preventing cancer stem cell differentiation compared to nf-RGE. PMID:25658580

  2. Labeling Stem Cells with Superparamagnetic Iron Oxide Nanoparticles: Analysis of the Labeling Efficacy by Microscopy and Magnetic Resonance Imaging

    PubMed Central

    Jasmin; Torres, Ana Luiza Machado; Jelicks, Linda; de Carvalho, Antonio Carlos Campos; Spray, David C.; Mendez-Otero, Rosalia

    2013-01-01

    Stem cell therapy has emerged as a potential therapeutic option for cell death-related heart diseases. Application of non-invasive cell tracking approaches is necessary to determine tissue distribution and lifetime of stem cells following their injection and will likely provide knowledge about poorly understood stem cells mechanisms of tissue repair. Magnetic resonance imaging (MRI) is a potentially excellent tool for high-resolution visualization of the fate of cells after transplantation and for evaluation of therapeutic strategies. The application of MRI for in vivo cell tracking requires contrast agents to achieve efficient cell labeling without causing any toxic cellular effects or eliciting any other side effects. For these reasons clinically approved contrast agents (e.g., ferumoxides) and incorporation facilitators (e.g., protamine) are currently the preferred materials for cell labeling and tracking. Here we describe how to use superparamag-netic iron oxide nanoparticles to label cells and to monitor cell fate in several disease models. PMID:22791437

  3. Investigations into agents for improving cell labeling with positron- and gamma-emitting radionuclides

    SciTech Connect

    Zoghbi, S.S.; Thakur, M.L.; Gottschalk, A.; Pande, S.; Srivastava, S.C.; Richards, P.

    1982-01-01

    It was possible to label leukocytes with Co-oxine, but a large proportion of the radioactivity was eluted from the cells upon washing. Ruthenium oxine labeled platelets efficiently in plasma while negligible proportion of radioactivity was eluted from the cells. Three factors influence the labeling efficiency of the cells: duration of the incubation periods; cell concentration; and ACD concentration.

  4. Dynamic quantitative microscopy and nanoscopy of red blood cells in sickle cell disease

    NASA Astrophysics Data System (ADS)

    Shaked, Natan T.; Satterwhite, Lisa L.; Telen, Marilyn J.; Truskey, George A.; Wax, Adam

    2012-03-01

    We have applied wide-field digital interferometric techniques to quantitatively image sickle red blood cells (RBCs) [1] in a noncontact label-free manner, and measure the nanometer-scale fluctuations in their thickness as an indication of their stiffness. The technique can simultaneously measure the fluctuations for multiple spatial points on the RBC and thus yields a map describing the stiffness of each RBC in the field of view. Using this map, the local rigidity regions of the RBC are evaluated quantitatively. Since wide-field digital interferometry is a quantitative holographic imaging technique rather than one-point measurement, it can be used to simultaneously evaluate cell transverse morphology plus thickness in addition to its stiffness profile. Using this technique, we examine the morphology and dynamics of RBCs from individuals who suffer from sickle cell disease, and find that the sickle RBCs are significantly stiffer than healthy RBCs. Furthermore, we show that the technique is sensitive enough to distinguish various classes of sickle RBCs, including sickle RBCs with visibly-normal morphology, compared to the stiffer crescent-shaped sickle RBCs.

  5. Spatial Distributions of Red Blood Cells Significantly Alter Local Haemodynamics

    PubMed Central

    Sherwood, Joseph M.; Holmes, David; Kaliviotis, Efstathios; Balabani, Stavroula

    2014-01-01

    Although bulk changes in red blood cell concentration between vessels have been well characterised, local distributions are generally overlooked. Red blood cells aggregate, deform and migrate within vessels, forming heterogeneous distributions which have considerable effect on local haemodynamics. The present study reports data on the local distribution of human red blood cells in a sequentially bifurcating microchannel, representing the branching geometry of the microvasculature. Imaging methodologies with simple extrapolations are used to infer three dimensional, time-averaged velocity and haematocrit distributions under a range of flow conditions. Strong correlation between the bluntness of the velocity and haematocrit profiles in the parent branch of the geometry is observed and red blood cell aggregation has a notable effect on the observed trends. The two branches of the first bifurcation show similar characteristics in terms of the shapes of the profiles and the extent of plasma skimming, despite the difference in geometric configuration. In the second bifurcation, considerable asymmetry between the branches in the plasma skimming relationship is observed, and elucidated by considering individual haematocrit profiles. The results of the study highlight the importance of considering local haematocrit distributions in the analysis of blood flow and could lead to more accurate computational models of blood flow in microvascular networks. The experimental approaches developed in this work provide a foundation for further examining the characteristics of microhaemodynamics. PMID:24950214

  6. Red Blood Cell Spectrin Skeleton in the Spotlight.

    PubMed

    Braun-Breton, Catherine; Abkarian, Manouk

    2016-02-01

    Das et al. recently reported a role for the major merozoite surface protein MSP1 in malarial parasite egress from the red blood cell (RBC). On the basis of these new data and physical considerations, we propose an updated model for the main steps of this essential process for parasite proliferation. PMID:26652974

  7. Further studies of sodium transport in feline red cells.

    PubMed

    Sha'afi, R I; Pascoe, E

    1973-06-01

    The transport of radioactive sodium in high sodium cat red blood cells has been studied under various experimental conditions. It was found that iodoacetate (IAA) and iodoacetamide (IAM) inhibit Na influx by 50% whereas NaF has no effect. Reversible dyes, such as methylene blue (Mb), also inhibit this influx by 60%. Both IAA and Mb effects show a lag period of about 40 min. Cell starvation abolishes the volume-dependent Na influx which is generally observed in these cells. IAA reduces significantly the volume-dependent Na influx but does not inhibit it completely. 5 mM magnesium chloride produces a twofold increase in Na influx. On the other hand, MgCl(2) has no effect on Na transport in human red cells or on potassium or sulfate transport in cat red cells. The effect of MgCl(2) is quite rapid and does not interfere with the volume-dependent Na influx. This effect is abolished in starved cells. Reincubation of previously stored cells in buffered solutions containing glucose and MgCl(2) causes more than one order of magnitude increase in Na influx. These several observations are discussed in terms of the possibility of a link between Na transport and Na-Mg-activated ATPase. PMID:4733097

  8. Red blood cell replacement, or nanobiotherapeutics with enhanced red blood cell functions?

    PubMed

    Chang, Thomas Ming Swi

    2015-06-01

    Why is this important? Under normal circumstances, donor blood is the best replacement for blood. However, there are exceptions: During natural epidemics (e.g., HIV, Ebola, etc.) or man-made epidemics (terrorism, war, etc.), there is a risk of donor blood being contaminated, and donors being disqualified because they have contracted disease. Unlike red blood cells (RBCs), blood substitutes can be sterilized to remove infective agents. Heart attack and stroke are usually caused by obstruction of arterial blood vessels. Unlike RBCs, which are particulate, blood substitutes are in the form of a solution that can perfuse through obstructed vessels with greater ease to reach the heart and brain, as has been demonstrated in animal studies. Severe blood loss from injuries sustained during accidents, disasters, or war may require urgent blood transfusion that cannot wait for transportation to the hospital for blood group testing. Unlike RBCs, blood substitutes do not have specific blood groups, and can be administered on the spot. RBCs have to be stored under refrigeration for up to 42 days, and are thus difficult to transport and store in times of disaster and at the battlefront. Blood substitutes can be stored at room temperature for more than 1 year, compared to the RBC shelf life of 1 day, at room temperature. In cases of very severe hemorrhagic shock, there is usually a safety window of 60 min for blood replacement, beyond which there could be problems related to irreversible shock. Animal studies show that a particular type of blood substitute, with enhanced RBC enzymes, may be able to prolong the duration of the safety window. PMID:26096663

  9. Red blood cell homeostasis: recognition of distinct types of damaged homologous red blood cells by a mouse macrophage cell line.

    PubMed

    Singer, J A; Morrison, M; Walker, W S

    1987-06-01

    The mouse macrophage (M phi) cell line IC-21 preferentially ingests a subpopulation of homologous red blood cells (MRBC) from normal mice. This subpopulation presumably bears the so-called transfusion lesion, a consequence of damage acquired during the drawing and processing of blood. To determine if all damaged MRBC were recognized by a common receptor site on IC-21 M phi, we prepared suspensions of MRBC damaged in vitro by treatment with tannic acid and compared the phagocytic uptake of these cells with those bearing the transfusion lesion. Trypsin treatment of IC-21 M phi rendered them unable to recognize MRBC bearing the transfusion lesion; but it had no effect on the uptake of tannic acid-damaged MRBC, showing that IC-21 M phi have separate recognition sites for these two populations of damaged MRBC. PMID:3474332

  10. Aggregation of red blood cells: From rouleaux to clot formation

    NASA Astrophysics Data System (ADS)

    Wagner, Christian; Steffen, Patrick; Svetina, Saša

    2013-06-01

    Red blood cells are known to form aggregates in the form of rouleaux. This aggregation process is believed to be reversible, but there is still no full understanding on the adhesion mechanism. There are at least two competing models, based either on bridging or on depletion. We review recent experimental results on the single cell level and theoretical analyses of the depletion model and of the influence of the cell shape on the adhesion strength. Another important aggregation mechanism is caused by activation of platelets. This leads to clot formation which is life-saving in the case of wound healing, but also a major cause of death in the case of a thrombus induced stroke. We review historical and recent results on the participation of red blood cells in clot formation.

  11. Calcium movements across the membrane of human red cells

    PubMed Central

    Schatzmann, H. J.; Vincenzi, F. F.

    1969-01-01

    1. A study has been made of the cellular content and movement of Ca across the membrane of human red blood cells. 2. The [Ca] in the cellular contents of fresh red cells is 4·09 × 10-2 mM. The intracellular concentration of free ionic Ca ([Ca2+]) is considered to be less than this value and therefore less than extracellular [Ca2+] under normal conditions. 3. Observation of unidirectional Ca fluxes with 45Ca confirms previous reports of low permeability of the red cell membrane for Ca. After nearly 1 week of loading in the cold, intracellular 45Ca content is 1·8% of extracellular 45Ca content. Appearance in extracellular fluid of 45Ca from coldloaded cells can be considered to arise from two compartments. Efflux of 45Ca from the `slower compartment' is accelerated by the addition of glucose. 4. Starved red cells, incubated at 37° C, after reversible haemolysis for loading with Ca and Mg-ATP, exhibit an outward net transport of Ca against an electrochemical gradient. The transport is associated with the appearance of inorganic phosphate (Pi). Cells treated similarly, but without ATP show no transport and no appearance of Pi. 5. During the initial phase of transport, 1·3 mole Pi appear per mole Ca transported. 6. The transport of Ca from ATP-loaded cells is highly temperature-dependent, with a Q10 of 3·5. 7. Cell membrane adenosine triphosphatase (ATPase) activity of reversibly haemolysed cells is stimulated only by intracellular, and not by extracellular Ca. 8. Neither Ca transport in reversibly haemolysed cells, nor the Ca-Mg activated ATPase of isolated cell membranes is sensitive to Na, K, ouabain or oligomycin. 9. Mg is not transported under the conditions which reveal Ca transport, but Mg appears to be necessary for Ca transport. 10. Sr is transported from reversibly haemolysed Mg-ATP-loaded cells. Sr also can substitute for Ca, but not for Mg, in the activation of membrane ATPase. 11. It is concluded that, in addition to a low passive permeability, an

  12. Red Blood Cell Polymorphism and Susceptibility to Plasmodium vivax

    PubMed Central

    Zimmerman, Peter A.; Ferreira, Marcelo U.; Howes, Rosalind E.; Mercereau-Puijalon, Odile

    2013-01-01

    Resistance to Plasmodium vivax blood-stage infection has been widely recognised to result from absence of the Duffy (Fy) blood group from the surface of red blood cells (RBCs) in individuals of African descent. Interestingly, recent studies from different malaria-endemic regions have begun to reveal new perspectives on the association between Duffy gene polymorphism and P. vivax malaria. In Papua New Guinea and the Americas, heterozygous carriers of a Duffy-negative allele are less susceptible to P. vivax infection than Duffy-positive homozygotes. In Brazil, studies show that the Fya antigen, compared to Fyb, is associated with lower binding to the P. vivax Duffy-binding protein and reduced susceptibility to vivax malaria. Additionally, it is interesting that numerous studies have now shown that P. vivax can infect RBCs and cause clinical disease in Duffy-negative people. This suggests that the relationship between P. vivax and the Duffy antigen is more complex than customarily described. Evidence of P. vivax Duffy-independent red cell invasion indicates that the parasite must be evolving alternative red cell invasion pathways. In this chapter, we review the evidence for P. vivax Duffy-dependent and Duffy-independent red cell invasion. We also consider the influence of further host gene polymorphism associated with malaria endemicity on susceptibility to vivax malaria. The interaction between the parasite and the RBC has significant potential to influence the effectiveness of P. vivax-specific vaccines and drug treatments. Ultimately, the relationships between red cell polymorphisms and P. vivax blood-stage infection will influence our estimates on the population at risk and efforts to eliminate vivax malaria. PMID:23384621

  13. Red blood cell and iron metabolism during space flight.

    PubMed

    Smith, Scott M

    2002-10-01

    Space flight anemia is a widely recognized phenomenon in astronauts. Reduction in circulating red blood cells and plasma volume results in a 10% to 15% decrement in circulatory volume. This effect appears to be a normal physiologic adaptation to weightlessness and results from the removal of newly released blood cells from the circulation. Iron availability increases, and (in the few subjects studied) iron stores increase during long-duration space flight. The consequences of these changes are not fully understood. PMID:12361780

  14. Red blood cell and iron metabolism during space flight

    NASA Technical Reports Server (NTRS)

    Smith, Scott M.

    2002-01-01

    Space flight anemia is a widely recognized phenomenon in astronauts. Reduction in circulating red blood cells and plasma volume results in a 10% to 15% decrement in circulatory volume. This effect appears to be a normal physiologic adaptation to weightlessness and results from the removal of newly released blood cells from the circulation. Iron availability increases, and (in the few subjects studied) iron stores increase during long-duration space flight. The consequences of these changes are not fully understood.

  15. Label-free quantitative cell division monitoring of endothelial cells by digital holographic microscopy

    NASA Astrophysics Data System (ADS)

    Kemper, Björn; Bauwens, Andreas; Vollmer, Angelika; Ketelhut, Steffi; Langehanenberg, Patrik; Müthing, Johannes; Karch, Helge; von Bally, Gert

    2010-05-01

    Digital holographic microscopy (DHM) enables quantitative multifocus phase contrast imaging for nondestructive technical inspection and live cell analysis. Time-lapse investigations on human brain microvascular endothelial cells demonstrate the use of DHM for label-free dynamic quantitative monitoring of cell division of mother cells into daughter cells. Cytokinetic DHM analysis provides future applications in toxicology and cancer research.

  16. Backward elastic light scattering of malaria infected red blood cells

    NASA Astrophysics Data System (ADS)

    Lee, Seungjun; Lu, Wei

    2011-08-01

    We investigated the backward light scattering pattern of healthy and malaria (Plasmodium falciparum) parasitized red blood cells. The spectrum could clearly distinguish between predominant ring stage infected blood cells and healthy blood cells. Further, we found that infected samples mixed with different stages of P. falciparum showed different signals, suggesting that even variance in parasite stages could also be detected by the spectrum. These results together with the backward scattering technique suggest the potential of non-invasive diagnosis of malaria through light scattering of blood cells near the surface of human body, such as using eyes or skin surface.

  17. Intracellular energetic units in red muscle cells.

    PubMed Central

    Saks, V A; Kaambre, T; Sikk, P; Eimre, M; Orlova, E; Paju, K; Piirsoo, A; Appaix, F; Kay, L; Regitz-Zagrosek, V; Fleck, E; Seppet, E

    2001-01-01

    The kinetics of regulation of mitochondrial respiration by endogenous and exogenous ADP in muscle cells in situ was studied in skinned cardiac and skeletal muscle fibres. Endogenous ADP production was initiated by addition of MgATP; under these conditions the respiration rate and ADP concentration in the medium were dependent on the calcium concentration, and 70-80% of maximal rate of respiration was achieved at ADP concentration below 20 microM in the medium. In contrast, when exogenous ADP was added, maximal respiration rate was observed only at millimolar concentrations. An exogenous ADP-consuming system consisting of pyruvate kinase (PK; 20-40 units/ml) and phosphoenolpyruvate (PEP; 5 mM), totally suppressed respiration activated by exogenous ADP, but the respiration maintained by endogenous ADP was not suppressed by more than 20-40%. Creatine (20 mM) further activated respiration in the presence of ATP and PK+PEP. Short treatment with trypsin (50-500 nM for 5 min) decreased the apparent K(m) for exogenous ADP from 300-350 microM to 50-60 microM, increased inhibition of respiration by PK+PEP system up to 70-80%, with no changes in MgATPase activity and maximal respiration rates. Electron-microscopic observations showed detachment of mitochondria and disordering of the regular structure of the sarcomere after trypsin treatment. Two-dimensional electrophoresis revealed a group of at least seven low-molecular-mass proteins in cardiac skinned fibres which were very sensitive to trypsin and not present in glycolytic fibres, which have low apparent K(m) for exogenous ADP. It is concluded that, in oxidative muscle cells, mitochondria are incorporated into functional complexes ('intracellular energetic units') with adjacent ADP-producing systems in myofibrils and in sarcoplasmic reticulum, probably due to specific interaction with cytoskeletal elements responsible for mitochondrial distribution in the cell. It is suggested that these complexes represent the basic

  18. Activated polymorphonuclear cells increase sickle red blood cell retention in lung: role of phospholipids.

    PubMed

    Haynes, Johnson; Obiako, Boniface

    2002-01-01

    This study investigates the role of the activated polymorphonuclear cell (APMN) products on sickle red blood cell (SRBC) retention/adherence in the pulmonary circulation. Isolated rat lungs were perfused with (51)Cr-labeled normal RBCs (NRBC) or SRBCs (10% hematocrit) suspensions +/- PMNs. Specific activities of lung and perfusate were measured and retention (the number of SRBC/g lung) was calculated. SRBC retention was 3.5 times greater than NRBC retention. PMN activation was required to increase SRBC retention. Supernatants from APMN increased SRBC retention, which suggested soluble products such as oxidants, PAF, and/or leukotriene (LTB(4)) are involved. Heat inactivation of PMN NADPH oxidase had no effect on retention. Whereas neither platelet-activating factor (PAF) nor LTB(4) (secreted by APMN) increased SRBC retention, PAF+LTB(4) did. The PAF antagonist, WEB-2170, attenuated SRBC retention mediated by PAF+LTB(4) and APMNs. Similarly, zileuton (5-lipoxygenase inhibitor) attenuated APMN-mediated SRBC retention. We conclude the concomitant release of PAF and LTB(4) from APMN is involved in the initiation of microvascular occlusion by SRBCs in the perfused rat lung. PMID:11748055

  19. Membranotropic photobiomodulation on red blood cell deformability

    NASA Astrophysics Data System (ADS)

    Luo, Gang-Yue; Zhao, Yan-Ping; Liu, Timon C.; Liu, Song-Hao

    2007-05-01

    To assess modulation of laser on erythrocyte permeability and deformability via cell morphology changes, healthy human echinocytes with shrinking size and high plasmic viscosity due to cellular dehydration were treated with 1 mW, 2 mW, 3 mW, and 5 mW laser power exposure respectively. Image analyzing system on single intact erythrocyte was applied for measuring comprehensive cell morphological parameters (surface area, external membrane perimeter, circle index and elongation index) that were determined by the modulation of erythrocyte water permeability and deformability to detect relationship between erythrocyte water permeability alteration and deformability. Our preliminary experiment showed that exposure under light dose of 5 mW for 5 min could induce more active erythrocyte swelling and deformation. water channel aquaporin-1(AQP-1) was inhibited by the incubation of HgCl II in the presence and absence of 5 mW laser irradiation. The result suggested that osmotic water permeability is a primary factor in the procedure of erythrocyte deformability. In addition, no modulation of laser(5mW) on erythrocyte deformability had been found when the echinocytes were cultured with GDP-β-S (G protein inhibitor).

  20. Broad-host-range plasmids for red fluorescent protein labeling of gram-negative bacteria for use in the zebrafish model system.

    PubMed

    Singer, John T; Phennicie, Ryan T; Sullivan, Matthew J; Porter, Laura A; Shaffer, Valerie J; Kim, Carol H

    2010-06-01

    To observe real-time interactions between green fluorescent protein-labeled immune cells and invading bacteria in the zebrafish (Danio rerio), a series of plasmids was constructed for the red fluorescent protein (RFP) labeling of a variety of fish and human pathogens. The aim of this study was to create a collection of plasmids that would express RFP pigments both constitutively and under tac promoter regulation and that would be nontoxic and broadly transmissible to a variety of Gram-negative bacteria. DNA fragments encoding the RFP dimeric (d), monomeric (m), and tandem dimeric (td) derivatives d-Tomato, td-Tomato, m-Orange, and m-Cherry were cloned into the IncQ-based vector pMMB66EH in Escherichia coli. Plasmids were mobilized into recipient strains by conjugal mating. Pigment production was inducible in Escherichia coli, Pseudomonas aeruginosa, Edwardsiella tarda, and Vibrio (Listonella) anguillarum strains by isopropyl-beta-d-thiogalactopyranoside (IPTG) treatment. A spontaneous mutant exconjugant of P. aeruginosa PA14 was isolated that expressed td-Tomato constitutively. Complementation analysis revealed that the constitutive phenotype likely was due to a mutation in lacI(q) carried on pMMB66EH. DNA sequence analysis confirmed the presence of five transitions, four transversions, and a 2-bp addition within a 14-bp region of lacI. Vector DNA was purified from this constitutive mutant, and structural DNA sequences for RFP pigments were cloned into the constitutive vector. Exconjugants of P. aeruginosa, E. tarda, and V. anguillarum expressed all pigments in an IPTG-independent fashion. Results from zebrafish infectivity studies indicate that RFP-labeled pathogens will be useful for the study of real-time interactions between host cells of the innate immune system and the infecting pathogen. PMID:20363780

  1. Broad-Host-Range Plasmids for Red Fluorescent Protein Labeling of Gram-Negative Bacteria for Use in the Zebrafish Model System▿ †

    PubMed Central

    Singer, John T.; Phennicie, Ryan T.; Sullivan, Matthew J.; Porter, Laura A.; Shaffer, Valerie J.; Kim, Carol H.

    2010-01-01

    To observe real-time interactions between green fluorescent protein-labeled immune cells and invading bacteria in the zebrafish (Danio rerio), a series of plasmids was constructed for the red fluorescent protein (RFP) labeling of a variety of fish and human pathogens. The aim of this study was to create a collection of plasmids that would express RFP pigments both constitutively and under tac promoter regulation and that would be nontoxic and broadly transmissible to a variety of Gram-negative bacteria. DNA fragments encoding the RFP dimeric (d), monomeric (m), and tandem dimeric (td) derivatives d-Tomato, td-Tomato, m-Orange, and m-Cherry were cloned into the IncQ-based vector pMMB66EH in Escherichia coli. Plasmids were mobilized into recipient strains by conjugal mating. Pigment production was inducible in Escherichia coli, Pseudomonas aeruginosa, Edwardsiella tarda, and Vibrio (Listonella) anguillarum strains by isopropyl-β-d-thiogalactopyranoside (IPTG) treatment. A spontaneous mutant exconjugant of P. aeruginosa PA14 was isolated that expressed td-Tomato constitutively. Complementation analysis revealed that the constitutive phenotype likely was due to a mutation in lacIq carried on pMMB66EH. DNA sequence analysis confirmed the presence of five transitions, four transversions, and a 2-bp addition within a 14-bp region of lacI. Vector DNA was purified from this constitutive mutant, and structural DNA sequences for RFP pigments were cloned into the constitutive vector. Exconjugants of P. aeruginosa, E. tarda, and V. anguillarum expressed all pigments in an IPTG-independent fashion. Results from zebrafish infectivity studies indicate that RFP-labeled pathogens will be useful for the study of real-time interactions between host cells of the innate immune system and the infecting pathogen. PMID:20363780

  2. Red blood cell sodium heteroexchange in familial primary hypertrophic cardiomyopathy.

    PubMed

    Semplicini, A; Mozzato, M G; Bongiovi, S; Marzola, M; Macor, F; Ceolotto, G; Serena, L; Pessina, A C

    1994-03-01

    The hallmark of primary hypertrophic cardiomyopathy is an inappropriate myocardial hypertrophy, linked to myofibril disarray of the left ventricle. Its variable clinical expression may be due to genetic heterogeneity and variable penetrance. Since we have recently shown that abnormalities of cation transport in the erythrocytes are associated with cardiac hypertrophy in essential hypertensives and insulin-dependent diabetics, we have investigated the relationship between cardiac anatomy and function and red cell Li+/Na+ and Na+/H+ exchange in 33 relatives of a patient who died of cardiac failure and was found to have a primary hypertrophic cardiomyopathy at autopsy. According to echocardiographic examination, 11 members of the family also had a hypertrophic cardiomyopathy, with a family distribution compatible with autosomal dominant genetic transmission and variable penetrance. Red cell Li+/Na+ and Na+/H+ exchange were not significantly different in the affected members as compared to the unaffected, but in the former, after correction for potentially confounding variables, interventricular septum thickness was positively correlated to Na+/H+ exchange and diastolic function (Area E/Area A and Vmax E/Vmax A) negatively correlated to Li+/Na+ exchange. Since a generalized overactivity of the cell membrane Na+/H+ exchange, reflected by increased Na+/H+ and Li+/Na+ exchanges in the red cells, could favour cellular growth and diastolic dysfunction, our data suggest that abnormalities of cell membrane cation transport could play a role in the phenotypic expression of hypertrophic cardiomyopathy. PMID:8013504

  3. Labeling Cytosolic Targets in Live Cells with Blinking Probes

    PubMed Central

    Xu, Jianmin; Chang, Jason; Yan, Qi; Dertinger, Thomas; Bruchez, Marcel; Weiss, Shimon

    2013-01-01

    With the advent of superresolution imaging methods, fast dynamic imaging of biological processes in live cells remains a challenge. A subset of these methods requires the cellular targets to be labeled with spontaneously blinking probes. The delivery and specific targeting of cytosolic targets and the control of the probes’ blinking properties are reviewed for three types of blinking probes: quantum dots, synthetic dyes, and fluorescent proteins. PMID:23930154

  4. Ultra-fast stem cell labelling using cationised magnetoferritin

    NASA Astrophysics Data System (ADS)

    Correia Carreira, S.; Armstrong, J. P. K.; Seddon, A. M.; Perriman, A. W.; Hartley-Davies, R.; Schwarzacher, W.

    2016-03-01

    Magnetic cell labelling with superparamagnetic iron oxide nanoparticles (SPIONs) facilitates many important biotechnological applications, such as cell imaging and remote manipulation. However, to achieve adequate cellular loading of SPIONs, long incubation times (24 hours and more) or laborious surface functionalisation are often employed, which can adversely affect cell function. Here, we demonstrate that chemical cationisation of magnetoferritin produces a highly membrane-active nanoparticle that can magnetise human mesenchymal stem cells (hMSCs) using incubation times as short as one minute. Magnetisation persisted for several weeks in culture and provided significant T2* contrast enhancement during magnetic resonance imaging. Exposure to cationised magnetoferritin did not adversely affect the membrane integrity, proliferation and multi-lineage differentiation capacity of hMSCs, which provides the first detailed evidence for the biocompatibility of magnetoferritin. The combination of synthetic ease and flexibility, the rapidity of labelling and absence of cytotoxicity make this novel nanoparticle system an easily accessible and versatile platform for a range of cell-based therapies in regenerative medicine.Magnetic cell labelling with superparamagnetic iron oxide nanoparticles (SPIONs) facilitates many important biotechnological applications, such as cell imaging and remote manipulation. However, to achieve adequate cellular loading of SPIONs, long incubation times (24 hours and more) or laborious surface functionalisation are often employed, which can adversely affect cell function. Here, we demonstrate that chemical cationisation of magnetoferritin produces a highly membrane-active nanoparticle that can magnetise human mesenchymal stem cells (hMSCs) using incubation times as short as one minute. Magnetisation persisted for several weeks in culture and provided significant T2* contrast enhancement during magnetic resonance imaging. Exposure to cationised

  5. Alterations of Red Cell Membrane Properties in Nneuroacanthocytosis

    PubMed Central

    Siegl, Claudia; Hamminger, Patricia; Jank, Herbert; Ahting, Uwe; Bader, Benedikt; Danek, Adrian; Gregory, Allison; Hartig, Monika; Hayflick, Susan; Hermann, Andreas; Prokisch, Holger; Sammler, Esther M.; Yapici, Zuhal; Prohaska, Rainer; Salzer, Ulrich

    2013-01-01

    Neuroacanthocytosis (NA) refers to a group of heterogenous, rare genetic disorders, namely chorea acanthocytosis (ChAc), McLeod syndrome (MLS), Huntington’s disease-like 2 (HDL2) and pantothenate kinase associated neurodegeneration (PKAN), that mainly affect the basal ganglia and are associated with similar neurological symptoms. PKAN is also assigned to a group of rare neurodegenerative diseases, known as NBIA (neurodegeneration with brain iron accumulation), associated with iron accumulation in the basal ganglia and progressive movement disorder. Acanthocytosis, the occurrence of misshaped erythrocytes with thorny protrusions, is frequently observed in ChAc and MLS patients but less prevalent in PKAN (about 10%) and HDL2 patients. The pathological factors that lead to the formation of the acanthocytic red blood cell shape are currently unknown. The aim of this study was to determine whether NA/NBIA acanthocytes differ in their functionality from normal erythrocytes. Several flow-cytometry-based assays were applied to test the physiological responses of the plasma membrane, namely drug-induced endocytosis, phosphatidylserine exposure and calcium uptake upon treatment with lysophosphatidic acid. ChAc red cell samples clearly showed a reduced response in drug-induced endovesiculation, lysophosphatidic acid-induced phosphatidylserine exposure, and calcium uptake. Impaired responses were also observed in acanthocyte-positive NBIA (PKAN) red cells but not in patient cells without shape abnormalities. These data suggest an “acanthocytic state” of the red cell where alterations in functional and interdependent membrane properties arise together with an acanthocytic cell shape. Further elucidation of the aberrant molecular mechanisms that cause this acanthocytic state may possibly help to evaluate the pathological pathways leading to neurodegeneration. PMID:24098554

  6. Alterations of red cell membrane properties in neuroacanthocytosis.

    PubMed

    Siegl, Claudia; Hamminger, Patricia; Jank, Herbert; Ahting, Uwe; Bader, Benedikt; Danek, Adrian; Gregory, Allison; Hartig, Monika; Hayflick, Susan; Hermann, Andreas; Prokisch, Holger; Sammler, Esther M; Yapici, Zuhal; Prohaska, Rainer; Salzer, Ulrich

    2013-01-01

    Neuroacanthocytosis (NA) refers to a group of heterogenous, rare genetic disorders, namely chorea acanthocytosis (ChAc), McLeod syndrome (MLS), Huntington's disease-like 2 (HDL2) and pantothenate kinase associated neurodegeneration (PKAN), that mainly affect the basal ganglia and are associated with similar neurological symptoms. PKAN is also assigned to a group of rare neurodegenerative diseases, known as NBIA (neurodegeneration with brain iron accumulation), associated with iron accumulation in the basal ganglia and progressive movement disorder. Acanthocytosis, the occurrence of misshaped erythrocytes with thorny protrusions, is frequently observed in ChAc and MLS patients but less prevalent in PKAN (about 10%) and HDL2 patients. The pathological factors that lead to the formation of the acanthocytic red blood cell shape are currently unknown. The aim of this study was to determine whether NA/NBIA acanthocytes differ in their functionality from normal erythrocytes. Several flow-cytometry-based assays were applied to test the physiological responses of the plasma membrane, namely drug-induced endocytosis, phosphatidylserine exposure and calcium uptake upon treatment with lysophosphatidic acid. ChAc red cell samples clearly showed a reduced response in drug-induced endovesiculation, lysophosphatidic acid-induced phosphatidylserine exposure, and calcium uptake. Impaired responses were also observed in acanthocyte-positive NBIA (PKAN) red cells but not in patient cells without shape abnormalities. These data suggest an "acanthocytic state" of the red cell where alterations in functional and interdependent membrane properties arise together with an acanthocytic cell shape. Further elucidation of the aberrant molecular mechanisms that cause this acanthocytic state may possibly help to evaluate the pathological pathways leading to neurodegeneration. PMID:24098554

  7. Label-free nonlinear optical microscopy detects early markers for osteogenic differentiation of human stem cells

    PubMed Central

    Hofemeier, Arne D.; Hachmeister, Henning; Pilger, Christian; Schürmann, Matthias; Greiner, Johannes F. W.; Nolte, Lena; Sudhoff, Holger; Kaltschmidt, Christian; Huser, Thomas; Kaltschmidt, Barbara

    2016-01-01

    Tissue engineering by stem cell differentiation is a novel treatment option for bone regeneration. Most approaches for the detection of osteogenic differentiation are invasive or destructive and not compatible with live cell analysis. Here, non-destructive and label-free approaches of Raman spectroscopy, coherent anti-Stokes Raman scattering (CARS) and second harmonic generation (SHG) microscopy were used to detect and image osteogenic differentiation of human neural crest-derived inferior turbinate stem cells (ITSCs). Combined CARS and SHG microscopy was able to detect markers of osteogenesis within 14 days after osteogenic induction. This process increased during continued differentiation. Furthermore, Raman spectroscopy showed significant increases of the PO43− symmetric stretch vibrations at 959 cm−1 assigned to calcium hydroxyapatite between days 14 and 21. Additionally, CARS microscopy was able to image calcium hydroxyapatite deposits within 14 days following osteogenic induction, which was confirmed by Alizarin Red-Staining and RT- PCR. Taken together, the multimodal label-free analysis methods Raman spectroscopy, CARS and SHG microscopy can monitor osteogenic differentiation of adult human stem cells into osteoblasts with high sensitivity and spatial resolution in three dimensions. Our findings suggest a great potential of these optical detection methods for clinical applications including in vivo observation of bone tissue–implant-interfaces or disease diagnosis. PMID:27225821

  8. Label-free nonlinear optical microscopy detects early markers for osteogenic differentiation of human stem cells

    NASA Astrophysics Data System (ADS)

    Hofemeier, Arne D.; Hachmeister, Henning; Pilger, Christian; Schürmann, Matthias; Greiner, Johannes F. W.; Nolte, Lena; Sudhoff, Holger; Kaltschmidt, Christian; Huser, Thomas; Kaltschmidt, Barbara

    2016-05-01

    Tissue engineering by stem cell differentiation is a novel treatment option for bone regeneration. Most approaches for the detection of osteogenic differentiation are invasive or destructive and not compatible with live cell analysis. Here, non-destructive and label-free approaches of Raman spectroscopy, coherent anti-Stokes Raman scattering (CARS) and second harmonic generation (SHG) microscopy were used to detect and image osteogenic differentiation of human neural crest-derived inferior turbinate stem cells (ITSCs). Combined CARS and SHG microscopy was able to detect markers of osteogenesis within 14 days after osteogenic induction. This process increased during continued differentiation. Furthermore, Raman spectroscopy showed significant increases of the PO43‑ symmetric stretch vibrations at 959 cm‑1 assigned to calcium hydroxyapatite between days 14 and 21. Additionally, CARS microscopy was able to image calcium hydroxyapatite deposits within 14 days following osteogenic induction, which was confirmed by Alizarin Red-Staining and RT- PCR. Taken together, the multimodal label-free analysis methods Raman spectroscopy, CARS and SHG microscopy can monitor osteogenic differentiation of adult human stem cells into osteoblasts with high sensitivity and spatial resolution in three dimensions. Our findings suggest a great potential of these optical detection methods for clinical applications including in vivo observation of bone tissue–implant-interfaces or disease diagnosis.

  9. Label-free nonlinear optical microscopy detects early markers for osteogenic differentiation of human stem cells.

    PubMed

    Hofemeier, Arne D; Hachmeister, Henning; Pilger, Christian; Schürmann, Matthias; Greiner, Johannes F W; Nolte, Lena; Sudhoff, Holger; Kaltschmidt, Christian; Huser, Thomas; Kaltschmidt, Barbara

    2016-01-01

    Tissue engineering by stem cell differentiation is a novel treatment option for bone regeneration. Most approaches for the detection of osteogenic differentiation are invasive or destructive and not compatible with live cell analysis. Here, non-destructive and label-free approaches of Raman spectroscopy, coherent anti-Stokes Raman scattering (CARS) and second harmonic generation (SHG) microscopy were used to detect and image osteogenic differentiation of human neural crest-derived inferior turbinate stem cells (ITSCs). Combined CARS and SHG microscopy was able to detect markers of osteogenesis within 14 days after osteogenic induction. This process increased during continued differentiation. Furthermore, Raman spectroscopy showed significant increases of the PO4(3-) symmetric stretch vibrations at 959 cm(-1) assigned to calcium hydroxyapatite between days 14 and 21. Additionally, CARS microscopy was able to image calcium hydroxyapatite deposits within 14 days following osteogenic induction, which was confirmed by Alizarin Red-Staining and RT- PCR. Taken together, the multimodal label-free analysis methods Raman spectroscopy, CARS and SHG microscopy can monitor osteogenic differentiation of adult human stem cells into osteoblasts with high sensitivity and spatial resolution in three dimensions. Our findings suggest a great potential of these optical detection methods for clinical applications including in vivo observation of bone tissue-implant-interfaces or disease diagnosis. PMID:27225821

  10. Fluorogenic Green-Inside Red-Outside (GIRO) Labeling Approach Reveals Adenylyl Cyclase-Dependent Control of BKα Surface Expression

    PubMed Central

    2015-01-01

    The regulation of surface levels of protein is critical for proper cell function and influences properties including cell adhesion, ion channel contributions to current flux, and the sensitivity of surface receptors to ligands. Here we demonstrate a two-color labeling system in live cells using a single fluorogen activating peptide (FAP) based fusion tag, which enables the rapid and simultaneous quantification of surface and internal proteins. In the nervous system, BK channels can regulate neural excitability and neurotransmitter release, and the surface trafficking of BK channels can be modulated by signaling cascades and assembly with accessory proteins. Using this labeling approach, we examine the dynamics of BK channel surface expression in HEK293 cells. Surface pools of the pore-forming BKα subunit were stable, exhibiting a plasma membrane half-life of >10 h. Long-term activation of adenylyl cyclase by forskolin reduced BKα surface levels by 30%, an effect that could not be attributed to increased bulk endocytosis of plasma membrane proteins. This labeling approach is compatible with microscopic imaging and flow cytometry, providing a solid platform for examining protein trafficking in living cells. PMID:26301573

  11. Fluorescent labeling of tetracysteine-tagged proteins in intact cells

    PubMed Central

    Hoffmann, Carsten; Gaietta, Guido; Zürn, Alexander; Adams, Stephen R; Terrillon, Sonia; Ellisman, Mark H; Tsien, Roger Y; Lohse, Martin J

    2011-01-01

    In this paper, we provide a general protocol for labeling proteins with the membrane-permeant fluorogenic biarsenical dye fluorescein arsenical hairpin binder–ethanedithiol (FlAsH-EDT2). Generation of the tetracysteine-tagged protein construct by itself is not described, as this is a protein-specific process. This method allows site-selective labeling of proteins in living cells and has been applied to a wide variety of proteins and biological problems. We provide here a generally applicable labeling procedure and discuss the problems that can occur as well as general considerations that must be taken into account when designing and implementing the procedure. The method can even be applied to proteins with expression below 1 pmol mg−1 of protein, such as G protein–coupled receptors, and it can be used to study the intracellular localization of proteins as well as functional interactions in fluorescence resonance energy transfer experiments. The labeling procedure using FlAsH-EDT2 as described takes 2–3 h, depending on the number of samples to be processed. PMID:20885379

  12. Label-free cell separation and sorting in microfluidic systems

    PubMed Central

    Gossett, Daniel R.; Weaver, Westbrook M.; Mach, Albert J.; Hur, Soojung Claire; Tse, Henry Tat Kwong; Lee, Wonhee; Amini, Hamed

    2010-01-01

    Cell separation and sorting are essential steps in cell biology research and in many diagnostic and therapeutic methods. Recently, there has been interest in methods which avoid the use of biochemical labels; numerous intrinsic biomarkers have been explored to identify cells including size, electrical polarizability, and hydrodynamic properties. This review highlights microfluidic techniques used for label-free discrimination and fractionation of cell populations. Microfluidic systems have been adopted to precisely handle single cells and interface with other tools for biochemical analysis. We analyzed many of these techniques, detailing their mode of separation, while concentrating on recent developments and evaluating their prospects for application. Furthermore, this was done from a perspective where inertial effects are considered important and general performance metrics were proposed which would ease comparison of reported technologies. Lastly, we assess the current state of these technologies and suggest directions which may make them more accessible. Figure A wide range of microfluidic technologies have been developed to separate and sort cells by taking advantage of differences in their intrinsic biophysical properties PMID:20419490

  13. Oxidative inhibition of red blood cell ATPases by glyceraldehyde.

    PubMed

    Mira, M L; Martinho, F; Azevedo, M S; Manso, C F

    1991-11-01

    Glyceraldehyde and other simple monosaccharides autoxidize under physiological conditions, forming dicarbonyl compounds and hydrogen peroxide via intermediate free radicals. These products may have deleterious effects on cell components. In this paper we study the effect of glyceraldehyde autoxidation on red-cell ATPase activities. The autoxidation of glyceraldehyde in imidazole-glycylglycine buffer, measured by oxygen consumption, depends on the buffer concentration and decreases in the presence of superoxide dismutase and catalase. The addition of DETAPAC inhibits the autoxidation almost completely. When human red-blood-cell membranes are incubated with glyceraldehyde, the red-blood-cell ATPase activities decrease significantly. The addition of DETAPAC, GSH and DTE (dithioerythritol) protects the enzyme from inactivation, but superoxide dismutase and catalase have no effect. Methylglyoxal (a dicarbonyl which is analogous to hydroxypyruvaldehyde derived from glyceraldehyde autoxidation) proved to have a powerful inhibitory action on ATPase activities. The addition of DTE completely protects the enzyme from inactivation, suggesting that the sulphydryl groups of the active site of the enzyme are the critical targets for dicarbonyl compounds. PMID:1836354

  14. Hypochromicity in red blood cells: an experimental and theoretical investigation

    PubMed Central

    Nonoyama, Akihisa; Garcia-Lopez, Alicia; Garcia-Rubio, Luis H.; Leparc, German F.; Potter, Robert L.

    2011-01-01

    Multiwavelength UV-visible transmission spectrophotometry is a useful tool for the examination of micron-size particle suspensions in the context of particle size and chemical composition. This paper reports the reliability of this method to characterize the spectra of purified red blood cells both in their physiological state and with modified hemoglobin content. Previous studies have suggested the contribution of hypochromism on the particle spectra caused by the close electronic interaction of the encapsulated chromophores. Our research shows, however, that this perceived hypochromism can be accounted for by considering two important issues: the acceptance angle of the instrument and the combined scattering and absorption effect of light on the particles. In order to establish these ideas, spectral analysis was performed on purified and modified red cells where the latter was accomplished with a modified hypotonic shock protocol that altered the hemoglobin concentration within the cells. Moreover, the Mie theory was used to successfully simulate the spectral features and trends of the red cells. With this combination of experimental and theoretical exploration, definition of hypochromism has been extended to two subcategories. PMID:21833353

  15. Method using CO for extending the useful shelf-life of refrigerated red blood cells

    DOEpatents

    Bitensky, Mark W.

    1995-01-01

    Method using CO for extending the useful shelf-life of refrigerated red blood cells. Carbon monoxide is utilized for stabilizing hemoglobin in red blood cells to be stored at low temperature. Changes observed in the stored cells are similar to those found in normal red cell aging in the body, the extent thereof being directly related to the duration of refrigerated storage. Changes in cell buoyant density, vesiculation, and the tendency of stored cells to bind autologous IgG antibody directed against polymerized band 3 IgG, all of which are related to red blood cell senescence and increase with refrigerated storage time, have been substantially slowed when red blood cells are treated with CO. Removal of the carbon monoxide from the red blood cells is readily and efficiently accomplished by photolysis in the presence of oxygen so that the stored red blood cells may be safely transfused into a recipient.

  16. Method using CO for extending the useful shelf-life of refrigerated red blood cells

    DOEpatents

    Bitensky, M.W.

    1995-12-19

    A method is disclosed using CO for extending the useful shelf-life of refrigerated red blood cells. Carbon monoxide is utilized for stabilizing hemoglobin in red blood cells to be stored at low temperature. Changes observed in the stored cells are similar to those found in normal red cell aging in the body, the extent thereof being directly related to the duration of refrigerated storage. Changes in cell buoyant density, vesiculation, and the tendency of stored cells to bind autologous IgG antibody directed against polymerized band 3 IgG, all of which are related to red blood cell senescence and increase with refrigerated storage time, have been substantially slowed when red blood cells are treated with CO. Removal of the carbon monoxide from the red blood cells is readily and efficiently accomplished by photolysis in the presence of oxygen so that the stored red blood cells may be safely transfused into a recipient. 5 figs.

  17. Commercial Nanoparticles for Stem Cell Labeling and Tracking

    PubMed Central

    Wang, Yaqi; Xu, Chenjie; Ow, Hooisweng

    2013-01-01

    Stem cell therapy provides promising solutions for diseases and injuries that conventional medicines and therapies cannot effectively treat. To achieve its full therapeutic potentials, the homing process, survival, differentiation, and engraftment of stem cells post transplantation must be clearly understood. To address this need, non-invasive imaging technologies based on nanoparticles (NPs) have been developed to track transplanted stem cells. Here we summarize existing commercial NPs which can act as contrast agents of three commonly used imaging modalities, including fluorescence imaging, magnetic resonance imaging and photoacoustic imaging, for stem cell labeling and tracking. Specifically, we go through their technologies, industry distributors, applications and existing concerns in stem cell research. Finally, we provide an industry perspective on the potential challenges and future for the development of new NP products. PMID:23946821

  18. Label-free electronic detection of target cells

    NASA Astrophysics Data System (ADS)

    Esfandyarpour, Rahim; Javanmard, Mehdi; Harris, James; Davis, Ronald W.

    2014-03-01

    In this manuscript we describe an electronic label-free method for detection of target cells, which has potential applications ranging from pathogen detection for food safety all the way to detection of circulating tumor cells for cancer diagnosis. The nanoelectronic platform consists of a stack of electrodes separated by a 30nm thick insulating layer. Cells binding to the tip of the sensor result in a decrease in the impedance at the sensing tip due to an increase in the fringing capacitance between the electrodes. As a proof of concept we demonstrate the ability to detect Saccharomyces Cerevisae cells with high specificity using a sensor functionalized with Concanavalin A. Ultimately we envision using this sensor in conjunction with a technology for pre-concentration of target cells to develop a fully integrated micro total analysis system.

  19. Labeling index in squamous cell carcinoma of the larynx

    SciTech Connect

    Balzi, M.; Ninu, B.M.; Becciolini, A.; Scubla, E.; Boanini, P.; Gallina, E.; Gallo, O.; Fini-Storchi, O.; Bondi, R. )

    1991-07-01

    Two cell kinetic parameters, the 3H-thymidine labeling index (TLI) and the mitotic index (MI), were studied in vitro on fragments of squamous cell carcinoma tissue of the larynx. They were evaluated to identify those elements able to characterize the growth of these solid tumors. The values of these parameters were analyzed as a function of the clinical stage and the involvement of the regional lymph nodes. Results showed a statistically significant increase in the TLI from stage T1 to T3. No statistically significant differences in the TLI values were observed between the patients with positive and negative lymph nodes.

  20. Functionalized nanopipettes: toward label-free, single cell biosensors

    PubMed Central

    Actis, Paolo; Mak, Andy C.

    2010-01-01

    Nanopipette technology has been proven to be a label-free biosensor capable of identifying DNA and proteins. The nanopipette can include specific recognition elements for analyte discrimination based on size, shape, and charge density. The fully electrical read-out and the ease and low-cost fabrication are unique features that give this technology an enormous potential. Unlike other biosensing platforms, nanopipettes can be precisely manipulated with submicron accuracy and used to study single cell dynamics. This review is focused on creative applications of nanopipette technology for biosensing. We highlight the potential of this technology with a particular attention to integration of this biosensor with single cell manipulation platforms. PMID:20730113

  1. A self-assembling fluorescent dipeptide conjugate for cell labelling.

    PubMed

    Kirkham, Steven; Hamley, Ian W; Smith, Andrew M; Gouveia, Ricardo M; Connon, Che J; Reza, Mehedi; Ruokolainen, Janne

    2016-01-01

    Derivatives of fluorophore FITC (fluorescein isothiocyanate) are widely used in bioassays to label proteins and cells. An N-terminal leucine dipeptide is attached to FITC, and we show that this simple conjugate molecule is cytocompatible and is uptaken by cells (human dermal and corneal fibroblasts) in contrast to FITC itself. Co-localisation shows that FITC-LL segregates in peri-nuclear and intracellular vesicle regions. Above a critical aggregation concentration, the conjugate is shown to self-assemble into beta-sheet nanostructures comprising molecular bilayers. PMID:25990811

  2. A new model to simulate and analyze proliferating cell populations in BrdU labeling experiments

    PubMed Central

    2013-01-01

    Background This paper presents a novel model for proliferating cell populations in labeling experiments. It is especially tailored to the technique of Bromodeoxyuridine (BrdU), which is taken up by dividing cells and thus accumulates with increasing division number during uplabeling. The study of the evolving label intensities of BrdU labeled cell populations is aimed at quantifying proliferation properties such as division and death rates. Results In contrast to existing models, our model considers a labeling efficacy that follows a distribution, rather than a uniform value. It thereby allows to account for noise as well as possibly space-dependent heterogeneity in the effective label uptake of the individual cells in a population. Furthermore, it enables more informative comparison with experimental data: The population-level label distribution is provided as a model output, thereby increasing the information content compared to existing models that give the fraction of labeled cells or the mean label intensity. We employ our model to study some naturally arising examples of heterogeneity in label uptake, which are not covered by existing models. With simulations of noisy and spacially heterogeneous label uptake, we demonstrate that our model contributes a more realistic quantitative description of labeling experiments. Conclusion The presented model is to our knowledge the first one that predicts the full label distribution for BrdU labeling experiments. Thus, it can exploit more information, namely the full intensity distribution, from labeling measurements, and thereby opens up new quantitative insights into cell proliferation. PMID:24268033

  3. Preparation of Fluorescent Dye-Doped Biocompatible Nanoparticles for Cell Labeling.

    PubMed

    Wang, Xiaohui; Peng, Hongshang; Huang, Shihua; You, Fangtian

    2016-04-01

    In this paper, we report a series of fluorescent biocompatible nanoparticles (NPs), prepared by a facile reprecipitation-encapsulation method, for cellular labeling. The as-prepared NPs exhibit a narrow size distribution of 70-110 nm, and a core-shell structure comprised of a hybrid core doped with different dyes and a poly-L-lysine (PLL) shell. With coumarin 6, nile red, and meso- tetraphenylporphyrin as the imaging agents, the fluorescent NPs gave green, orange, and red emissions respectively. Due to the positively charged PLL shell, the fluorescent NPs exhibit neglected cytotoxicity and efficient cellular uptake. After incubation with living cells, the results obtained by laser confocal microscope from green, orange, and red channels all clearly show that the fluores- cent NPs are inhomogenously localized inside the cytoplasm without penetrating into the nucleus. Since such PLL-modified NPs can encapsulate other hydrophobic dyes, a wide spectrum of nanoimaging agents is thus expected. Furthermore, the surface amino groups on the PLL shell afford an anchoring site for further bioconjugation, and targeted imaging is also very promising. PMID:27451673

  4. Online biomedical resources for malaria-related red cell disorders.

    PubMed

    Piel, Frédéric B; Howes, Rosalind E; Nyangiri, Oscar A; Moyes, Catherine L; Williams, Thomas N; Weatherall, David J; Hay, Simon I

    2013-07-01

    Warnings about the expected increase of the global public health burden of malaria-related red cell disorders are accruing. Past and present epidemiological data are necessary to track spatial and temporal changes in the frequencies of these genetic disorders. A number of open access biomedical databases including data on malaria-related red cell disorders have been launched over the last two decades. Here, we review the content of these databases, most of which focus on genetic diversity, and we describe a new epidemiological resource developed by the Malaria Atlas Project. To tackle upcoming public health challenges, the integration of epidemiological and genetic data is important. As many countries are considering implementing national screening programs, strategies to make such data more accessible are also needed. PMID:23568771

  5. Automated microscopy system for detection and genetic characterization of fetal nucleated red blood cells on slides

    NASA Astrophysics Data System (ADS)

    Ravkin, Ilya; Temov, Vladimir

    1998-04-01

    The detection and genetic analysis of fetal cells in maternal blood will permit noninvasive prenatal screening for genetic defects. Applied Imaging has developed and is currently evaluating a system for semiautomatic detection of fetal nucleated red blood cells on slides and acquisition of their DNA probe FISH images. The specimens are blood smears from pregnant women (9 - 16 weeks gestation) enriched for nucleated red blood cells (NRBC). The cells are identified by using labeled monoclonal antibodies directed to different types of hemoglobin chains (gamma, epsilon); the nuclei are stained with DAPI. The Applied Imaging system has been implemented with both Olympus BX and Nikon Eclipse series microscopes which were equipped with transmission and fluorescence optics. The system includes the following motorized components: stage, focus, transmission, and fluorescence filter wheels. A video camera with light integration (COHU 4910) permits low light imaging. The software capabilities include scanning, relocation, autofocusing, feature extraction, facilities for operator review, and data analysis. Detection of fetal NRBCs is achieved by employing a combination of brightfield and fluorescence images of nuclear and cytoplasmic markers. The brightfield and fluorescence images are all obtained with a single multi-bandpass dichroic mirror. A Z-stack of DNA probe FISH images is acquired by moving focus and switching excitation filters. This stack is combined to produce an enhanced image for presentation and spot counting.

  6. Color contrast of red blood cells on solid substrate

    NASA Astrophysics Data System (ADS)

    Paiziev, Adkham A.

    2013-02-01

    In present study we developed the new method of colour visualization of red blood cells without using any chemical staining. The method based on physical phenomena a white light interference on thin transparent films. It is shown that in the case of thin human blood smears colour interference contrast occurs on solid polished substrates. The best contrast shows substrates with maximal refractive index (Mo, W, Si). These materials have been selected as substrate instead of ordinary microscopic slide in reflected light microscopy. It is shown that reflection of incident white light from blood cell surface and boundary cell-substrate generate two coherent lights. The second one (object signal) after passing through red blood cell gathers additional phase and after interference interaction with reference signal (light reflected from outer cell surface) enables cell image in colour. Number of blood smears of healthy persons (control) and patients who were diagnosed with cancer are presented. It is concluded that the offered method may be used as an effective diagnostic tool to detect early stage blood cells lesion by its interference painting in white light. Offered method may be used in research laboratories, hospitals, diagnostic centres, emergency medicine and other as complementary diagnostic tool to present convenient optical and electron microscopy technique.

  7. Characterization of red blood cells (RBCs) using dual Brillouin/Raman micro-spectroscopy

    NASA Astrophysics Data System (ADS)

    Meng, Zhaokai; Bustamante-Lopez, Sandra C.; Yakovlev, Vladislav V.; Meissner, Kenith E.

    2016-04-01

    Erythrocytes, or red blood cells, transport oxygen to and carbon dioxide from the body's tissues and organs. Red blood cell mechanical properties are altered in a number of diseases such as sickle cell anaemia and malaria. Additionally, mechanically modified red blood cell ghosts are being considered as a long-term, biocompatible carrier for drug delivery and for blood analyte sensing. Brillouin spectroscopy enables viscoelastic characterization of samples at the microscale. In this report, Brillouin spectroscopy is applied to characterize the mechanical properties of red blood cells and red blood cell ghosts.

  8. Quantitative microscopy and nanoscopy of sickle red blood cells performed by wide field digital interferometry

    NASA Astrophysics Data System (ADS)

    Shaked, Natan T.; Satterwhite, Lisa L.; Telen, Marilyn J.; Truskey, George A.; Wax, Adam

    2011-03-01

    We have applied wide-field digital interferometry (WFDI) to examine the morphology and dynamics of live red blood cells (RBCs) from individuals who suffer from sickle cell anemia (SCA), a genetic disorder that affects the structure and mechanical properties of RBCs. WFDI is a noncontact, label-free optical microscopy approach that can yield quantitative thickness profiles of RBCs and measurements of their membrane fluctuations at the nanometer scale reflecting their stiffness. We find that RBCs from individuals with SCA are significantly stiffer than those from a healthy control. Moreover, we show that the technique is sensitive enough to distinguish classes of RBCs in SCA, including sickle RBCs with apparently normal morphology, compared to the stiffer crescent-shaped sickle RBCs. We expect that this approach will be useful for diagnosis of SCA and for determining efficacy of therapeutic agents.

  9. Hoechst fluorescence intensity can be used to separate viable bromodeoxyuridine-labeled cells from viable non-bromodeoxyuridine-labeled cells

    NASA Technical Reports Server (NTRS)

    Mozdziak, P. E.; Pulvermacher, P. M.; Schultz, E.; Schell, K.

    2000-01-01

    BACKGROUND: 5-Bromo-2'-deoxyuridine (BrdU) is a powerful compound to study the mitotic activity of a cell. Most techniques that identify BrdU-labeled cells require conditions that kill the cells. However, the fluorescence intensity of the membrane-permeable Hoechst dyes is reduced by the incorporation of BrdU into DNA, allowing the separation of viable BrdU positive (BrdU+) cells from viable BrdU negative (BrdU-) cells. METHODS: Cultures of proliferating cells were supplemented with BrdU for 48 h and other cultures of proliferating cells were maintained without BrdU. Mixtures of viable BrdU+ and viable BrdU- cells from the two proliferating cultures were stained with Hoechst 33342. The viable BrdU+ and BrdU- cells were sorted into different fractions from a mixture of BrdU+ and BrdU- cells based on Hoechst fluorescence intensity and the ability to exclude the vital dye, propidium iodide. Subsequently, samples from the original mixture, the sorted BrdU+ cell population, and the sorted BrdU- cell population were immunostained using an anti-BrdU monoclonal antibody and evaluated using flow cytometry. RESULTS: Two mixtures consisting of approximately 55% and 69% BrdU+ cells were sorted into fractions consisting of greater than 93% BrdU+ cells and 92% BrdU- cells. The separated cell populations were maintained in vitro after sorting to demonstrate their viability. CONCLUSIONS: Hoechst fluorescence intensity in combination with cell sorting is an effective tool to separate viable BrdU+ from viable BrdU- cells for further study. The separated cell populations were maintained in vitro after sorting to demonstrate their viability. Copyright 2000 Wiley-Liss, Inc.

  10. [Management of feto-maternal red cell allo-immunizations].

    PubMed

    Bricca, P; Guinchard, E; Guitton Bliem, C

    2011-04-01

    Feto-maternal red cell alloimmunization is defined by the presence in a pregnant woman of alloantibodies directed against blood group antigens present on the red blood cells of the fetus and inherited from the father. It arises from the immune response to a first contact to these same antigens during a prior transfusion, transplant or pregnancy. The placental transfer and the fixation of the antibodies on the fetal red cells antigenic targets lead to a haemolysis in the fetus and the newborn. The resulting haemolytic disease can show different clinical forms, from a mild anaemia with neonatal hyperbilirubinemia to a major fetal damage with stillbirth caused by hydrops fetalis. The objective of management strategies of feto-maternal alloimmunization is to detect and monitor maternal alloimmunization and to appreciate the effects on the fetus or the newborn. Since a few years, some new non-invasive techniques of surveillance are used, for instance fetal RHD genotyping on maternal plasma and evaluation of fetal anaemia through velocimetry measurement of the blood flow in the middle cerebral artery. The need for a careful postnatal surveillance has to be emphasized due to the neonatal anaemia, which can be prolonged, and to the resurgence of cases of severe neonatal icteruses recently reported by the Académie de Médecine. The policy of prevention of anti-RH1 alloimmunization should also benefit from the evolution of biological techniques by allowing an improved targeting of concerned women. PMID:21397546

  11. Enhanced detection of fluorescence quenching in labeled cells

    SciTech Connect

    Crissman, H.A.; Steinkamp, J.A.

    1992-01-28

    This patent describes a method for relatively quantifying BrdU-labeled DNA in cells in an S-phase during a selected interval within a cell cycle. It comprises: incorporating the BrdU into the DNA for a time of about 5 minutes to thirty minutes; staining the DNA with a first fluorochrome having a fluorescence which is quenchable by BrdU; staining the DNA with a second fluorochrome having a fluorescence which is substantially unaffected by BrdU; measuring fluorescence from the first and second fluorochromes to obtain first and second output signals, respectively, on a cell-by-cell basis; and subtracting the first output signal from the second output signal to obtain a different signal functionally related to the quantity of incorporated BrdU.

  12. Computational cell analysis for label-free detection of cell properties in a microfluidic laminar flow.

    PubMed

    Zhang, Alex Ce; Gu, Yi; Han, Yuanyuan; Mei, Zhe; Chiu, Yu-Jui; Geng, Lina; Cho, Sung Hwan; Lo, Yu-Hwa

    2016-06-20

    Although a flow cytometer, being one of the most popular research and clinical tools for biomedicine, can analyze cells based on the cell size, internal structures such as granularity, and molecular markers, it provides little information about the physical properties of cells such as cell stiffness and physical interactions between the cell membrane and fluid. In this paper, we propose a computational cell analysis technique using cells' different equilibrium positions in a laminar flow. This method utilizes a spatial coding technique to acquire the spatial position of the cell in a microfluidic channel and then uses mathematical algorithms to calculate the ratio of cell mixtures. Most uniquely, the invented computational cell analysis technique can unequivocally detect the subpopulation of each cell type without labeling even when the cell type shows a substantial overlap in the distribution plot with other cell types, a scenario limiting the use of conventional flow cytometers and machine learning techniques. To prove this concept, we have applied the computation method to distinguish live and fixed cancer cells without labeling, count neutrophils from human blood, and distinguish drug treated cells from untreated cells. Our work paves the way for using computation algorithms and fluidic dynamic properties for cell classification, a label-free method that can potentially classify over 200 types of human cells. Being a highly cost-effective cell analysis method complementary to flow cytometers, our method can offer orthogonal tests in companion with flow cytometers to provide crucial information for biomedical samples. PMID:27163941

  13. Label-free detection of immune complexes with myeloid cells.

    PubMed

    Szittner, Z; Bentlage, A E H; Rovero, P; Migliorini, P; Lóránd, V; Prechl, J; Vidarsson, G

    2016-07-01

    The aim of this study was to provide proof-of-concept for quantitative and qualitative label-free detection of immune complexes through myeloid cells with imaging surface plasmon resonance. Surface plasmon resonance imaging was first applied to monitor the binding of human sera from healthy and rheumatoid arthritis (RA) patients to immobilized citrullinated RA-specific peptide antigens, histone citrullinated peptide 2 (HCP2) and viral citrullinated peptide 2 (VCP2). Next, the binding of monocytoid cell line U937 to the resulting immune complexes on the sensor surface was monitored. As control, binding of U937 was monitored to immunoglobulin (Ig)G subclasses simultaneously. Cell response results were compared to results of cyclic citrullinated peptide 2 (CCP2) enzyme-linked immunosorbent assay (ELISA), clinical RA diagnosis and antigen-specific antibody distribution of the samples. Human IgG3 triggered the most pronounced response, followed by IgG1 and IgG4, while IgG2 did not result in U937 cell binding. Serum samples obtained from RA patients resulted in a significantly increased cell response to VCP2 compared to healthy controls. The strength of cell response towards VCP2 immune complexes showed significant correlation with levels of antigen-specific IgA, IgG and IgG3. Cellular responses on VCP2 immune complexes showed significant association with both CCP2-based serological positivity and European League Against Rheumatism (EULAR) criteria-based clinical RA diagnosis. Immunoglobulin-triggered binding of monocytoid cells can be monitored using a label-free multiplex technology. Because these binding events are presumably initiated by Fc receptors, the system provides a tool for biological detection of autoantibodies with diagnostic value, here exemplified by anti-citrullinated antibodies. This provides added information to antibody levels, as interaction with Fc-receptor-expressing cells is also affected by post-translational modification of the immunoglobulins

  14. Hemoglobin Aggregation in Single Red Blood Cells of Sickle Cell Anemia

    NASA Astrophysics Data System (ADS)

    Nishio, Izumi; Tanaka, Toyoichi; Sun, Shao-Tang; Imanishi, Yuri; Tsuyoshi Ohnishi, S.

    1983-06-01

    A laser light scattering technique was used to observe the extent of hemoglobin aggregation in solitary red blood cells of sickle cell anemia. Hemoglobin aggregation was confirmed in deoxygenated cells. The light scattering technique can also be applied to cytoplasmic studies of any biological cell.

  15. Live-cell protein labelling with nanometre precision by cell squeezing

    PubMed Central

    Kollmannsperger, Alina; Sharei, Armon; Raulf, Anika; Heilemann, Mike; Langer, Robert; Jensen, Klavs F.; Wieneke, Ralph; Tampé, Robert

    2016-01-01

    Live-cell labelling techniques to visualize proteins with minimal disturbance are important; however, the currently available methods are limited in their labelling efficiency, specificity and cell permeability. We describe high-throughput protein labelling facilitated by minimalistic probes delivered to mammalian cells by microfluidic cell squeezing. High-affinity and target-specific tracing of proteins in various subcellular compartments is demonstrated, culminating in photoinduced labelling within live cells. Both the fine-tuned delivery of subnanomolar concentrations and the minimal size of the probe allow for live-cell super-resolution imaging with very low background and nanometre precision. This method is fast in probe delivery (∼1,000,000 cells per second), versatile across cell types and can be readily transferred to a multitude of proteins. Moreover, the technique succeeds in combination with well-established methods to gain multiplexed labelling and has demonstrated potential to precisely trace target proteins, in live mammalian cells, by super-resolution microscopy. PMID:26822409

  16. SCF increases in utero-labeled stem cells migration and improves wound healing.

    PubMed

    Zgheib, Carlos; Xu, Junwang; Mallette, Andrew C; Caskey, Robert C; Zhang, Liping; Hu, Junyi; Liechty, Kenneth W

    2015-01-01

    Diabetic skin wounds lack the ability to heal properly and constitute a major and significant complication of diabetes. Nontraumatic lower extremity amputations are the number one complication of diabetic skin wounds. The complexity of their pathophysiology requires an intervention at many levels to enhance healing and wound closure. Stem cells are a promising treatment for diabetic skin wounds as they have the ability to correct abnormal healing. Stem cell factor (SCF), a chemokine expressed in the skin, can induce stem cells migration, however the role of SCF in diabetic skin wound healing is still unknown. We hypothesize that SCF would correct the impairment and promote the healing of diabetic skin wounds. Our results show that SCF improved wound closure in diabetic mice and increased HIF-1α and vascular endothelial growth factor (VEGF) expression levels in these wounds. SCF treatment also enhanced the migration of red fluorescent protein (RFP)-labeled skin stem cells via in utero intra-amniotic injection of lenti-RFP at E8. Interestingly these RFP+ cells are present in the epidermis, stain negative for K15, and appear to be distinct from the already known hair follicle stem cells. These results demonstrate that SCF improves diabetic wound healing in part by increasing the recruitment of a unique stem cell population present in the skin. PMID:26032674

  17. Red blood cell clustering in Poiseuille microcapillary flow

    NASA Astrophysics Data System (ADS)

    Tomaiuolo, Giovanna; Lanotte, Luca; Ghigliotti, Giovanni; Misbah, Chaouqi; Guido, Stefano

    2012-05-01

    Red blood cells (RBC) flowing in microcapillaries tend to associate into clusters, i.e., small trains of cells separated from each other by a distance comparable to cell size. This process is usually attributed to slower RBCs acting to create a sequence of trailing cells. Here, based on the first systematic investigation of collective RBC flow behavior in microcapillaries in vitro by high-speed video microscopy and numerical simulations, we show that RBC size polydispersity within the physiological range does not affect cluster stability. Lower applied pressure drops and longer residence times favor larger RBC clusters. A limiting cluster length, depending on the number of cells in a cluster, is found by increasing the applied pressure drop. The insight on the mechanism of RBC clustering provided by this work can be applied to further our understanding of RBC aggregability, which is a key parameter implicated in clotting and thrombus formation.

  18. Shape anisotropy induces rotations in optically trapped red blood cells

    NASA Astrophysics Data System (ADS)

    Bambardekar, Kapil; Dharmadhikari, Jayashree A.; Dharmadhikari, Aditya K.; Yamada, Toshihoro; Kato, Tsuyoshi; Kono, Hirohiko; Fujimura, Yuichi; Sharma, Shobhona; Mathur, Deepak

    2010-07-01

    A combined experimental and theoretical study is carried out to probe the rotational behavior of red blood cells (RBCs) in a single beam optical trap. We induce shape changes in RBCs by altering the properties of the suspension medium in which live cells float. We find that certain shape anisotropies result in the rotation of optically trapped cells. Indeed, even normal (healthy) RBCs can be made to rotate using linearly polarized trapping light by altering the osmotic stress the cells are subjected to. Hyperosmotic stress is found to induce shape anisotropies. We also probe the effect of the medium's viscosity on cell rotation. The observed rotations are modeled using a Langevin-type equation of motion that takes into account frictional forces that are generated as RBCs rotate in the medium. We observe good correlation between our measured data and calculated results.

  19. Extremes of urine osmolality - Lack of effect on red blood cell survival

    NASA Technical Reports Server (NTRS)

    Leon, H. A.; Fleming, J. E.

    1980-01-01

    Rats were allowed a third of normal water intake for 20 days, and food consumption decreased. The reticulocyte count indicated a suppression of erythropoiesis. Urine osmolality increased from 2,000 mosmol/kg to 3,390 mosmol/kg. Random hemolysis and senescence of a cohort of red blood cell (RBC) previously labeled with (2-(C-14)) glycine was monitored via the production of (C-14)O. Neither hemolysis nor senescence was affected. Following water restriction, the polydipsic rats generated a hypotonic urine. Urine osmolality decreased to 1,300 mosmol/kg for at least 6 days; a reticulocytosis occurred, but RBC survival was unaffected. These results contradict those previously reported, which suggest that RBC survival is influenced by the osmotic stress imposed on the RBC by extremes of urine tonicity. This discrepancy, it is concluded, is due to differences in the methods employed for measuring RBC survival. The random-labeling technique employed previously assumes a steady state between RBC production and destruction. The cohort-labeling technique used here measures hemolysis and senescence independent of changes in RBC production, which is known to be depressed by fasting.

  20. Red Blood Cell Membrane-Cloaked Nanoparticles For Drug Delivery

    NASA Astrophysics Data System (ADS)

    Carpenter, Cody Westcott

    Herein we describe the development of the Red Blood Cell coated nanoparticle, RBC-NP. Purified natural erythrocyte membrane is used to coat drug-loaded poly(lacticco-glycolic acid) (PLGA). Synthetic PLGA co-polymer is biocompatible and biodegradable and has already received US FDA approval for drug-delivery and diagnostics. This work looks specifically at the retention of immunosuppressive proteins on RBC-NPs, right-sidedness of natural RBC membranes interfacing with synthetic polymer nanoparticles, sustained and retarded drug release of RBC-NPs as well as further surface modification of RBC-NPs for increased targeting of model cancer cell lines.

  1. Monoclonal gammopathy-associated pure red cell aplasia.

    PubMed

    Korde, Neha; Zhang, Yong; Loeliger, Kelsey; Poon, Andrea; Simakova, Olga; Zingone, Adriana; Costello, Rene; Childs, Richard; Noel, Pierre; Silver, Samuel; Kwok, Mary; Mo, Clifton; Young, Neal; Landgren, Ola; Sloand, Elaine; Maric, Irina

    2016-06-01

    Pure red cell aplasia (PRCA) is a rare disorder characterized by inhibition of erythroid precursors in the bone marrow and normochromic, normocytic anaemia with reticulocytopenia. Among 51 PRCA patients, we identified 12 (24%) patients having monoclonal gammopathy, monoclonal gammopathy of undetermined significance or smouldering multiple myeloma, with presence of monoclonal protein or abnormal serum free light chains and atypical bone marrow features of clonal plasmacytosis, hypercellularity and fibrosis. Thus far, three patients treated with anti-myeloma based therapeutics have responded with reticulocyte recovery and clinical transfusion independence, suggesting plasma cells play a key role in the pathogenesis of this specific monoclonal gammopathy-associated PRCA. PMID:26999424

  2. Home improvements: malaria and the red blood cell.

    PubMed

    Foley, M; Tilley, L

    1995-11-01

    In real-estate agent's terms, the red blood cell is a renovator's dream. The mature human erythrocyte has no internal organelles, no protein synthesis machinery and no infrastructure for protein trafficking. The malaria parasite invades this empty shell and effectively converts the erythrocyte back into a fully functional eukaryotic cell. In this article, Michael Foley and Leann Tilley examine the Plasmodium falciparum proteins that interact with the membrane skeleton at different stages of the infection and speculate on the roles of these proteins in the remodelling process. PMID:15275396

  3. Flow of Red Blood Cells in Stenosed Microvessels

    NASA Astrophysics Data System (ADS)

    Vahidkhah, Koohyar; Balogh, Peter; Bagchi, Prosenjit

    2016-06-01

    A computational study is presented on the flow of deformable red blood cells in stenosed microvessels. It is observed that the Fahraeus-Lindqvist effect is significantly enhanced due to the presence of a stenosis. The apparent viscosity of blood is observed to increase by several folds when compared to non-stenosed vessels. An asymmetric distribution of the red blood cells, caused by geometric focusing in stenosed vessels, is observed to play a major role in the enhancement. The asymmetry in cell distribution also results in an asymmetry in average velocity and wall shear stress along the length of the stenosis. The discrete motion of the cells causes large time-dependent fluctuations in flow properties. The root-mean-square of flow rate fluctuations could be an order of magnitude higher than that in non-stenosed vessels. Several folds increase in Eulerian velocity fluctuation is also observed in the vicinity of the stenosis. Surprisingly, a transient flow reversal is observed upstream a stenosis but not downstream. The asymmetry and fluctuations in flow quantities and the flow reversal would not occur in absence of the cells. It is concluded that the flow physics and its physiological consequences are significantly different in micro- versus macrovascular stenosis.

  4. Flow of Red Blood Cells in Stenosed Microvessels.

    PubMed

    Vahidkhah, Koohyar; Balogh, Peter; Bagchi, Prosenjit

    2016-01-01

    A computational study is presented on the flow of deformable red blood cells in stenosed microvessels. It is observed that the Fahraeus-Lindqvist effect is significantly enhanced due to the presence of a stenosis. The apparent viscosity of blood is observed to increase by several folds when compared to non-stenosed vessels. An asymmetric distribution of the red blood cells, caused by geometric focusing in stenosed vessels, is observed to play a major role in the enhancement. The asymmetry in cell distribution also results in an asymmetry in average velocity and wall shear stress along the length of the stenosis. The discrete motion of the cells causes large time-dependent fluctuations in flow properties. The root-mean-square of flow rate fluctuations could be an order of magnitude higher than that in non-stenosed vessels. Several folds increase in Eulerian velocity fluctuation is also observed in the vicinity of the stenosis. Surprisingly, a transient flow reversal is observed upstream a stenosis but not downstream. The asymmetry and fluctuations in flow quantities and the flow reversal would not occur in absence of the cells. It is concluded that the flow physics and its physiological consequences are significantly different in micro- versus macrovascular stenosis. PMID:27319318

  5. Effects of ethanol on red blood cell rheological behavior

    PubMed Central

    Rabai, M.; Detterich, J.A.; Wenby, R.B.; Toth, K.; Meiselman, H.J.

    2016-01-01

    Consumption of red wine is associated with a decreased risk of several cardiovascular diseases (e.g., coronary artery disease, stroke), but unfortunately literature reports regarding ethanol’s effects on hemorheological parameters are not concordant. In the present study, red blood cell (RBC) deformability was tested via laser ektacytometry (LORCA, 0.3 – 30 Pa) using two approaches: 1) addition of ethanol to whole blood at 0.25% – 2% followed by incubation and testing in ethanol-free LORCA medium; 2) addition of ethanol to the LORCA medium at 0.25% – 6% then testing untreated native RBC in these media. The effects of ethanol on deformability for oxidatively stressed RBC were investigated as were changes of RBC aggregation (Myrenne Aggregometer) for cells in autologous plasma or 3% 70 kDa dextran. Significant dose-related increases of RBC deformability were observed at 0.25% (p<0.05) and higher concentrations only if ethanol was in the LORCA medium; no changes occurred for cells previously incubated with ethanol then tested in ethanol-free medium. The impaired deformability of cells pre-exposed to oxidative stress was improved only if ethanol was in the LORCA medium. RBC aggregation decreased with concentration at 0.25% and higher for cells in both autologous plasma and dextran 70. Our results indicate that ethanol reversibly improves erythrocyte deformability and irreversibly decreases erythrocyte aggregation; the relevance of these results to the health benefits of moderate wine consumption require further investigation. PMID:23089886

  6. Flow of Red Blood Cells in Stenosed Microvessels

    PubMed Central

    Vahidkhah, Koohyar; Balogh, Peter; Bagchi, Prosenjit

    2016-01-01

    A computational study is presented on the flow of deformable red blood cells in stenosed microvessels. It is observed that the Fahraeus-Lindqvist effect is significantly enhanced due to the presence of a stenosis. The apparent viscosity of blood is observed to increase by several folds when compared to non-stenosed vessels. An asymmetric distribution of the red blood cells, caused by geometric focusing in stenosed vessels, is observed to play a major role in the enhancement. The asymmetry in cell distribution also results in an asymmetry in average velocity and wall shear stress along the length of the stenosis. The discrete motion of the cells causes large time-dependent fluctuations in flow properties. The root-mean-square of flow rate fluctuations could be an order of magnitude higher than that in non-stenosed vessels. Several folds increase in Eulerian velocity fluctuation is also observed in the vicinity of the stenosis. Surprisingly, a transient flow reversal is observed upstream a stenosis but not downstream. The asymmetry and fluctuations in flow quantities and the flow reversal would not occur in absence of the cells. It is concluded that the flow physics and its physiological consequences are significantly different in micro- versus macrovascular stenosis. PMID:27319318

  7. 660 nm red light-enhanced bone marrow mesenchymal stem cell transplantation for hypoxic-ischemic brain damage treatment

    PubMed Central

    Li, Xianchao; Hou, Wensheng; Wu, Xiaoying; Jiang, Wei; Chen, Haiyan; Xiao, Nong; Zhou, Ping

    2014-01-01

    Bone marrow mesenchymal stem cell transplantation is an effective treatment for neonatal hypoxic-ischemic brain damage. However, the in vivo transplantation effects are poor and their survival, colonization and differentiation efficiencies are relatively low. Red or near-infrared light from 600–1,000 nm promotes cellular migration and prevents apoptosis. Thus, we hypothesized that the combination of red light with bone marrow mesenchymal stem cell transplantation would be effective for the treatment of hypoxic-ischemic brain damage. In this study, the migration and colonization of cultured bone marrow mesenchymal stem cells on primary neurons after oxygen-glucose deprivation were detected using Transwell assay. The results showed that, after a 40-hour irradiation under red light-emitting diodes at 660 nm and 60 mW/cm2, an increasing number of green fluorescence-labeled bone marrow mesenchymal stem cells migrated towards hypoxic-ischemic damaged primary neurons. Meanwhile, neonatal rats with hypoxic-ischemic brain damage were given an intraperitoneal injection of 1 × 106 bone marrow mesenchymal stem cells, followed by irradiation under red light-emitting diodes at 660 nm and 60 mW/cm2 for 7 successive days. Shuttle box test results showed that, after phototherapy and bone marrow mesenchymal stem cell transplantation, the active avoidance response rate of hypoxic-ischemic brain damage rats was significantly increased, which was higher than that after bone marrow mesenchymal stem cell transplantation alone. Experimental findings indicate that 660 nm red light emitting diode irradiation promotes the migration of bone marrow mesenchymal stem cells, thereby enhancing the contribution of cell transplantation in the treatment of hypoxic-ischemic brain damage. PMID:25206807

  8. Cytoplasmic calcium buffers in intact human red cells.

    PubMed Central

    Tiffert, T; Lew, V L

    1997-01-01

    1. Precise knowledge of the cytoplasmic Ca2+ buffering behaviour in intact human red cells is essential for the characterization of their [Ca2+]i-dependent functions. This was investigated by using a refined method and experimental protocols which allowed continuity in the estimates of [Ca2+]i, from nanomolar to millimolar concentrations, in the presence and absence of external Ca2+ chelators. 2. The study was carried out in human red cells whose plasma membrane Ca2+ pump was inhibited either by depleting the cells of ATP or by adding vanadate to the cell suspension. Cytoplasmic Ca2+ buffering was analysed from plots of total cell calcium content vs. ionized cytoplasmic Ca2+ concentration ([CaT]i vs. [Ca2+]i) obtained from measurements of the equilibrium distribution of 45Ca2+ at different external Ca2+ concentrations ([Ca2+]o), in conditions known to clamp cell volume and pH. The equilibrium distribution of 45Ca2+ was induced by the divalent cation ionophore A23187. 3. The results showed the following. (i) The known red cell Ca2+ buffer represented by alpha, with a large capacity and low Ca2+ affinity, was the main cytoplasmic Ca2+ binding agent. (ii) The value of alpha was remarkably constant; the means for each of four donors ranged from 0.33 to 0.35, with a combined value of all independent measurements of 0.34 +/- 0.01 (mean +/- S.E.M., n = 16). This contrasts with the variability previously reported. (iii) There was an additional Ca2+ buffering complex with a low capacity (approximately 80 micromol (340 g Hb)(-1)) and intermediate Ca2+ affinity (apparent dissociation constant, K(D,app) approximately 4-50 microM) whose possible identity is discussed. (iv) The cell content of putative Ca2+ buffers with submicromolar Ca2+ dissociation constants was below the detection limit of the methods used here (less than 2 micromol (340 g Hb)(-1)). 4. Vanadate (1 mM) inhibited the Vmax of the Ca2+ pump in inosine-fed cells by 99.7%. The cytoplasmic Ca2+ buffering behaviour

  9. Changes in En(a-) human red blood cell membranes during in vivo ageing.

    PubMed

    Shinozuka, T; Miyata, Y; Takei, S; Yoshida, R; Ogamo, A; Nakagawa, Y; Kuroda, N; Yanagida, J

    1996-01-01

    The human red blood cells with phenotype En(a-) were characterized by the lack of MN antigens. The red blood cells with phenotype En(a-) which were found in a Japanese family were tested to clarify the changes in membrane surfaces of the red blood cells during in vivo ageing. The contents of sialic acid, glucose, mannose, galactose, fucose, N-acetylglucosamine and N-acetylgalactosamine of the red blood cell membranes obtained from the old red blood cells with phenotype En(a-) were significantly lower than those of the young red blood cell membranes. Neither the young nor the old red blood cells with phenotype En(a-) showed the agglutination with Arachis hypogaea (PNA) which was capable of binding to T agglutinogen. It is presumed that En(a-) red blood cells are not exposed to sialidase in vivo. In comparison with the young En(a-) red blood cell membranes, the number and the distribution density of lectin receptor sites on the old ones for Limulus polyphemus (LPA), Canavalia ensiformis (Con A), Triticum vulgaris (WGA) and Bauhinia purpurea (BPA) were significantly lower. It is thought that En(a-) red blood cell ageing is accompanied by elimination of some sialoglycoconjugates which have affinity for LPA, Con A, WGA and BPA, whereas En(a-) red blood cells lack glycophorin A. PMID:8866734

  10. Metabolic pathways that correlate with post-transfusion circulation of stored murine red blood cells.

    PubMed

    de Wolski, Karen; Fu, Xiaoyoun; Dumont, Larry J; Roback, John D; Waterman, Hayley; Odem-Davis, Katherine; Howie, Heather L; Zimring, James C

    2016-05-01

    Transfusion of red blood cells is a very common inpatient procedure, with more than 1 in 70 people in the USA receiving a red blood cell transfusion annually. However, stored red blood cells are a non-uniform product, based upon donor-to-donor variation in red blood cell storage biology. While thousands of biological parameters change in red blood cells over storage, it has remained unclear which changes correlate with function of the red blood cells, as opposed to being co-incidental changes. In the current report, a murine model of red blood cell storage/transfusion is applied across 13 genetically distinct mouse strains and combined with high resolution metabolomics to identify metabolic changes that correlated with red blood cell circulation post storage. Oxidation in general, and peroxidation of lipids in particular, emerged as changes that correlated with extreme statistical significance, including generation of dicarboxylic acids and monohydroxy fatty acids. In addition, differences in anti-oxidant pathways known to regulate oxidative stress on lipid membranes were identified. Finally, metabolites were identified that differed at the time the blood was harvested, and predict how the red blood cells perform after storage, allowing the potential to screen donors at time of collection. Together, these findings map out a new landscape in understanding metabolic changes during red blood cell storage as they relate to red blood cell circulation. PMID:26921359

  11. Resonance Raman Probes for Organelle-Specific Labeling in Live Cells.

    PubMed

    Kuzmin, Andrey N; Pliss, Artem; Lim, Chang-Keun; Heo, Jeongyun; Kim, Sehoon; Rzhevskii, Alexander; Gu, Bobo; Yong, Ken-Tye; Wen, Shangchun; Prasad, Paras N

    2016-01-01

    Raman microspectroscopy provides for high-resolution non-invasive molecular analysis of biological samples and has a breakthrough potential for dissection of cellular molecular composition at a single organelle level. However, the potential of Raman microspectroscopy can be fully realized only when novel types of molecular probes distinguishable in the Raman spectroscopy modality are developed for labeling of specific cellular domains to guide spectrochemical spatial imaging. Here we report on the design of a next generation Raman probe, based on BlackBerry Quencher 650 compound, which provides unprecedentedly high signal intensity through the Resonance Raman (RR) enhancement mechanism. Remarkably, RR enhancement occurs with low-toxic red light, which is close to maximum transparency in the biological optical window. The utility of proposed RR probes was validated for targeting lysosomes in live cultured cells, which enabled identification and subsequent monitoring of dynamic changes in this organelle by Raman imaging. PMID:27339882

  12. Resonance Raman Probes for Organelle-Specific Labeling in Live Cells

    NASA Astrophysics Data System (ADS)

    Kuzmin, Andrey N.; Pliss, Artem; Lim, Chang-Keun; Heo, Jeongyun; Kim, Sehoon; Rzhevskii, Alexander; Gu, Bobo; Yong, Ken-Tye; Wen, Shangchun; Prasad, Paras N.

    2016-06-01

    Raman microspectroscopy provides for high-resolution non-invasive molecular analysis of biological samples and has a breakthrough potential for dissection of cellular molecular composition at a single organelle level. However, the potential of Raman microspectroscopy can be fully realized only when novel types of molecular probes distinguishable in the Raman spectroscopy modality are developed for labeling of specific cellular domains to guide spectrochemical spatial imaging. Here we report on the design of a next generation Raman probe, based on BlackBerry Quencher 650 compound, which provides unprecedentedly high signal intensity through the Resonance Raman (RR) enhancement mechanism. Remarkably, RR enhancement occurs with low-toxic red light, which is close to maximum transparency in the biological optical window. The utility of proposed RR probes was validated for targeting lysosomes in live cultured cells, which enabled identification and subsequent monitoring of dynamic changes in this organelle by Raman imaging.

  13. Resonance Raman Probes for Organelle-Specific Labeling in Live Cells

    PubMed Central

    Kuzmin, Andrey N.; Pliss, Artem; Lim, Chang-Keun; Heo, Jeongyun; Kim, Sehoon; Rzhevskii, Alexander; Gu, Bobo; Yong, Ken-Tye; Wen, Shangchun; Prasad, Paras N.

    2016-01-01

    Raman microspectroscopy provides for high-resolution non-invasive molecular analysis of biological samples and has a breakthrough potential for dissection of cellular molecular composition at a single organelle level. However, the potential of Raman microspectroscopy can be fully realized only when novel types of molecular probes distinguishable in the Raman spectroscopy modality are developed for labeling of specific cellular domains to guide spectrochemical spatial imaging. Here we report on the design of a next generation Raman probe, based on BlackBerry Quencher 650 compound, which provides unprecedentedly high signal intensity through the Resonance Raman (RR) enhancement mechanism. Remarkably, RR enhancement occurs with low-toxic red light, which is close to maximum transparency in the biological optical window. The utility of proposed RR probes was validated for targeting lysosomes in live cultured cells, which enabled identification and subsequent monitoring of dynamic changes in this organelle by Raman imaging. PMID:27339882

  14. New integrative modules for multicolor-protein labeling and live-cell imaging in Saccharomyces cerevisiae.

    PubMed

    Malcova, Ivana; Farkasovsky, Marian; Senohrabkova, Lenka; Vasicova, Pavla; Hasek, Jiri

    2016-05-01

    Live-imaging analysis is performed in many laboratories all over the world. Various tools have been developed to enable protein labeling either in plasmid or genomic context in live yeast cells. Here, we introduce a set of nine integrative modules for the C-terminal gene tagging that combines three fluorescent proteins (FPs)-ymTagBFP, mCherry and yTagRFP-T with three dominant selection markers: geneticin, nourseothricin and hygromycin. In addition, the construction of two episomal modules for Saccharomyces cerevisiae with photostable yTagRFP-T is also referred to. Our cassettes with orange, red and blue FPs can be combined with other fluorescent probes like green fluorescent protein to prepare double- or triple-labeled strains for multicolor live-cell imaging. Primers for PCR amplification of the cassettes were designed in such a way as to be fully compatible with the existing PCR toolbox representing over 50 various integrative modules and also with deletion cassettes either for single or repeated usage to enable a cost-effective and an easy exchange of tags. New modules can also be used for biochemical analysis since antibodies are available for all three fluorescent probes. PMID:26994102

  15. Membrane stress increases cation permeability in red cells.

    PubMed

    Johnson, R M

    1994-11-01

    The human red cell is known to increase its cation permeability when deformed by mechanical forces. Light-scattering measurements were used to quantitate the cell deformation, as ellipticity under shear. Permeability to sodium and potassium was not proportional to the cell deformation. An ellipticity of 0.75 was required to increase the permeability of the membrane to cations, and flux thereafter increased rapidly as the limits of cell extension were reached. Induction of membrane curvature by chemical agents also did not increase cation permeability. These results indicate that membrane deformation per se does not increase permeability, and that membrane tension is the effector for increased cation permeability. This may be relevant to some cation permeabilities observed by patch clamping. PMID:7858123

  16. Diffusional solute flux during osmotic water flow across the human red cell membrane.

    PubMed

    Brahm, J; Galey, W R

    1987-05-01

    The effect of solvent drag on the unidirectional efflux of labeled water, urea, and chloride from human red cells was studied by means of the continuous flow tube method under conditions of osmotic equilibrium and net volume flow. Solvent (water) flow out of cells was created by mixing cells equilibrated in 100 mM salt solution with a 200-mM or 250-mM salt solution, while flow of water into cells was obtained by equilibrating the cells in the higher concentration and mixing them with the 100-mM solution. Control experiments constitute measurements of efflux of [14C]ethanol in normal cells and 3H2O in cells treated with p-chloromercuribenzosulfonate under the conditions described above. In both instances, the solute is known to penetrate the membrane through nonporous pathways. As anticipated, the tracer flux of neither urea nor chloride showed any dependence on net solvent flow, regardless of the direction. If one assumes the recently reported reflection coefficient for urea of 0.7, the urea tracer flux should change by at least 24% under volume flow conditions. Since such changes would be easily detected with our method, we conclude that the pathways for water, for urea, and for chloride are functionally separated. PMID:3037007

  17. Red cell glycolytic enzyme disorders caused by mutations: an update.

    PubMed

    Climent, Fernando; Roset, Feliu; Repiso, Ada; Pérez de la Ossa, Pablo

    2009-06-01

    Glycolysis is one of the principle pathways of ATP generation in cells and is present in all cell tissues; in erythrocytes, glycolysis is the only pathway for ATP synthesis since mature red cells lack the internal structures necessary to produce the energy vital for life. Red cell deficiencies have been detected in all erythrocyte glycolytic pathways, although their frequencies differ owing to diverse causes, such as the affected enzyme and severity of clinical manifestations. The number of enzyme deficiencies known is endless. The most frequent glycolysis abnormality is pyruvate kinase deficiency, since around 500 cases are known, the first of which was reported in 1961. However, only approximately 200 cases were due to mutations. In contrast, only one case of phosphoglycerate mutase BB type mutation, described in 2003, has been detected. Most mutations are located in the coding sequences of genes, while others, missense, deletions, insertions, splice defects, premature stop codons and promoter mutations, are also frequent. Understanding of the crystal structure of enzymes permits molecular modelling studies which, in turn, reveal how mutations can affect enzyme structure and function. PMID:19519368

  18. Creation of Primary Cell Lines from Lineage-Labeled Mouse Models of Cancer

    PubMed Central

    Rhim, Andrew D.

    2015-01-01

    Frequently, it is necessary to isolate pure populations of cancer cells for downstream assays, such as transcriptional analysis, signaling studies, and the creation of noncontaminated primary cell lines. Genetic lineage labeling with fluorescent reporter alleles allows for the identification of epithelial-derived cells within tumors. This protocol describes a method to isolate lineage-labeled pancreatic epithelial cells for ex vivo analysis, but it can be adapted for any type of lineage-labeled tumor. PMID:25934932

  19. Deformation of red blood cells using acoustic radiation forces

    PubMed Central

    Mishra, Puja; Hill, Martyn; Glynne-Jones, Peter

    2014-01-01

    Acoustic radiation forces have been used to manipulate cells and bacteria in a number of recent microfluidic applications. The net force on a cell has been subject to careful investigation over a number of decades. We demonstrate that the radiation forces also act to deform cells. An ultrasonic standing wave field is created in a 0.1 mm glass capillary at a frequency of 7.9 MHz. Using osmotically swollen red-blood cells, we show observable deformations up to an aspect ratio of 1.35, comparable to deformations created by optical tweezing. In contrast to optical technologies, ultrasonic devices are potentially capable of deforming thousands of cells simultaneously. We create a finite element model that includes both the acoustic environment of the cell, and a model of the cell membrane subject to forces resulting from the non-linear aspects of the acoustic field. The model is found to give reasonable agreement with the experimental results, and shows that the deformation is the result of variation in an acoustic force that is directed outwards at all points on the cell membrane. We foresee applications in diagnostic devices, and in the possibility of mechanically stimulating cells to promote differentiation and physiological effects. PMID:25379070

  20. Evaluating the efficacy of subcellular fractionation of blast cells using live cell labeling and 2D DIGE.

    PubMed

    Ho, Yin Ying; Penno, Megan; Perugini, Michelle; Lewis, Ian; Hoffmann, Peter

    2012-01-01

    Labeling of exposed cell surface proteins of live cells using CyDye DIGE fluor minimal dyes is an efficient strategy for cell surface proteome profiling and quantifying differentially expressed proteins in diseases. Here we describe a strategy to evaluate a two-step detergent-based protein fractionation method using live cell labeling followed by visualization of the fluorescently labeled cell surface proteins and fractionated proteins within a single 2D gel. PMID:22311770

  1. Nanoscale Label-free Bioprobes to Detect Intracellular Proteins in Single Living Cells

    NASA Astrophysics Data System (ADS)

    Hong, Wooyoung; Liang, Feng; Schaak, Diane; Loncar, Marko; Quan, Qimin

    2014-08-01

    Fluorescent labeling techniques have been widely used in live cell studies; however, the labeling processes can be laborious and challenging for use in non-transfectable cells, and labels can interfere with protein functions. While label-free biosensors have been realized by nanofabrication, a method to track intracellular protein dynamics in real-time, in situ and in living cells has not been found. Here we present the first demonstration of label-free detection of intracellular p53 protein dynamics through a nanoscale surface plasmon-polariton fiber-tip-probe (FTP).

  2. Metabolic profiling of hematopoietic stem and progenitor cells during proliferation and differentiation into red blood cells.

    PubMed

    Daud, Hasbullah; Browne, Susan; Al-Majmaie, Rasoul; Murphy, William; Al-Rubeai, Mohamed

    2016-01-25

    An understanding of the metabolic profile of cell proliferation and differentiation should support the optimization of culture conditions for hematopoietic stem and progenitor cell (HSPC) proliferation, differentiation, and maturation into red blood cells. We have evaluated the key metabolic parameters during each phase of HSPC culture for red blood cell production in serum-supplemented (SS) and serum-free (SF) conditions. A simultaneous decrease in growth rate, total protein content, cell size, and the percentage of cells in the S/G2 phase of cell cycle, as well as an increase in the percentage of cells with a CD71(-)/GpA(+) surface marker profile, indicates HSPC differentiation into red blood cells. Compared with proliferating HSPCs, differentiating HSPCs showed significantly lower glucose and glutamine consumption rates, lactate and ammonia production rates, and amino acid consumption and production rates in both SS and SF conditions. Furthermore, extracellular acidification was associated with late proliferation phase, suggesting a reduced cellular metabolic rate during the transition from proliferation to differentiation. Under both SS and SF conditions, cells demonstrated a high metabolic rate with a mixed metabolism of both glycolysis and oxidative phosphorylation (OXPHOS) in early and late proliferation, an increased dependence on OXPHOS activity during differentiation, and a shift to glycolytic metabolism only during maturation phase. These changes indicate that cell metabolism may have an important impact on the ability of HSPCs to proliferate and differentiate into red blood cells. PMID:26013297

  3. Influence of increased membrane cholesterol on membrane fluidity and cell function in human red blood cells.

    PubMed

    Cooper, R A

    1978-01-01

    Cholesterol and phospholipid are the two major lipids of the red cell membrane. Cholesterol is insoluble in water but is solubilized by phospholipids both in membranes and in plasma lipoproteins. Morever, cholesterol exchanges between membranes and lipoproteins. An equilibrium partition is established based on the amount of cholesterol relative to phospholipid (C/PL) in these two compartments. Increases in the C/PL of red cell membranes have been studied under three conditions: First, spontaneous increases in vivo have been observed in the spur red cells of patients with severe liver disease; second, similar red cell changes in vivo have been induced by the administration of cholesterol-enriched diets to rodents and dogs; third, increases in membrane cholesterol have been induced in vitro by enriching the C/PL of the lipoprotein environment with cholesterol-phospholipid dispersions (liposomes) having a C/PL of greater than 1.0. In each case, there is a close relationship between the C/PL of the plasma environment and the C/PL of the red cell membrane. In vivo, the C/PL mole ratio of red cell membranes ranges from a normal value of 0.09--1.0 to values which approach but do not reach 2.0. In vitro, this ratio approaches 3.0. Cholesterol enrichment of red cell membranes directly influences membrane lipid fluidity, as assessed by the rotational diffusion of hydrophobic fluorescent probes such as diphenyl hexatriene (DPH). A close correlation exists between increases in red cell membrane C/PL and decreases in membrane fluidity over the range of membrane C/PL from 1.0 to 2.0; however, little further change in fluidity occurs when membrane C/PL is increased to 2.0--3.0. Cholesterol enrichment of red cell membranes is associated with the transformation of cell contour to one which is redundant and folded, and this is associated with a decrease in red cell filterability in vitro. Circulation in vivo in the presence of the spleen further modifies cell shape to a spiny

  4. Full-field velocity imaging of red blood cells in capillaries with spatiotemporal demodulation autocorrelation

    NASA Astrophysics Data System (ADS)

    Wang, Mingyi; Zeng, Yaguang; Dong, Nannan; Liao, Riwei; Yang, Guojian

    2016-03-01

    We propose a full-field optical method for the label-free and quantitative mapping of the velocities of red blood cells (RBCs) in capillaries. It integrates spatiotemporal demodulation and an autocorrelation algorithm, and measures RBC velocity according to the ratio of RBC length to lag time. Conventionally, RBC length is assumed to be a constant and lag time is taken as a variable, while our method treats both of them as variables. We use temporal demodulation and the Butterworth spatial filter to separate RBC signal from background signal, based on which we obtain the RBC length by image segmentation and lag time by autocorrelation analysis. The RBC velocity calculated now is more accurate. The validity of our method is verified by an in vivo experiment on a mouse ear. Owing to its higher image signal-to-noise ratio, our method can be used for mapping RBC velocity in the turbid tissue case.

  5. Quantum dot-labeled aptamer nanoprobes specifically targeting glioma cells

    NASA Astrophysics Data System (ADS)

    Chen, Xue-Chai; Deng, Yu-Lin; Lin, Yi; Pang, Dai-Wen; Qing, Hong; Qu, Feng; Xie, Hai-Yan

    2008-06-01

    Two new techniques, aptamer-based specific recognition and quantum dot (QD)-based fluorescence labeling, are becoming increasingly important in biosensing. In this study, these two techniques have been coupled together to construct a new kind of fluorescent QD-labeled aptamer (QD-Apt) nanoprobe by conjugating GBI-10 aptamer to the QD surface. GBI-10 is a single-stranded DNA (ssDNA) aptamer for tenascin-C, which distributes on the surface of glioma cells as a dominant extracellular matrix protein. The QD-Apt nanoprobe can recognize the tenascin-C on the human glioma cell surface, which will be helpful for the development of new convenient and sensitive in vitro diagnostic assays for glioma. The QD-Apt nanoprobe has particular features such as strong fluorescence, stability, monodispersity and uniformity. In addition, this probe preparation method is universal, so it is expected to provide a new type of stable nanoprobe for high-throughput and fast biosensing detection and bioimaging. New methods for real-time and dynamic tracking and imaging can be accordingly developed.

  6. Nanoparticle-labeled stem cells: a novel therapeutic vehicle

    PubMed Central

    El-Sadik, Abir O; El-Ansary, Afaf; Sabry, Sherif M

    2010-01-01

    Nanotechnology has been described as a general purpose technology. It has already generated a range of inventions and innovations. Development of nanotechnology will provide clinical medicine with a range of new diagnostic and therapeutic opportunities such as medical imaging, medical diagnosis, drug delivery, and cancer detection and management. Nanoparticles such as manganese, polystyrene, silica, titanium oxide, gold, silver, carbon, quantum dots, and iron oxide have received enormous attention in the creation of new types of analytical tools for biotechnology and life sciences. Labeling of stem cells with nanoparticles overcame the problems in homing and fixing stem cells to their desired site and guiding extension of stem cells to specific directions. Although the biologic effects of some nanoparticles have already been assessed, information on toxicity and possible mechanisms of various particle types remains inadequate. The aim of this review is to give an overview of the mechanisms of internalization and distribution of nanoparticles inside stem cells, as well as the influence of different types of nanoparticles on stem cell viability, proliferation, differentiation, and cytotoxicity, and to assess the role of nanoparticles in tracking the fate of stem cells used in tissue regeneration. PMID:22291483

  7. Cell-selective labelling of proteomes in Drosophila melanogaster

    PubMed Central

    Erdmann, Ines; Marter, Kathrin; Kobler, Oliver; Niehues, Sven; Abele, Julia; Müller, Anke; Bussmann, Julia; Storkebaum, Erik; Ziv, Tamar; Thomas, Ulrich; Dieterich, Daniela C.

    2015-01-01

    The specification and adaptability of cells rely on changes in protein composition. Nonetheless, uncovering proteome dynamics with cell-type-specific resolution remains challenging. Here we introduce a strategy for cell-specific analysis of newly synthesized proteomes by combining targeted expression of a mutated methionyl-tRNA synthetase (MetRS) with bioorthogonal or fluorescent non-canonical amino-acid-tagging techniques (BONCAT or FUNCAT). Substituting leucine by glycine within the MetRS-binding pocket (MetRSLtoG) enables incorporation of the non-canonical amino acid azidonorleucine (ANL) instead of methionine during translation. Newly synthesized proteins can thus be labelled by coupling the azide group of ANL to alkyne-bearing tags through ‘click chemistry'. To test these methods for applicability in vivo, we expressed MetRSLtoG cell specifically in Drosophila. FUNCAT and BONCAT reveal ANL incorporation into proteins selectively in cells expressing the mutated enzyme. Cell-type-specific FUNCAT and BONCAT, thus, constitute eligible techniques to study protein synthesis-dependent processes in complex and behaving organisms. PMID:26138272

  8. 99mTc red blood cell scintigraphy in evaluating focal liver lesions

    SciTech Connect

    Rabinowitz, S.A.; McKusick, K.A.; Strauss, H.W.

    1984-07-01

    To determine the accuracy of blood-pool imaging in the diagnosis of hepatic hemangiomas, 39 patients with various focal hepatic lesions were studied. The diagnoses in these patients were made by biopsy, angiography, surgical exploration, or clinical stability for a minimum of 14 months. The diagnoses were: hemangiomas (13 patients), hepatoma (three), metastases (19), abscesses (two), and liver cysts (two). After modified in vivo labeling of red blood cells with 20 mCi (740 MBq) of 99mTc pertechnetate, an initial flow study and early (1-15 min) and delayed (1-2 hr) static images were obtained. Increased blood-pool activity with a discordant flow pattern was seen in 11 of 13 patients with hemangiomas. False-negative scans occurred in two hemangiomas with extensive fibrosis. Two of three hepatomas had increased blood-pool activity associated with increased flow in a pattern identical to the increased blood-pool activity. None of the metastatic, abscess, or cystic lesions had increased blood-pool activity at any time after injection. It is concluded that 99mTc red blood cell imaging can distinguish hemangiomas from other focal liver lesions.

  9. /sup 99m/Tc red blood cell scintigraphy in evaluating focal liver lesions

    SciTech Connect

    Rabinowitz, S.A.; McKusick, K.A.; Strauss, H.W.

    1984-07-01

    To determine the accuracy of blood-pool imaging in the diagnosis of hepatic hemangiomas, 39 patients with various focal hepatic lesions were studied. The diagnoses in these patients were made by biopsy, angiography, surgical exploration, or clinical stability for a minimum of 14 months. The diagnoses were: hemangiomas (13 patients), hepatoma (three), metastases (19), abscesses (two), and liver cysts (two). After modified in vivo labeling of red blood cells with 20 mCi (740 MBq) of /sup 99m/Tc pertechnetate, an initial flow study and early (1-15 min) and delayed (1-2 hr) static images were obtained. Increased blood-pool activity with a discordant flow pattern was seen in 11 of 13 patients with hemangiomas. False-negative scans occurred in two hemangiomas with extensive fibrosis. None of the metastatic, abscess, or cystic lesions had increased blood-pool activity at any time after injection. It is concluded that /sup 99m/Tc red blood cell imaging can distinguish hemangiomas from other focal liver lesions.

  10. Methods for quantitative detection of antibody-induced complement activation on red blood cells.

    PubMed

    Meulenbroek, Elisabeth M; Wouters, Diana; Zeerleder, Sacha

    2014-01-01

    Antibodies against red blood cells (RBCs) can lead to complement activation resulting in an accelerated clearance via complement receptors in the liver (extravascular hemolysis) or leading to intravascular lysis of RBCs. Alloantibodies (e.g. ABO) or autoantibodies to RBC antigens (as seen in autoimmune hemolytic anemia, AIHA) leading to complement activation are potentially harmful and can be - especially when leading to intravascular lysis - fatal(1). Currently, complement activation due to (auto)-antibodies on RBCs is assessed in vitro by using the Coombs test reflecting complement deposition on RBC or by a nonquantitative hemolytic assay reflecting RBC lysis(1-4). However, to assess the efficacy of complement inhibitors, it is mandatory to have quantitative techniques. Here we describe two such techniques. First, an assay to detect C3 and C4 deposition on red blood cells that is induced by antibodies in patient serum is presented. For this, FACS analysis is used with fluorescently labeled anti-C3 or anti-C4 antibodies. Next, a quantitative hemolytic assay is described. In this assay, complement-mediated hemolysis induced by patient serum is measured making use of spectrophotometric detection of the released hemoglobin. Both of these assays are very reproducible and quantitative, facilitating studies of antibody-induced complement activation. PMID:24514151

  11. From stem cell to red blood cells in vitro: "the 12 labors of Hercules".

    PubMed

    Douay, Luc

    2010-06-01

    This article describes the research in progress that will permit the large-scale production of human red blood cells from hematopoietic stem cells. It also discusses the current state of this research, suggests the obstacles to be overcome to pass from the laboratory model to clinical practice, and analyzes the possible indications in the medium and long term. The potential interest of pluripotent stem cells as an unlimited source of red blood cells is considered. If it succeeds, this new approach could mark a considerable advance in the field of transfusion. PMID:20513558

  12. Automatic analysis of microscopic images of red blood cell aggregates

    NASA Astrophysics Data System (ADS)

    Menichini, Pablo A.; Larese, Mónica G.; Riquelme, Bibiana D.

    2015-06-01

    Red blood cell aggregation is one of the most important factors in blood viscosity at stasis or at very low rates of flow. The basic structure of aggregates is a linear array of cell commonly termed as rouleaux. Enhanced or abnormal aggregation is seen in clinical conditions, such as diabetes and hypertension, producing alterations in the microcirculation, some of which can be analyzed through the characterization of aggregated cells. Frequently, image processing and analysis for the characterization of RBC aggregation were done manually or semi-automatically using interactive tools. We propose a system that processes images of RBC aggregation and automatically obtains the characterization and quantification of the different types of RBC aggregates. Present technique could be interesting to perform the adaptation as a routine used in hemorheological and Clinical Biochemistry Laboratories because this automatic method is rapid, efficient and economical, and at the same time independent of the user performing the analysis (repeatability of the analysis).

  13. Measurement of red blood cell mechanics during morphological changes

    NASA Astrophysics Data System (ADS)

    Popescu, Gabriel; Park, Yongkeun; Best, Catherine; Dasari, Ramachandra; Feld, Michael; Kuriabova, Tatiana; Henle, Mark; Levine, Alex

    2010-03-01

    The human red blood cell (RBC) membrane, a fluid lipid bilayer tethered to an elastic 2D spectrin network, provides the principal control of the cell's morphology and mechanics. These properties, in turn, influence the ability of RBCs to transport oxygen in circulation. Current mechanical measurements of RBCs rely on external loads. Here we apply a Noncontact optical interferometric technique to quantify the thermal fluctuations of RBC membranes with 3 nm accuracy over a broad range of spatial and temporal frequencies. Combining this technique with a new mathematical model describing RBC membrane undulations, we measure the mechanical changes of RBCs as they undergo a transition from the normal discoid shape to the abnormal echinocyte and spherical shapes. These measurements indicate that, coincident with this morphological transition, there is a significant increase in the membrane's shear and bending moduli. This mechanical transition can alter cell circulation and impede oxygen delivery.

  14. Depletion induced clustering of red blood cells in microchannels

    NASA Astrophysics Data System (ADS)

    Wagner, Christian; Brust, Mathias; Podgorski, Thomas; Coupier, Gwennou

    2012-11-01

    The flow properties of blood are determined by the physical properties of its main constituents, the red blood cells (RBC's). At low shear rates RBC's form aggregates, so called rouleaux. Higher shear rates can break them up and the viscosity of blood shows a shear thinning behavior. The physical origin of the rouleaux formation is not yet fully resolved and there are two competing models available. One predicts that the adhesion is induced by bridging of the plasma (macromolecular) proteins in-between two RBC's. The other is based on the depletion effect and thus predicts the absence of macromolecules in-between the cells of a rouleaux. Recent single cell force measurements by use of an AFM support strongly the depletion model. By varying the concentration of Dextran at different molecular weights we can control the adhesions strength. Measurements at low hematocrit in a microfluidic channel show that the number of size of clusters is determined by the depletion induced adhesion strength.

  15. Transport of diseased red blood cells in the spleen

    NASA Astrophysics Data System (ADS)

    Peng, Zhangli; Pivkin, Igor; Dao, Ming

    2012-11-01

    A major function of the spleen is to remove old and diseased red blood cells (RBCs) with abnormal mechanical properties. We investigated this mechanical filtering mechanism by combining experiments and computational modeling, especially for red blood cells in malaria and sickle cell disease (SCD). First, utilizing a transgenic line for 3D confocal live imaging, in vitro capillary assays and 3D finite element modeling, we extracted the mechanical properties of both the RBC membrane and malaria parasites for different asexual malaria stages. Secondly, using a non-invasive laser interferometric technique, we optically measured the dynamic membrane fluctuations of SCD RBCs. By simulating the membrane fluctuation experiment using the dissipative particle dynamics (DPD) model, we retrieved mechanical properties of SCD RBCs with different shapes. Finally, based on the mechanical properties obtained from these experiments, we simulated the full fluid-structure interaction problem of diseased RBCs passing through endothelial slits in the spleen under different fluid pressure gradients using the DPD model. The effects of the mechanical properties of the lipid bilayer, the cytoskeleton and the parasite on the critical pressure of splenic passage of RBCs were investigated separately. This work is supported by NIH and Singapore-MIT Alliance for Science and Technology (SMART).

  16. Vibrational modes of hemoglobin in red blood cells.

    PubMed Central

    Martel, P; Calmettes, P; Hennion, B

    1991-01-01

    Equine red blood cells were washed in saline heavy water (2H2O) to exchange the hydrogen atoms of the non-hemoglobin components with deuterons. This led to novel neutron scattering measurements of protein vibrations within a cellular system and permitted a comparison with inelastic neutron scattering measurements on purified horse hemoglobin, either dry or wetted with 2H2O. As a function of wavevector transfer Q and the frequency transfer v the neutron response typified by the dynamic structure factor S(Q, v) was found to be similar for extracted and cellular hemoglobin at low and high temperatures. At 77 K, in the cells, a peak in S(Q, v) due to the protein was found near 0.7 THz, approximately half the frequency of a strong peak in the aqueous medium. Measurements at higher temperatures (170 and 230 K) indicated similar small shifts downwards in the peak frequencies of both components. At 260 K the low frequency component became predominantly quasielastic, but a significant inelastic component could still be ascribed to the aqueous scattering. Near 295 K the frequency responses of both components were similar and centered near zero. When scattering due to water is taken into account it appears that the protein neutron response in, or out of, red blood cells is little affected by hydration in the low frequency regime where Van der Waals forces are thought to be effective. PMID:1849028

  17. Pure red cell aplasia induced by epoetin zeta.

    PubMed

    Panichi, Vincenzo; Ricchiuti, Guido; Scatena, Alessia; Del Vecchio, Lucia; Locatelli, Francesco

    2016-08-01

    Pure red cell aplasia (PRCA) may develop in patients with chronic kidney disease receiving erythropoiesis-stimulating agents (ESA). We report on a 72-year-old patient who developed hypo-proliferative anaemia unresponsive to ESA following the administration of epoetin zeta subcutaneously for 7 months. On the basis of severe isolated hypoplasia of the erythroid line in the bone marrow and high-titre neutralizing anti-erythropoietin antibodies (Ab), a diagnosis of Ab-mediated PRCA was made. Epoetin zeta was discontinued and the patient was given steroids. This was associated with anaemia recovery. To our knowledge this is the first PRCA case related to epoetin zeta. PMID:27478604

  18. The nature of multiphoton fluorescence from red blood cells

    NASA Astrophysics Data System (ADS)

    Saytashev, Ilyas; Murphy, Michael; Osseiran, Sam; Spence, Dana M.; Evans, Conor L.; Dantus, Marcos

    2016-03-01

    We report on the nature of multiphoton excited fluorescence observed from human erythrocytes (red blood cells RBC's) and their "ghosts" following 800nm sub-15 fs excitation. The detected optical signal is assigned as two-photon excited fluorescence from hemoglobin. Our findings are supported by wavelength-resolved fluorescence lifetime decay measurements using time-correlated single photon counting system from RBC's, their ghosts as well as in vitro samples of various fluorophores including riboflavin, NADH, NAD(P)H, hemoglobin. We find that low-energy and short-duration pulses allow two-photon imaging of RBC's, but longer more intense pulses lead to their destruction.

  19. Pure red cell aplasia induced by epoetin zeta

    PubMed Central

    Panichi, Vincenzo; Ricchiuti, Guido; Scatena, Alessia; Del Vecchio, Lucia; Locatelli, Francesco

    2016-01-01

    Pure red cell aplasia (PRCA) may develop in patients with chronic kidney disease receiving erythropoiesis-stimulating agents (ESA). We report on a 72-year-old patient who developed hypo-proliferative anaemia unresponsive to ESA following the administration of epoetin zeta subcutaneously for 7 months. On the basis of severe isolated hypoplasia of the erythroid line in the bone marrow and high-titre neutralizing anti-erythropoietin antibodies (Ab), a diagnosis of Ab-mediated PRCA was made. Epoetin zeta was discontinued and the patient was given steroids. This was associated with anaemia recovery. To our knowledge this is the first PRCA case related to epoetin zeta. PMID:27478604

  20. Cytoskeleton confinement of red blood cell membrane fluctuations

    NASA Astrophysics Data System (ADS)

    Gov, Nir; Zilman, Anton; Safran, Samuel

    2003-03-01

    We analyze theoretically both the static and dynamic fluctuation spectrum of the red-blood cell in a unified manner, using a simple model of the composite membrane. In this model, the two-dimensional spectrin network that forms the cytoskeleton is treated as a rigid shell, located at a fixed, average distance from the lipid bilayer. The cytoskeleton thereby confines both the static and dynamic fluctuations of the lipid bilayer. The predictions of the model account for the wavevector and frequency dependence of the experimental data.

  1. Cytoskeleton Confinement and Tension of Red Blood Cell Membranes

    NASA Astrophysics Data System (ADS)

    Gov, N.; Zilman, A. G.; Safran, S.

    2003-06-01

    We analyze theoretically both the static and dynamic fluctuation spectra of the red blood cell in a unified manner, using a simple model of the composite membrane. In this model, the two-dimensional spectrin network that forms the cytoskeleton is treated as a rigid shell, located at a fixed, average distance from the lipid bilayer. The cytoskeleton thereby confines both the static and dynamic fluctuations of the lipid bilayer. The sparse connections of the cytoskeleton and bilayer induce a surface tension, for wavelengths larger than the bilayer persistence length. The predictions of the model give a consistent account for both the wave vector and frequency dependence of the experimental data.

  2. Aptamer-mediated indirect quantum dot labeling and fluorescent imaging of target proteins in living cells

    NASA Astrophysics Data System (ADS)

    Liu, Jianbo; Zhang, Pengfei; Yang, Xiaohai; Wang, Kemin; Guo, Qiuping; Huang, Jin; Li, Wei

    2014-12-01

    Protein labeling for dynamic living cell imaging plays a significant role in basic biological research, as well as in clinical diagnostics and therapeutics. We have developed a novel strategy in which the dynamic visualization of proteins within living cells is achieved by using aptamers as mediators for indirect protein labeling of quantum dots (QDs). With this strategy, the target protein angiogenin was successfully labeled with fluorescent QDs in a minor intactness model, which was mediated by the aptamer AL6-B. Subsequent living cell imaging analyses indicated that the QDs nanoprobes were selectively bound to human umbilical vein endothelial cells, gradually internalized into the cytoplasm, and mostly localized in the lysosome organelle, indicating that the labeled protein retained high activity. Compared with traditional direct protein labeling methods, the proposed aptamer-mediated strategy is simple, inexpensive, and provides a highly selective, stable, and intact labeling platform that has shown great promise for future biomedical labeling and intracellular protein dynamic analyses.

  3. Red blood cell orientation in orbit C = 0.

    PubMed Central

    Bitbol, M

    1986-01-01

    Two modes of behavior of single human red cells in a shear field have been described. It is known that in low viscosity media and at shear rates less than 20 s-1, the cells rotate with a periodically varying angular velocity, in accord with the theory of Jeffery (1922) for oblate spheroids. In media of viscosity greater than approximately 5 mPa s and sufficiently high shear rates, the cells align themselves at a constant angle to the direction of flow with the membrane undergoing tank-tread motion. Also, in low viscosity media, as the shear rate is increased, more and more cells lie in the plane of shear, undergoing spin with their axes of symmetry aligned with the vorticity axis of the shear field in an orbit "C = 0" (Goldsmith and Marlow, 1972). We have explored this latter phenomenon using two experimental methods. First, the erythrocytes were observed in the rheoscope and their diameters measured. Forward light scattering patterns were correlated with the red cell orientation mode. Light flux variations after flow onset or stop were measured, and the characteristic times of erythrocyte orientation and disorientation were assessed. The characteristic time of erythrocyte orientation in Orbit C = 0 is proportional to the inverse of the shear rate. The corresponding coefficient of proportionality depends on the suspending medium viscosity eta o. The disorientation time tau D, after flow has been stopped, is such that the ratio tau D/eta o is independent of the initial applied shear stress. However, tau D is much shorter than one would expect if pure Brownian motion were involved. The proportion of erythrocytes in orbit C = 0 was also measured. It was found that this proportion is a function of both the shear rate and eta o. At low values of eta o, the proportion increases with increasing shear rate and then reaches a plateau. For higher values of eta o (5 to 10 mPa s), the proportion of RBC in orbit C = 0 is a decreasing function of the shear stress. A critical

  4. Blood volume and red cell mass in children with moderate and severe malaria measured by chromium-53 dilution and gas chromatography/mass spectrometric analysis.

    PubMed

    Macallan, Derek C; Abaye, Daniel A; Dottin, Simone; Onanga, Myriam; Kombila, Maryvonne; Dzeing-Ella, Arnaud; Kremsner, Peter G; Krishna, Sanjeev; Planche, Timothy

    2009-08-30

    Understanding blood volume changes in children with malaria is important for managing fluid status. Traditionally, blood/red cell volume measurements have used radioactive chromium isotopes. We applied an alternative approach, using non-radioactive chromium-53 labelling and mass spectrometry to investigate red cell volume (RCV) in Gabonese children with malaria. Nineteen children with malaria participated (10 severe, 9 moderately severe; ages 15 months to 7 years). Blood labelled with (53)Cr-chromate ex vivo was re-injected, then sampled 30 min later. Pre- and post-injection (53)Cr content were measured by gas chromatography/electron ionisation mass spectrometry of the chromium-trifluoroacetylacetone (TFA) chelate, calibrated against (50)Cr standards. Blood and red cell volumes were calculated from isotopic dilution in 15 of 19 children (in four, insufficient signal mitigated analysis). In this small pilot study, there were no significant differences between moderate and severe cases. Including all subjects, the mean RCV was reduced compared with predicted values (184 vs. 269 mL; p = 0.016) but blood volume, 71 +/- 33 mL/kg (normalised for weight), was close to predicted, approximately 77 mL/kg, commensurate with reduced haematocrit. Blood lactate concentration correlated negatively with RCV/weight (r = -0.56, p = 0.028), consistent with anaemia. In one case, sequential samples over 42 days gave an estimated rate of (53)Cr disappearance of 1.4%/day (equivalent half-life: 70 days). (53)Cr-labelling of red cells may be used to estimate blood and red cell volumes and can be used as an investigative tool in situations such as childhood diseases and resource-constrained settings. Although the red cell mass is depleted in malaria, the blood volume appears relatively well preserved. PMID:19603467

  5. [Red Blood Cells Raman Spectroscopy Comparison of Type Two Diabetes Patients and Rats].

    PubMed

    Wang, Lei; Liu, Gui-dong; Mu, Xin; Xiao, Hong-bin; Qi, Chao; Zhang, Si-qi; Niu Wen-ying; Jiang, Guang-kun; Feng, Yue-nan; Bian, Jing-qi

    2015-10-01

    By using confocal Raman spectroscopy, Raman spectra were measured in normal rat red blood cells, normal human red blood cells, STZ induced diabetetic rats red blood cells, Alloxan induced diabetetic rats red blood cells and human type 2 diabetes red blood cells. Then principal component analysis (PCA) with support vector machine (SVM) classifier was used for data analysis, and then the distance between classes was used to judge the degree of close to two kinds of rat model with type 2 diabetes. The results found significant differences in the Raman spectra of red blood cell in diabetic and normal red blood cells. To diabetic red blood cells, the peak in the amide VI C=O deformation vibration band is obvious, and amide V N-H deformation vibration band spectral lines appear deviation. Belong to phospholipid fatty acyl C-C skeleton, the 1 130 cm(-1) spectral line is enhanced and the 1 088 cm(-1) spectral line is abated, which show diabetes red cell membrane permeability increased. Raman spectra of PCA combined with SVM can well separate 5 types of red blood cells. Classifier test results show that the classification accuracy is up to 100%. Through the class distance between the two induced method and human type 2 diabetes, it is found that STZ induced model is more close to human type 2 diabetes. In conclusion, Raman spectroscopy can be used for diagnosis of diabetes and rats STZ induced diabetes method is closer to human type 2 diabetes. PMID:26904817

  6. Bio-inspired Cryo-ink Preserves Red Blood Cell Phenotype and Function during Nanoliter Vitrification

    PubMed Central

    Assal, Rami El; Guven, Sinan; Gurkan, Umut Atakan; Gozen, Irep; Shafiee, Hadi; Dalbeyber, Sedef; Abdalla, Noor; Thomas, Gawain; Fuld, Wendy; Illigens, Ben M.W.; Estanislau, Jessica; Khoory, Joseph; Kaufman, Richard; Zylberberg, Claudia; Lindeman, Neal; Wen, Qi; Ghiran, Ionita; Demirci, Utkan

    2014-01-01

    Current red blood cell cryopreservation methods utilize bulk volumes, causing cryo-injury of cells, which results in irreversible disruption of cell morphology, mechanics, and function. An innovative approach to preserve human red blood cell morphology, mechanics, and function following vitrification in nanoliter volumes is developed using a novel cryo-ink integrated with a bio-printing approach. PMID:25047246

  7. Multiplexed labeling system for high-throughput cell sorting.

    PubMed

    Shin, Seung Won; Park, Kyung Soo; Song, In Hyun; Shin, Woo Jung; Kim, Byung Woo; Kim, Dong-Ik; Um, Soong Ho

    2016-09-01

    Flow cytometry and fluorescence activated cell sorting techniques were designed to realize configurable classification and separation of target cells. A number of cell phenotypes with different functionalities have recently been revealed. Before simultaneous selective capture of cells, it is desirable to label different samples with the corresponding dyes in a multiplexing manner to allow for a single analysis. However, few methods to obtain multiple fluorescent colors for various cell types have been developed. Even when restricted laser sources are employed, a small number of color codes can be expressed simultaneously. In this study, we demonstrate the ability to manifest DNA nanostructure-based multifluorescent colors formed by a complex of dyes. Highly precise self-assembly of fluorescent dye-conjugated oligonucleotides gives anisotropic DNA nanostructures, Y- and tree-shaped DNA (Y-DNA and T-DNA, respectively), which may be used as platforms for fluorescent codes. As a proof of concept, we have demonstrated seven different fluorescent codes with only two different fluorescent dyes using T-DNA. This method provides maximum efficiency for current flow cytometry. We are confident that this system will provide highly efficient multiplexed fluorescent detection for bioanalysis compared with one-to-one fluorescent correspondence for specific marker detection. PMID:27181032

  8. A system for counting fetal and maternal red blood cells.

    PubMed

    Ge, Ji; Gong, Zheng; Chen, Jun; Liu, Jun; Nguyen, John; Yang, Zongyi; Wang, Chen; Sun, Yu

    2014-12-01

    The Kleihauer-Betke (KB) test is the standard method for quantitating fetal-maternal hemorrhage in maternal care. In hospitals, the KB test is performed by a certified technologist to count a minimum of 2000 fetal and maternal red blood cells (RBCs) on a blood smear. Manual counting suffers from inherent inconsistency and unreliability. This paper describes a system for automated counting and distinguishing fetal and maternal RBCs on clinical KB slides. A custom-adapted hardware platform is used for KB slide scanning and image capturing. Spatial-color pixel classification with spectral clustering is proposed to separate overlapping cells. Optimal clustering number and total cell number are obtained through maximizing cluster validity index. To accurately identify fetal RBCs from maternal RBCs, multiple features including cell size, roundness, gradient, and saturation difference between cell and whole slide are used in supervised learning to generate feature vectors, to tackle cell color, shape, and contrast variations across clinical KB slides. The results show that the automated system is capable of completing the counting of over 60,000 cells (versus ∼2000 by technologists) within 5 min (versus ∼15 min by technologists). The throughput is improved by approximately 90 times compared to manual reading by technologists. The counting results are highly accurate and correlate strongly with those from benchmarking flow cytometry measurement. PMID:24879644

  9. ESR measurement of time-dependent and equilibrium volumes in red cells.

    PubMed

    Moronne, M M; Mehlhorn, R J; Miller, M P; Ackerson, L C; Macey, R I

    1990-04-01

    Red cell water volumes were measured using ESR methods during transient osmotic perturbation, and under equilibrium conditions. Cell water contents were determined using the spin label Tempone (2,2,6,6-tetramethyl piperidine-N-oxyl) and the membrane impermeable quencher potassium chromium oxalate. With appropriate corrections for intracellular viscosity and changes in cavity sensitivity, equilibrium cell water measured both by electron spin resonance (ESR) and wet minus dry weight methods gave excellent agreement in solutions from 243-907 mOsm. Intracellular viscosities determined from the Tempone correlation times in the same cells gave values ranging from 9-47 centipoise at 21 degrees C. Osmotically induced transient volume changes were measured using Tempone and an ESR stopped-flow configuration. The Tempone response time was estimated at 17 msec compared to 250-350 msec for normal water relaxations. Nonlinear least square solutions to the Kedem-Katchalsky equations including a correction for the finite Tempone permeability gave 0.029 and 0.030 cm/sec for the osmotic permeability of RBCs in swell and shrink experiments, respectively. In stopped-flow experiments accurate water flux data are obtained very soon after challenging cells and do not require baseline subtractions. These results represent significant improvements over conventional light scattering techniques which necessitate corrections for long lasting optical artifacts (200-300 msec), and baseline drifts. PMID:2159519

  10. Red blood cell distribution width and cardiovascular diseases

    PubMed Central

    Danese, Elisa; Lippi, Giuseppe

    2015-01-01

    Background The red blood cell distribution width (RDW) is a rather simple measure of red blood cell (RBC) size heterogeneity (i.e., anisocytosis), which is easily calculated by dividing the standard deviation (SD) of erythrocyte volumes for the mean corpuscular volume (MCV). Emerging evidence suggests that, besides RBC abnormalities, many human disorders may be frequently associated with a high degree of anisocytosis. Methods In this narrative review, we analyzed the current scientific literature about the putative role and the potential epidemiologic association between RDW and cardiovascular diseases. The findings of the most representative epidemiological studies were summarized and discussed. Results Overall, considerable and convincing evidence has been brought that an increased RDW value is associated with acute coronary syndrome (ACS) [including acute myocardial infarction (AMI)], ischemic cerebrovascular disease (including stroke), peripheral artery disease (PAD), as well as with atrial fibrillation (AF), heart failure (HF) and hypertension. Higher anisocytosis also significantly and independently predicts adverse outcomes in patients with these conditions. Conclusions Although the role of anisocytosis in the pathogenesis of cardiovascular diseases remains uncertain, the considerable evidence available so far suggests that the clinical use of RDW may be broadened beyond the conventional boundaries of erythrocyte disorders, in particular for assisting the diagnosis and prognostication of patients with ACS, ischemic cerebrovascular disease, PAD, HF and AF. PMID:26623117

  11. Existence of a Flat Phase in Red Cell Membrane Skeletons

    NASA Astrophysics Data System (ADS)

    Schmidt, Christoph F.; Svoboda, Karel; Lei, Ning; Petsche, Irena B.; Berman, Lonny E.; Safinya, Cyrus R.; Grest, Gary S.

    1993-02-01

    Biomolecular membranes display rich statistical mechanical behavior. They are classified as liquid in the absence of shear elasticity in the plane of the membrane and tethered (solid) when the neighboring molecules or subunits are connected and the membranes exhibit solid-like elastic behavior in the plane of the membrane. The spectrin skeleton of red blood cells was studied as a model tethered membrane. The static structure factor of the skeletons, measured by small-angle x-ray and light scattering, was fitted with a structure factor predicted with a model calculation. The model describes tethered membrane sheets with free edges in a flat phase, which is a locally rough but globally flat membrane configuration. The fit was good for large scattering vectors. The membrane roughness exponent, zeta, defined through h propto L^zeta, where h is the average amplitude of out-of-plane fluctuations and L is the linear membrane dimension, was determined to be 0.65 ± 0.10. Computer simulations of model red blood cell skeletons also showed this flat phase. The value for the roughness exponent, which was determined from the scaling properties of membranes of different sizes, was consistent with that from the experiments.

  12. Superparamagnetic iron oxide nanoparticles for direct labeling of stem cells and in vivo MRI tracking.

    PubMed

    Kim, Saejeong J; Lewis, Bobbi; Steiner, Mark-Steven; Bissa, Ursula V; Dose, Christian; Frank, Joseph A

    2016-01-01

    To develop effective stem cell therapies, it is important to track therapeutic cells non-invasively and monitor homing to areas of pathology. The purpose of this study was to design and evaluate the labeling efficiency of commercially available dextran-coated superparamagnetic iron oxide nanoparticles, FeraTrack Direct (FTD), in various stem and immune cells; assess the cytotoxicity and tolerability of the FTD in stem cells; and monitor stem cell homing using FTD-labeled bone-marrow-derived mesenchymal stromal cells (BMSCs) and neural stem cells (NSCs) in a tumor model by in vivo MRI. BMSCs, NSCs, hematopoietic stem cells (HSCs), T-lymphocytes, and monocytes were labeled effectively with FTD without the need for transfection agents, and Prussian blue (PB) staining and transmission electron microscopy (TEM) confirmed intracellular uptake of the agent. The viability, proliferation, and functionality of the labeled cells were minimally or not affected after labeling. When 10(6) FTD-labeled BMSCs or NSCs were injected into C6 glioma bearing nude mice, the cells homing to the tumors were detected as hypointense regions within the tumor using 3 T clinical MRI up to 10 days post injection. Histological analysis confirmed the homing of injected cells to the tumor by the presence of PB positive cells that are not macrophages. Labeling of stem cells or immune cells with FTD was non-toxic, and should facilitate the translation of this agent to clinical trials for evaluation of trafficking of cells by MRI. PMID:26234504

  13. Flow cytometry-based characterization of label-retaining stem cells following transplacental BrdU labelling.

    PubMed

    Poojan, Shiv; Kumar, Sushil

    2011-02-01

    A method to characterize and culture stem cells from neonate mouse epidermis after transplacental BrdU (bromo-deoxyuridine) administration is described. We have characterized stem cells by their properties viz. to retain BrdU label, adhere rapidly onto collagen-fibronectin substratum and express a specific biomarker beta-1-integrin. BrdU-labelled cells (detected using monoclonal antibody) constituted a sum of 18% of the total number of cells. The ability of freshly isolated keratinocytes [LRCs (label-retaining cells)] to bind to primary BrdU antibody or to pick up PI (propidium iodide) stain was distinguishable. Viable LRCs did not retain PI. Such cells, termed EpSC (epidermis stem cell), were PI negative and BrdU positive. EpSC constituted 6% of the total cell yield. Culture in low Ca2+ medium and susceptibility to differentiation in the presence of high Ca2+ levels further characterized the stem cells. This protocol is useful for studying transplacental carcinogenesis. PMID:21261598

  14. Interaction of injectable neurotropic drugs with the red cell membrane.

    PubMed

    Reinhart, Walter H; Lubszky, Szabina; Thöny, Sandra; Schulzki, Thomas

    2014-10-01

    The normal red blood cell (RBC) shape is a biconcave discocyte. An intercalation of a drug in the outer half of the membrane lipid bilayer leads to echinocytosis, an intercalation in the inner half to stomatocytosis. We have used the shape transforming capacity of RBCs as a model to analyse the membrane interaction potential of various neurotropic drugs. Chlorpromazine, clomipramine, citalopram, clonazepam, and diazepam induced a reversible stomatocytosis, phenytoin induced echinocytosis, while the anticonvulsants levetiracetam, valproic acid and phenobarbital had no effect. This diversity of RBC shape transformations suggests that the pharmacological action is not linked to the membrane interaction. We conclude that this simple RBC shape transformation assay could be a useful tool to screen for potential drug interactions with cell membranes. PMID:24997296

  15. Anisotropic light scattering of individual sickle red blood cells

    NASA Astrophysics Data System (ADS)

    Kim, Youngchan; Higgins, John M.; Dasari, Ramachandra R.; Suresh, Subra; Park, YongKeun

    2012-04-01

    We present the anisotropic light scattering of individual red blood cells (RBCs) from a patient with sickle cell disease (SCD). To measure light scattering spectra along two independent axes of elongated-shaped sickle RBCs with arbitrary orientation, we introduce the anisotropic Fourier transform light scattering (aFTLS) technique and measured both the static and dynamic anisotropic light scattering. We observed strong anisotropy in light scattering patterns of elongated-shaped sickle RBCs along its major axes using static aFTLS. Dynamic aFTLS analysis reveals the significantly altered biophysical properties in individual sickle RBCs. These results provide evidence that effective viscosity and elasticity of sickle RBCs are significantly different from those of the healthy RBCs.

  16. Measurement of the nonlinear elasticity of red blood cell membranes

    NASA Astrophysics Data System (ADS)

    Park, Yongkeun; Best, Catherine A.; Kuriabova, Tatiana; Henle, Mark L.; Feld, Michael S.; Levine, Alex J.; Popescu, Gabriel

    2011-05-01

    The membranes of human red blood cells (RBCs) are a composite of a fluid lipid bilayer and a triangular network of semiflexible filaments (spectrin). We perform cellular microrheology using the dynamic membrane fluctuations of the RBCs to extract the elastic moduli of this composite membrane. By applying known osmotic stresses, we measure the changes in the elastic constants under imposed strain and thereby determine the nonlinear elastic properties of the membrane. We find that the elastic nonlinearities of the shear modulus in tensed RBC membranes can be well understood in terms of a simple wormlike chain model. Our results show that the elasticity of the spectrin network can mostly account for the area compression modulus at physiological osmolality, suggesting that the lipid bilayer has significant excess area. As the cell swells, the elastic contribution from the now tensed lipid membrane becomes dominant.

  17. Red Blood Cells Motion in a Glass Microchannel

    NASA Astrophysics Data System (ADS)

    Pinho, Diana; Pereira, Ana I.; Lima, Rui

    2010-09-01

    The motion of the red blood cells (RBCs) flowing in microvessels and microchannels depend on several effects, such as hematocrit (Hct), geometry, and temperature. According to our knowledge, the effect of the temperature on RBC motion was never investigated at a microscale level. Hence, the aim of the present work is to determine the effect of the temperature on the RBC's trajectories and to investigate the best approximation of the trajectories through a nonlinear optimization. In vitro human blood was pumped through a 100 μm circular microchannel and by using a confocal micro-PTV system the RBC's trajectories were measured at different temperatures, i.e., 25° C and 37° C. In this study we measured the motion of forty cells flowing in the middle of the microchannel and applied different functions to approximate its behavior.

  18. Adipose derived stem cells: efficiency, toxicity, stability of BrdU labeling and effects on self-renewal and adipose differentiation.

    PubMed

    Lequeux, Charlotte; Oni, Georgette; Mojallal, Ali; Damour, Odile; Brown, Spencer A

    2011-05-01

    5-bromo-2-deoxyurudine (BrdU) can be used as a methodological tool for in vivo investigations following in vitro prelabeling of isolated stem cells for subsequent cell tracking within the recipient host. The objective of this study was to determine how useful BrdU may be as a labeling modality for adipose derived stem cells (ASC) by examining BrdU toxicity, BrdU intracellular stability, and potential effects on ASC differentiation. Porcine and human ASC (pASC and hASC, respectively) were labeled with BrdU at 5 or 10 μM for 2, 6, 24, and 48 h. BrdU toxicity and stability over time in monolayer cultures, in 3-D collagen scaffolds implanted to a porcine model and after thawing from long-term storage were evaluated by MTT assays and immunohistochemistry. ASC differentiation was evaluated by Oil Red O staining. BrdU was not cytotoxic at all tested concentrations and incubation times. BrdU color intensity within each cell and the number of ASC labeled with BrdU decreased as a function of both incubation time and BrdU concentrations. Labeling intensities decreased over time and were undetectable after 6 passages for pASC and 4 passages for hASC. In 3-D scaffolds, BrdU-labeled ASC were identifiable after 90 days of in vitro cultures and for 30 days in a porcine model. BrdU did not prevent preadipocyte differentiation and BrdU labeling was still detectable after subsequent thawing after long-term storage of ASC. BrdU is an excellent candidate reagent to label and track ASC that will allow distinction between BrdU-labeled donor cells and host cells. The data provides a foundation for conducting future tissue engineering projects using BrdU-labeled ASC. PMID:21246262

  19. Genetic Method for Labeling Electrically Coupled Cells: Application to Retina

    PubMed Central

    Qiao, Mu; Sanes, Joshua R.

    2016-01-01

    Understanding how the nervous system functions requires mapping synaptic connections between neurons. Several methods are available for imaging neurons connected by chemical synapses, but few enable marking neurons connected by electrical synapses. Here, we demonstrate that a peptide transporter, Pept2, can be used for this purpose. Pept2 transports a gap junction-permeable fluorophore-coupled dipeptide, beta-alanine-lysine-N-7-amino-4-methyl coumarin-3-acid (βALA). Cre-dependent expression of pept2 in specific neurons followed by incubation in βALA labeled electrically coupled synaptic partners. Using this method, we analyze light-dependent modulation of electrical connectivity among retinal horizontal cells. PMID:26778956

  20. Long-term stem cell labeling by collagen-functionalized single-walled carbon nanotubes

    NASA Astrophysics Data System (ADS)

    Mao, Hongli; Cai, Rong; Kawazoe, Naoki; Chen, Guoping

    2014-01-01

    The monitoring of grafted stem cells is crucial to assess the efficiency, effectiveness and safety of such stem cell-based therapies. In this regard, a reliable and cytocompatible labeling method for stem cells is critically needed. In this study, the collagen-functionalized single-walled carbon nanotubes (Col-SWCNTs) were used as imaging probes for labeling of human mesenchymal stem cells (hMSCs) and the inherent Raman scattering of SWCNTs was used to image the SWCNT-labeled cells. The results showed that the Col-SWCNTs exhibit efficient cellular internalization by hMSCs without affecting their proliferation and differentiation. The prolonged dwell time of Col-SWCNTs in cells ensured the long-term labeling for up to 2 weeks. This work reveals the potential of Col-SWCNTs as probes for long-term stem cell labeling.

  1. Stretching and relaxation of malaria-infected red blood cells.

    PubMed

    Ye, Ting; Phan-Thien, Nhan; Khoo, Boo Cheong; Lim, Chwee Teck

    2013-09-01

    The invasion of red blood cells (RBCs) by malaria parasites is a complex dynamic process, in which the infected RBCs gradually lose their deformability and their ability to recover their original shape is greatly reduced with the maturation of the parasites. In this work, we developed two types of cell model, one with an included parasite, and the other without an included parasite. The former is a representation of real malaria-infected RBCs, in which the parasite is treated as a rigid body. In the latter, where the parasite is absent, the membrane modulus and viscosity are elevated so as to produce the same features present in the parasite model. In both cases, the cell membrane is modeled as a viscoelastic triangular network connected by wormlike chains. We studied the transient behaviors of stretching deformation and shape relaxation of malaria-infected RBCs based on these two models and found that both models can generate results in agreement with those of previously published studies. With the parasite maturation, the shape deformation becomes smaller and smaller due to increasing cell rigidity, whereas the shape relaxation time becomes longer and longer due to the cell's reduced ability to recover its original shape. PMID:24010653

  2. IMAGING RED BLOOD CELL DYNAMICS BY QUANTITATIVE PHASE MICROSCOPY

    PubMed Central

    Popescu, Gabriel; Park, YoungKeun; Choi, Wonshik; Dasari, Ramachandra R.; Feld, Michael S.; Badizadegan, Kamran

    2008-01-01

    Red blood cells (RBCs) play a crucial role in health and disease, and structural and mechanical abnormalities of these cells have been associated with important disorders such as Sickle cell disease and hereditary cytoskeletal abnormalities. Although several experimental methods exist for analysis of RBC mechanical properties, optical methods stand out as they enable collecting mechanical and dynamic data from live cells without physical contact and without the need for exogenous contrast agents. In this report, we present quantitative phase microscopy techniques that enable imaging RBC membrane fluctuations with nanometer sensitivity at arbitrary time scales from milliseconds to hours. We further provide a theoretical framework for extraction of membrane mechanical and dynamical properties using time series of quantitative phase images. Finally, we present an experimental approach to extend quantitative phase imaging to 3-dimensional space using tomographic methods. By providing non-invasive methods for imaging mechanics of live cells, these novel techniques provide an opportunity for high-throughput analysis and study of RBC mechanical properties in health and disease. PMID:18387320

  3. Measurement of interaction forces between red blood cells in aggregates by optical tweezers

    SciTech Connect

    Maklygin, A Yu; Priezzhev, A V; Karmenian, A; Nikitin, Sergei Yu; Obolenskii, I S; Lugovtsov, Andrei E; Kisun Li

    2012-06-30

    We have fabricated double-beam optical tweezers and demonstrated the possibility of their use for measuring the interaction forces between red blood cells (erythrocytes). It has been established experimentally that prolonged trapping of red blood cells in a tightly focused laser beam does not cause any visible changes in their shape or size. We have measured the interaction between red blood cells in the aggregate, deformed by optical tweezers.

  4. Research opportunities in loss of red blood cell mass in space flight

    NASA Technical Reports Server (NTRS)

    Talbot, J. M.; Fisher, K. D.

    1985-01-01

    Decreases of red blood cell mass and plasma volume have been observed consistently following manned space flights. Losses of red cell mass by United States astronauts have averaged 10 to 15% (range: 2 to 21%). Based on postflight estimates of total hemoglobin, Soviet cosmonauts engaged in space missions lasting from 1 to 7 months have exhibited somewhat greater losses. Restoration of red cell mass requires from 4 to 6 weeks following return to Earth, regardless of the duration of space flight.

  5. Measuring skewness of red blood cell deformability distribution by laser ektacytometry

    SciTech Connect

    Nikitin, S Yu; Priezzhev, A V; Lugovtsov, A E; Ustinov, V D

    2014-08-31

    An algorithm is proposed for measuring the parameters of red blood cell deformability distribution based on laser diffractometry of red blood cells in shear flow (ektacytometry). The algorithm is tested on specially prepared samples of rat blood. In these experiments we succeeded in measuring the mean deformability, deformability variance and skewness of red blood cell deformability distribution with errors of 10%, 15% and 35%, respectively. (laser biophotonics)

  6. A model for oxygen-dependent backscattering spectroscopic contrast from single red blood cells (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Liu, Rongrong; Yi, Ji; Chen, Siyu; Zhang, Hao F.; Backman, Vadim

    2016-03-01

    The oxygen-dependent absorption of hemoglobin provides the fundamental contrast for all label-free techniques measuring blood oxygenation. When hemoglobin is packaged into red blood cells (RBCs), the structure of the cells creates light scattering which also depends on the absorption based on the Kramers-Kronig relationship. Thus a proper characterization of the optical behaviors of blood has been a key to any accurate measurement of blood oxygenation, particularly at the capillary level where RBCs are dispersed individually in contrast to a densely packed whole blood. Here we provided a theoretical model under Born Approximation to characterize the oxygen dependent backscattering spectroscopic contrast from single RBCs. Using this theoretical model, we conducted simulations on both oxygenated and deoxygenated single RBCs with different sizes for standard and possible deformed cell geometries in blood flow, all which suggested similar backscattering spectroscopic contrast and were confirmed by Mie Theory and experiments using visible Optical Coherence Tomography (visOCT). As long as the cell size satisfies Gaussian distribution with a coefficient variance (C.V.) large enough, there is clear absorption contrast between the backscattering spectra of oxygenated and deoxygenated single RBCs calculated by this model, so oxygen saturation can then be characterized. Thus, this theoretical model can be extended to extract absorption features of other scattering particles as long as they satisfy Born Approximation.

  7. Thyroid hormone stimulation in vitro of red blood cell Ca2+-ATPase activity: interspecies variation.

    PubMed

    Davis, F B; Kite, J H; Davis, P J; Blas, S D

    1982-01-01

    In vitro susceptibility to thyroid hormone stimulation of membrane-associated Ca2+-ATPase activity has been examined in red blood cells from rat, rabbit, dog, monkey, and man. Monkey and human red cell Ca2+-ATPase activities responded comparably to 10(-10)M T4 or T3. Basal and thyroid hormone-stimulated Ca2+-ATPase activity in rabbit erythrocytes was four-fold higher than in primate red cells. Rat and dog red cell Ca2+-ATPase did not respond to iodothyronines in vitro. PMID:6459228

  8. Acute hydrodynamic damage induced by SPLITT fractionation and centrifugation in red blood cells.

    PubMed

    Urbina, Adriana; Godoy-Silva, Ruben; Hoyos, Mauricio; Camacho, Marcela

    2016-05-01

    Though blood bank processing traditionally employs centrifugation, new separation techniques may be appealing for large scale processes. Split-flow fractionation (SPLITT) is a family of techniques that separates in absence of labelling and uses very low flow rates and force fields, and is therefore expected to minimize cell damage. However, the hydrodynamic stress and possible consequent damaging effects of SPLITT fractionation have not been yet examined. The aim of this study was to investigate the hydrodynamic damage of SPLITT fractionation to human red blood cells, and to compare these effects with those induced by centrifugation. Peripheral whole blood samples were collected from healthy volunteers. Samples were diluted in a buffered saline solution, and were exposed to SPLITT fractionation (flow rates 1-10ml/min) or centrifugation (100-1500g) for 10min. Cell viability, shape, diameter, mean corpuscular hemoglobin, and membrane potential were measured. Under the operating conditions employed, both SPLITT and centrifugation maintained cell viability above 98%, but resulted in significant sublethal damage, including echinocyte formation, decreased cell diameter, decreased mean corpuscular hemoglobin, and membrane hyperpolarization which was inhibited by EGTA. Wall shear stress and maximum energy dissipation rate showed significant correlation with lethal and sublethal damage. Our data do not support the assumption that SPLITT fractionation induces very low shear stress and is innocuous to cell function. Some changes in SPLITT channel design are suggested to minimize cell damage. Measurement of membrane potential and cell diameter could provide a new, reliable and convenient basis for evaluation of hydrodynamic effects on different cell models, allowing identification of optimal operating conditions on different scales. PMID:27023157

  9. Frequency-domain flow cytometry: fluorescence-lifetime-based sensing technology for analyzing cells and chromosomes labeled with fluorescent probes

    NASA Astrophysics Data System (ADS)

    Steinkamp, John A.; Crissman, Harry A.; Lehnert, Bruce E.; Lehnert, Nancy M.; Deka, Chiranjit

    1997-05-01

    A flow cytometer has been developed that combines flow cytometry (FCM) and fluorescence lifetime spectroscopy measurement principles to provide unique capabilities for making frequency-domain, excited-state lifetime measurements on cells/chromosomes labeled with fluorescent probes, while preserving conventional FCM capabilities. Cells are analyzed as they intersect a high-frequency, intensity-modulated (sine-wave) laser excitation beam. Fluorescence signals are processed by (1) low-pass filtering to obtain conventional FCM dc-excited signals and (2) phase-sensitive detection electronics to resolve heterogeneous fluorescence based on differences in lifetimes expressed as phase-shifts and to quantify fluorescence lifetimes in real time. Processed signals are displayed as frequency distribution histograms and bivariate contour diagrams. Recent examples of biological applications include: (1) lifetime histograms recorded on autofluorescent human lung fibroblasts, murine thymus cells labeled with antibodies conjugated to fluorophores for studying fluorescence quenching as a function of antibody dilution and F/P ratio, and on cultured cells, nuclei, and chromosomes stained with DNA-binding fluorochromes and (2) phase-resolved, fluorescence signal- intensity histograms recorded on autofluorescent HLFs labeled with immunofluorescence markers and on murine thymus cells labeled with Red 613-antiThy 1.2 and propidium iodide (PI positive `dead' cells) to demonstrate the resolution of signals from highly overlapping emission spectra. This technology will increase the number of fluorescent markers usable in multilabeling studies and lifetimes can be used as spectroscopic probes to study the interaction of markers with their targets, each other, and the surrounding microenvironment.

  10. Minimal RED cell pairs markedly improve electrode kinetics and power production in microbial reverse electrodialysis cells.

    PubMed

    Cusick, Roland D; Hatzell, Marta; Zhang, Fang; Logan, Bruce E

    2013-12-17

    Power production from microbial reverse electrodialysis cell (MRC) electrodes is substantially improved compared to microbial fuel cells (MFCs) by using ammonium bicarbonate (AmB) solutions in multiple RED cell pair stacks and the cathode chamber. Reducing the number of RED membranes pairs while maintaining enhanced electrode performance could help to reduce capital costs. We show here that using only a single RED cell pair (CP), created by operating the cathode in concentrated AmB, dramatically increased power production normalized to cathode area from both acetate (Acetate: from 0.9 to 3.1 W/m(2)-cat) and wastewater (WW: 0.3 to 1.7 W/m(2)), by reducing solution and charge transfer resistances at the cathode. A second RED cell pair increased RED stack potential and reduced anode charge transfer resistance, further increasing power production (Acetate: 4.2 W/m(2); WW: 1.9 W/m(2)). By maintaining near optimal electrode power production with fewer membranes, power densities normalized to total membrane area for the 1-CP (Acetate: 3.1 W/m(2)-mem; WW: 1.7 W/m(2)) and 2-CP (Acetate: 1.3 W/m(2)-mem; WW: 0.6 W/m(2)) reactors were much higher than previous MRCs (0.3-0.5 W/m(2)-mem with acetate). While operating at peak power, the rate of wastewater COD removal, normalized to reactor volume, was 30-50 times higher in 1-CP and 2-CP MRCs than that in a single chamber MFC. These findings show that even a single cell pair AmB RED stack can significantly enhance electrical power production and wastewater treatment. PMID:24224718

  11. Modeling of Red Blood Cells and Related Spleen Function

    NASA Astrophysics Data System (ADS)

    Peng, Zhangli; Pivkin, Igor; Dao, Ming

    2011-11-01

    A key function of the spleen is to clear red blood cells (RBCs) with abnormal mechanical properties from the circulation. These abnormal mechanical properties may be due to RBC aging or RBC diseases, e.g., malaria and sickle cell anemia. Specifically, 10% of RBCs passing through the spleen are forced to squeeze into the narrow slits between the endothelial cells, and stiffer cells which get stuck are killed and digested by macrophages. To investigate this important physiological process, we employ three different approaches to study RBCs passage through these small slits, including analytical theory, Dissipative Particle Dynamics (DPD) simulation and Multiscale Finite Element Method (MS-FEM). By applying the analytical theory, we estimate the critical limiting geometries RBCs can pass. By using the DPD method, we study the full fluid-structure interaction problem, and compute RBC deformation under different pressure gradients. By employing the MS-FEM approach, we model the lipid bilayer and the cytoskeleton as two distinct layers, and focus on the cytoskeleton deformation and the bilayer-skeleton interaction force at the molecular level. Finally the results of these three approaches are compared to each other and correlated to the experimental observations.

  12. Immunoregulatory function of neonatal nucleated red blood cells in humans.

    PubMed

    Cui, Lili; Takada, Hidetoshi; Takimoto, Tomohito; Fujiyoshi, Junko; Ishimura, Masataka; Hara, Toshiro

    2016-08-01

    We found that human cord blood nucleated red blood cells (NRBCs) have a regulatory function in the innate immune reaction. These cells suppressed the production of inflammatory cytokines including TNF-α and IL-1β from monocytes in response to lipopolysaccharide (LPS). The NRBCs exerted their regulatory function even without cell-to-cell contact with the monocytes. However, IL-10 production from the monocytes by LPS stimulation in the presence of NRBCs was higher than that from LPS-stimulated monocytes cultured in the absence of NRBCs. Addition of an anti-IL-10 receptor blocking antibody restored the inflammatory cytokine production from the monocytes, suggesting that the functional change of the monocytes caused by the interaction with NRBCs was mediated by the increased IL-10 production. A whole-genome microarray analysis revealed that the monocytes expressed increased amounts of IL-10 superfamily genes after interacting with NRBCs. IL-19, which is a member of the IL-10 superfamily, enhanced IL-10 production from the monocytes, which suggested a cooperative role of the IL-10 superfamily in the suppression of inflammatory cytokine production from monocytes. Arginase, which was reported to play an important role in the suppressive function of NRBCs in mice monocytes, was found to have no significant role in human monocytes. The NRBCs seem to have a regulatory role through the induction of IL-10/IL-19 production by monocytes to suppress a vigorous innate immune reaction, which can be harmful to fetuses. PMID:27117669

  13. Dynamic deformability of sickle red blood cells in microphysiological flow

    PubMed Central

    Alapan, Y.; Matsuyama, Y.; Little, J. A.; Gurkan, U. A.

    2016-01-01

    In sickle cell disease (SCD), hemoglobin molecules polymerize intracellularly and lead to a cascade of events resulting in decreased deformability and increased adhesion of red blood cells (RBCs). Decreased deformability and increased adhesion of sickle RBCs lead to blood vessel occlusion (vaso-occlusion) in SCD patients. Here, we present a microfluidic approach integrated with a cell dimensioning algorithm to analyze dynamic deformability of adhered RBC at the single-cell level in controlled microphysiological flow. We measured and compared dynamic deformability and adhesion of healthy hemoglobin A (HbA) and homozygous sickle hemoglobin (HbS) containing RBCs in blood samples obtained from 24 subjects. We introduce a new parameter to assess deformability of RBCs: the dynamic deformability index (DDI), which is defined as the time-dependent change of the cell’s aspect ratio in response to fluid flow shear stress. Our results show that DDI of HbS-containing RBCs were significantly lower compared to that of HbA-containing RBCs. Moreover, we observed subpopulations of HbS containing RBCs in terms of their dynamic deformability characteristics: deformable and non-deformable RBCs. Then, we tested blood samples from SCD patients and analyzed RBC adhesion and deformability at physiological and above physiological flow shear stresses. We observed significantly greater number of adhered non-deformable sickle RBCs than deformable sickle RBCs at flow shear stresses well above the physiological range, suggesting an interplay between dynamic deformability and increased adhesion of RBCs in vaso-occlusive events. PMID:27437432

  14. A red/far-red light-responsive bi-stable toggle switch to control gene expression in mammalian cells

    PubMed Central

    Müller, Konrad; Engesser, Raphael; Metzger, Stéphanie; Schulz, Simon; Kämpf, Michael M.; Busacker, Moritz; Steinberg, Thorsten; Tomakidi, Pascal; Ehrbar, Martin; Nagy, Ferenc; Timmer, Jens; Zubriggen, Matias D.; Weber, Wilfried

    2013-01-01

    Growth and differentiation of multicellular systems is orchestrated by spatially restricted gene expression programs in specialized subpopulations. The targeted manipulation of such processes by synthetic tools with high-spatiotemporal resolution could, therefore, enable a deepened understanding of developmental processes and open new opportunities in tissue engineering. Here, we describe the first red/far-red light-triggered gene switch for mammalian cells for achieving gene expression control in time and space. We show that the system can reversibly be toggled between stable on- and off-states using short light pulses at 660 or 740 nm. Red light-induced gene expression was shown to correlate with the applied photon number and was compatible with different mammalian cell lines, including human primary cells. The light-induced expression kinetics were quantitatively analyzed by a mathematical model. We apply the system for the spatially controlled engineering of angiogenesis in chicken embryos. The system’s performance combined with cell- and tissue-compatible regulating red light will enable unprecedented spatiotemporally controlled molecular interventions in mammalian cells, tissues and organisms. PMID:23355611

  15. P2X and P2Y receptor signaling in red blood cells

    PubMed Central

    Sluyter, Ronald

    2015-01-01

    Purinergic signaling involves the activation of cell surface P1 and P2 receptors by extracellular nucleosides and nucleotides such as adenosine and adenosine triphosphate (ATP), respectively. P2 receptors comprise P2X and P2Y receptors, and have well-established roles in leukocyte and platelet biology. Emerging evidence indicates important roles for these receptors in red blood cells. P2 receptor activation stimulates a number of signaling pathways in progenitor red blood cells resulting in microparticle release, reactive oxygen species formation, and apoptosis. Likewise, activation of P2 receptors in mature red blood cells stimulates signaling pathways mediating volume regulation, eicosanoid release, phosphatidylserine exposure, hemolysis, impaired ATP release, and susceptibility or resistance to infection. This review summarizes the distribution of P2 receptors in red blood cells, and outlines the functions of P2 receptor signaling in these cells and its implications in red blood cell biology. PMID:26579528

  16. Determination of single and repeated red cell volumes by the indicator dilution method using carbon monoxide as the indicator

    SciTech Connect

    Fukui, M.; Shigemi, K. )

    1989-11-01

    The use of radioactive isotopes limits clinical applications of blood volume measurement in the ICU. We measured red cell volumes with carbon monoxide-labeled RBC in six dogs and five human volunteers. The measured values obtained on the dogs were compared with the simultaneous measurements with the {sup 51}Cr method; the ratio of the carbon monoxide to {sup 51}Cr values ranged from 0.86 to 1.17, and the mean ratio was 1.0 +/- 0.1 (SD), r = .93. We infer from these results that the carbon monoxide method has several advantages over the {sup 51}Cr method: (a) the short labeling time (about 1 min), (b) rapidly decreasing background levels of carbon monoxide with FIO2 1.0, and (c) repeatability at intervals of several hours.

  17. Fluorescent liposomes to probe how DOTAP lipid concentrations can change red blood cells homeostasis

    NASA Astrophysics Data System (ADS)

    Matos, Anna L. L.; Pereira, Goreti; Santos, Beate S.; Fontes, Adriana

    2015-06-01

    Liposomes have been used to deliver DNA, drugs and, more recently, nanoparticles such as quantum dots, into living cells. Their electrostatic interaction with cell's surface (negatively charged) can lead to membrane destabilization and/or fusion, facilitating intracellular release of those compounds. Nevertheless, cationic lipids can modify living cells homeostasis, depending on their concentration. In this study, we observed that the DOTAP cationic lipid concentrations influence the red blood cells (RBCs) homeostasis. We used fluorescent fusogenic liposomes composed by three lipids: DOPE, DOTAP and DPPE-Rhodamine (1:0.1/0.3/0.5/0.8/1:0.1 mM respectively), varying DOTAP from 0.1 to 1 mM. To probe liposomes ability to fuse with cells, RBCs (1% in saline) were utilized. Liposomes were characterized by zeta potential, dynamic light scattering (DLS), fluorescence and transmission electron microscopy. Their interaction with RBCs was evaluated by fluorescence microscopy and flow cytometry. Zeta potential results showed that, from 0.1 to 1 mM concentration, the charge increases, due to the addition of DOTAP. Liposomes' diameter does not vary significantly when more DOTAP was added, except for the one containing 0.1 mM of DOTAP, according to DLS results. Flow cytometry and microscopy analysis showed that for all DOTAP' concentration applied, the liposomes were capable to label RBCs. However, as higher the amount of DOTAP in liposomes, the more harmful they were to cells. Thus, the results showed that it is possible to use lower concentrations of DOTAP keeping the fusogenic liposomes's ability and cell homeostasis. This is important to guarantee a greater efficiency in the delivery of nanoparticles or other active samples into cells.

  18. Bacterial glycosidases for the production of universal red blood cells.

    PubMed

    Liu, Qiyong P; Sulzenbacher, Gerlind; Yuan, Huaiping; Bennett, Eric P; Pietz, Greg; Saunders, Kristen; Spence, Jean; Nudelman, Edward; Levery, Steven B; White, Thayer; Neveu, John M; Lane, William S; Bourne, Yves; Olsson, Martin L; Henrissat, Bernard; Clausen, Henrik

    2007-04-01

    Enzymatic removal of blood group ABO antigens to develop universal red blood cells (RBCs) was a pioneering vision originally proposed more than 25 years ago. Although the feasibility of this approach was demonstrated in clinical trials for group B RBCs, a major obstacle in translating this technology to clinical practice has been the lack of efficient glycosidase enzymes. Here we report two bacterial glycosidase gene families that provide enzymes capable of efficient removal of A and B antigens at neutral pH with low consumption of recombinant enzymes. The crystal structure of a member of the alpha-N-acetylgalactosaminidase family reveals an unusual catalytic mechanism involving NAD+. The enzymatic conversion processes we describe hold promise for achieving the goal of producing universal RBCs, which would improve the blood supply while enhancing the safety of clinical transfusions. PMID:17401360

  19. Mobility Enhancement of Red Blood Cells with Biopolymers

    NASA Astrophysics Data System (ADS)

    Tahara, Daiki; Oikawa, Noriko; Kurita, Rei

    2016-03-01

    Adhesion of red blood cells (RBC) to substrates are one of crucial problems for a blood clot. Here we investigate the mobility of RBC between two glass substrates in saline with polymer systems. We find that RBCs are adhered to the glass substrate with PEG, however the mobility steeply increases with fibrinogen and dextran, which are biopolymers. We also find that the mobility affects an aggregation dynamics of RBCs, which is related with diseases such as influenza, blood clot and so on. The Brownian motion helps to increase probability of contact with each other and to find a more stable condition of the aggregation. Thus the biopolymers play important roles not only for preventing the adhesion but also for the aggregation.

  20. SEM analysis of red blood cells in aged human bloodstains.

    PubMed

    Hortolà, P

    1992-08-01

    Mammal red blood cells (RBC) in bloodstains have been previously detected by light microscopy on stone tools from as early as 100,000 +/- 25,000 years ago. In order to evaluate the degree of morphological preservation of erythrocytes in bloodstains, an accidental human blood smear on white chert and several experimental bloodstains on hard substrates (the same stone-white chert; another type of stone-graywacke; a non-stone support-stainless steel), were stored in a room, in non-sterile and fluctuating conditions, for lengths of time ranging from 3 to 18 months. Afterwards, the specimens were coated with gold and examined by a Cambridge Stereoscan 120 scanning electron microscope. Results revealed a high preservation of RBC integrity, with the maintenance of several discocytary shapes, a low tendency to echinocytosis and a frequent appearance of a moon-like erythrocytary shape in the thinner areas of the bloodstains. PMID:1398371

  1. Image-based red cell counting for wild animals blood.

    PubMed

    Mauricio, Claudio R M; Schneider, Fabio K; Dos Santos, Leonilda Correia

    2010-01-01

    An image-based red blood cell (RBC) automatic counting system is presented for wild animals blood analysis. Images with 2048×1536-pixel resolution acquired on an optical microscope using Neubauer chambers are used to evaluate RBC counting for three animal species (Leopardus pardalis, Cebus apella and Nasua nasua) and the error found using the proposed method is similar to that obtained for inter observer visual counting method, i.e., around 10%. Smaller errors (e.g., 3%) can be obtained in regions with less grid artifacts. These promising results allow the use of the proposed method either as a complete automatic counting tool in laboratories for wild animal's blood analysis or as a first counting stage in a semi-automatic counting tool. PMID:21096766

  2. Iron-Deficiency Anemia Enhances Red Blood Cell Oxidative Stress

    PubMed Central

    Nagababu, Enika; Gulyani, Seema; Earley, Christopher J.; Cutler, Roy G.; Mattson, Mark P.; Rifkind, Joseph M.

    2009-01-01

    Oxidative stress associated with iron deficiency anemia in a murine model was studied feeding an iron deficient diet. Anemia was monitored by a decrease in hematocrit and hemoglobin. For the 9 week study an increase in total iron binding capacity was also demonstrated. Anemia resulted in an increase in red blood cells (RBC) oxidative stress as indicated by increased levels of fluorescent heme degradation products (1.24 fold after 5 weeks; 2.1 fold after 9 weeks). The increase in oxidative stress was further confirmed by elevated levels of methemoglobin for mice fed an iron deficient diet. Increased hemoglobin autoxidation and subsequent generation of ROS can account for the shorter RBC lifespan and other pathological changes associated with iron deficiency anemia. PMID:19051108

  3. Dynamics of flat membranes and flickering in red blood cells

    NASA Astrophysics Data System (ADS)

    Frey, Erwin; Nelson, David R.

    1991-12-01

    A theory of the dynamics of polymerized membranes in the flat phase is presented. The dynamics of dilute membrane solutions is strongly influenced by long-ranged hydrodynamic interactions among the monomers, mediated by the intervening solvent. We discuss the renormalization of the kinetic coefficients for the undulation and phonon modes due to hydrodynamic “backflow” (Zimm behavior). The dynamics is also studied for free draining membranes (Rouse dynamics) corresponding to the Brownian dynamics method used in Monte Carlo simulations. The long time behavior of the dynamic structure factor is given by stretched exponentials with stretching exponents determined by the exponents of the elastic coefficients and the wave vector dependence of the Oseen tensor. We also study the dynamics of the thickness fluctuations in red blood cells (flicker phenomenon) taking into account the underlying polymerized spectrin skeleton. Qualitatively different dynamical behavior is predicted for spectrin skeletons isolated from heir natural lipid environment.

  4. Identification of Putative Bovine Mammary Epithelial Stem Cells by Their Retention of Labeled DNA Strands

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Stem cells characteristically retain labeled DNA for extended periods due to their selective segregation of template DNA strands during mitosis. In this study, proliferating cells in the prepubertal bovine mammary gland were labeled using five daily-injections of 5-bromo-2-deoxyuridine (BrdU). Fiv...

  5. Anemia and red blood cell transfusion in neurocritical care

    PubMed Central

    Kramer, Andreas H; Zygun, David A

    2009-01-01

    Introduction Anemia is one of the most common medical complications to be encountered in critically ill patients. Based on the results of clinical trials, transfusion practices across the world have generally become more restrictive. However, because reduced oxygen delivery contributes to 'secondary' cerebral injury, anemia may not be as well tolerated among neurocritical care patients. Methods The first portion of this paper is a narrative review of the physiologic implications of anemia, hemodilution, and transfusion in the setting of brain-injury and stroke. The second portion is a systematic review to identify studies assessing the association between anemia or the use of red blood cell transfusions and relevant clinical outcomes in various neurocritical care populations. Results There have been no randomized controlled trials that have adequately assessed optimal transfusion thresholds specifically among brain-injured patients. The importance of ischemia and the implications of anemia are not necessarily the same for all neurocritical care conditions. Nevertheless, there exists an extensive body of experimental work, as well as human observational and physiologic studies, which have advanced knowledge in this area and provide some guidance to clinicians. Lower hemoglobin concentrations are consistently associated with worse physiologic parameters and clinical outcomes; however, this relationship may not be altered by more aggressive use of red blood cell transfusions. Conclusions Although hemoglobin concentrations as low as 7 g/dl are well tolerated in most critical care patients, such a severe degree of anemia could be harmful in brain-injured patients. Randomized controlled trials of different transfusion thresholds, specifically in neurocritical care settings, are required. The impact of the duration of blood storage on the neurologic implications of transfusion also requires further investigation. PMID:19519893

  6. Pure red cell aplasia secondary to treatment with erythropoietin.

    PubMed

    Locatelli, Francesco; Del Vecchio, Lucia

    2003-01-01

    Pure red cell aplasia (PRCA) is a rare condition defined as severe anemia secondary to the virtual absence of red blood cell precursors in the bone marrow. In the setting of patients treated with rHuEPO, the disease is generated by epoetin-induced antibodies that neutralise all the exogenous rHuEPO and cross-react with endogenous erythropoietin. As a result, serum erythropoietin levels are undetectable and erythropoiesis becomes ineffective. Only 4 cases of PRCA associated with rh-EPO have been reported before 1998. Thereafter, a sharp increase in the incidence of this rare condition has been reported, mainly associated with epoetin alpha use outside the United States. A number of possible mechanisms leading to PRCA development have been identified. Among these, modification of drug formulation and down stream processing probably has had a major role. Indeed, in 1998 the formulation of epoetin alpha in Europe was modified because of the fear of the "mad cow" syndrome. However, differences in molecule structure and glycosylation among different epoetins can not be excluded. It should also be underlined that the rise in the incidence of PRCA cases has been coincident with a major shift from intravenous to subcutaneous administration of rHuEPO. The abrupt rise in the incidence of PRCA cases observed in the last few years, deserves particular attention; however, we have to balance its severity, but extreme rarity, with the high number of chronic kidney disease patients who die each year because of cardiovascular disease that could partially be reduced by anemia treatment. PMID:14696747

  7. Enhanced detection of fluorescence quenching in labeled cells

    DOEpatents

    Crissman, Harry A.; Steinkamp, John A.

    1992-01-01

    A method is provided for quantifying BrdU labeled DNA in cells. The BrdU is incorporated into the DNA and the DNA is stained with a first fluorochrome having a fluorescence which is quenchable by BrdU. The first fluorochrome is preferably a thymidine base halogen analogue, such as a Hoechst fluorochrome. The DNA is then stained with a second fluorochrome having a fluorescence that is substantially uneffected by BrdU. The second fluorochrome may be selected from the group consisting of mithramycin, chromomycin A3, olivomycin, propidium iodide and ethidium bromine. The fluorescence from the first and second fluorochromes is then measured to obtain first and second output signals, respectively. The first output signal is substracted from the second output signal to obtain a difference signal which is functionally related to the quantity of BrdU incorporated into DNA. The technique is particularly useful for quantifying the synthesis of DNA during the S-phase of the cell cycle.

  8. Enhanced detection of fluorescence quenching in labeled cells

    DOEpatents

    Crissman, H.A.; Steinkamp, J.A.

    1987-11-30

    A method is provided for quantifying BrdU labeled DNA in cells. The BrdU is substituted onto the DNA and the DNA is stained with a first fluorochrome having a fluorescence which is quenchable by BrdU. The first fluorochrome is preferably a thymidine base halogen analogue, such as a Hoechst fluorochrome. The DNA is then stained with a second fluorochrome having a fluorescence which is substantially uneffected by BrdU. The second fluorochrome may be selected from the group consisting of mithramycin, chromomycin A3, olivomycin, propidium iodide and ethidium bromine. The fluorescence from the first and second fluorochromes is then measured to obtain first and second output signals, respectively. The first output signal is subtracted from the second output signal to obtain a difference signal which is functionally related to the quantity of BrdU incorporated into DNA. The technique is particularly useful for quantifying the synthesis of DNA during the S-phase of the cell cycle. 2 figs.

  9. Red blood cells do not contribute to removal of K+ released from exhaustively working forearm muscle.

    PubMed

    Maassen, N; Foerster, M; Mairbäurl, H

    1998-07-01

    K+ released from exercising muscle via K+ channels needs to be removed from the interstitium into the blood to maintain high muscle cell membrane potential and allow normal muscle contractility. Uptake by red blood cells has been discussed as one mechanism that would also serve to regulate red blood cell volume, which was found to be constant despite increased plasma osmolality and K+ concentration ([K+pl]). We evaluated exercise-related changes in [K+pl], pH, osmolality, mean cellular Hb concentration, cell water, and red blood cell K+ concentration during exhaustive handgrip exercise. Unidirectional 86Rb+ (K+) uptake by red blood cells was measured in media with elevated extracellular K+, osmolarity, and catecholamines to simulate particularly those exercise-related changes in plasma composition that are known to stimulate K+ uptake. During exercise [K+pl] increased from 4.4 +/- 0.7 to 7.1 +/- 0.5 mmol/l plasma water and red blood cell K+ concentration increased from 137.2 +/- 6.0 to 144.6 +/- 4.6 mmol/l cell water (P red blood cells was increased by approximately 20% on stimulation, caused by activation of the Na+-K+ pump and Na+-K+-2Cl- cotransport. Results indicate the K+ content of red blood cells did not change as cells passed the exhaustively exercising forearm muscle despite the elevated [K+pl]. The tendency for an increase in intracellular K+ concentration was due to a slight, although statistically not significant, decrease in red blood cell volume. K+ uptake, although elevated, was too small to move significant amounts of K+ into red blood cells. Our results suggest that red blood cells do not contribute to the removal of K+ released from muscle and do not regulate their volume by K+ uptake during exhaustive forearm exercise. PMID:9655793

  10. Infrared fluorescent protein 1.4 genetic labeling tracks engrafted cardiac progenitor cells in mouse ischemic hearts.

    PubMed

    Chen, Lijuan; Phillips, M Ian; Miao, Hui-Lai; Zeng, Rong; Qin, Gangjian; Kim, Il-man; Weintraub, Neal L; Tang, Yaoliang

    2014-01-01

    Stem cell therapy has a potential for regenerating damaged myocardium. However, a key obstacle to cell therapy's success is the loss of engrafted cells due to apoptosis or necrosis in the ischemic myocardium. While many strategies have been developed to improve engrafted cell survival, tools to evaluate cell efficacy within the body are limited. Traditional genetic labeling tools, such as GFP-like fluorescent proteins (eGFP, DsRed, mCherry), have limited penetration depths in vivo due to tissue scattering and absorption. To circumvent these limitations, a near-infrared fluorescent mutant of the DrBphP bacteriophytochrome from Deinococcus radiodurans, IFP1.4, was developed for in vivo imaging, but it has yet to be used for in vivo stem/progenitor cell tracking. In this study, we incorporated IFP1.4 into mouse cardiac progenitor cells (CPCs) by a lentiviral vector. Live IFP1.4-labeled CPCs were imaged by their near-infrared fluorescence (NIRF) using an Odyssey scanner following overnight incubation with biliverdin. A significant linear correlation was observed between the amount of cells and NIRF signal intensity in in vitro studies. Lentiviral mediated IFP1.4 gene labeling is stable, and does not impact the apoptosis and cardiac differentiation of CPC. To assess efficacy of our model for engrafted cells in vivo, IFP1.4-labeled CPCs were intramyocardially injected into infarcted hearts. NIRF signals were collected at 1-day, 7-days, and 14-days post-injection using the Kodak in vivo multispectral imaging system. Strong NIRF signals from engrafted cells were imaged 1 day after injection. At 1 week after injection, 70% of the NIRF signal was lost when compared to the intensity of the day 1 signal. The data collected 2 weeks following transplantation showed an 88% decrease when compared to day 1. Our studies have shown that IFP1.4 gene labeling can be used to track the viability of transplanted cells in vivo. PMID:25357000

  11. Integration of red cell genotyping into the blood supply chain: a population-based study

    PubMed Central

    Flegel, Willy A; Gottschall, Jerome L; Denomme, Gregory A

    2015-01-01

    Background When problems with compatibility arise, transfusion services often perform time-consuming serologic testing to locate antigen-negative red cell units for safe transfusion. New technologies enabled red cell genotyping for all clinically relevant blood group antigens. We performed mass-scale genotyping and provided access to a large red cell database to meet the demand for antigen-negative red cell units beyond ABO and Rh. Methods A red cell genotype database was established in 2010. Hospitals were given online access to a web-based antigen query portal in 2013 to find antigen-negative units in their inventories. Findings Genotype data were analyzed for 43,066 blood donors covering a set of 42 clinically relevant red cell antigens. Requests were filled for 5661 of 5672 patient encounters (99.8%) requiring antigen-negative red cell units in a multi-ethnic and multi-racial population. Red cell genotyping met the demand for antigen-negative blood in 5339 of 5672 (95%) patient encounters, while 333 remaining requests were filled using serologic data. In a pilot phase, seven community and rural transfusion services searched their local inventories using an online antigen query portal. Interpretation Red cell genotyping has the potential to transform the way antigen-negative red cell units are provided. An antigen query portal may reduce the need to ship blood or perform serologic screening. The wealth of genotype data, easily accessible online, facilitates the supply of affordable antigen-negative red cell units for patient safety. Physicians may recognize these new efficiencies for patient transfusion support. Funding BloodCenter of Wisconsin Diagnostic Laboratories Strategic Initiative and the NIH Clinical Center Intramural Research Program. PMID:26207259

  12. Peripheral red blood cell split chimerism as a consequence of intramedullary selective apoptosis of recipient red blood cells in a case of sickle cell disease.

    PubMed

    Marziali, Marco; Isgrò, Antonella; Sodani, Pietro; Gaziev, Javid; Fraboni, Daniela; Paciaroni, Katia; Gallucci, Cristiano; Alfieri, Cecilia; Roveda, Andrea; De Angelis, Gioia; Cardarelli, Luisa; Ribersani, Michela; Andreani, Marco; Lucarelli, Guido

    2014-01-01

    Allogeneic cellular gene therapy through hematopoietic stem cell transplantation is the only radical cure for congenital hemoglobinopathies like thalassemia and sickle cell anemia. Persistent mixed hematopoietic chimerism (PMC) has been described in thalassemia and sickle cell anemia. Here, we describe the clinical course of a 6-year-old girl who had received bone marrow transplant for sickle cell anemia. After the transplant, the patient showed 36% donor hematopoietic stem cells in the bone marrow, whereas in the peripheral blood there was evidence of 80% circulating donor red blood cells (RBC). The analysis of apoptosis at the Bone Marrow level suggests that Fas might contribute to the cell death of host erythroid precursors. The increase in NK cells and the regulatory T cell population observed in this patient suggests that these cells might contribute to the condition of mixed chimerism. PMID:25408852

  13. Peripheral Red Blood Cell Split Chimerism as a Consequence of Intramedullary Selective Apoptosis of Recipient Red Blood Cells in a Case of Sickle Cell Disease

    PubMed Central

    Marziali, Marco; Isgrò, Antonella; Sodani, Pietro; Gaziev, Javid; Fraboni, Daniela; Paciaroni, Katia; Gallucci, Cristiano; Alfieri, Cecilia; Roveda, Andrea; De Angelis, Gioia; Cardarelli, Luisa; Ribersani, Michela; Andreani, Marco; Lucarelli, Guido

    2014-01-01

    Allogeneic cellular gene therapy through hematopoietic stem cell transplantation is the only radical cure for congenital hemoglobinopathies like thalassemia and sickle cell anemia. Persistent mixed hematopoietic chimerism (PMC) has been described in thalassemia and sickle cell anemia. Here, we describe the clinical course of a 6-year-old girl who had received bone marrow transplant for sickle cell anemia. After the transplant, the patient showed 36% donor hematopoietic stem cells in the bone marrow, whereas in the peripheral blood there was evidence of 80% circulating donor red blood cells (RBC). The analysis of apoptosis at the Bone Marrow level suggests that Fas might contribute to the cell death of host erythroid precursors. The increase in NK cells and the regulatory T cell population observed in this patient suggests that these cells might contribute to the condition of mixed chimerism. PMID:25408852

  14. Detection and quantification of subtle changes in red blood cell density using a cell phone.

    PubMed

    Felton, Edward J; Velasquez, Anthony; Lu, Shulin; Murphy, Ryann O; ElKhal, Abdala; Mazor, Ofer; Gorelik, Pavel; Sharda, Anish; Ghiran, Ionita C

    2016-08-16

    Magnetic levitation has emerged as a technique that offers the ability to differentiate between cells with different densities. We have developed a magnetic levitation system for this purpose that distinguishes not only different cell types but also density differences in cells of the same type. This small-scale system suspends cells in a paramagnetic medium in a capillary placed between two rare earth magnets, and cells levitate to an equilibrium position determined solely by their density. Uniform reference beads of known density are used in conjunction with the cells as a means to quantify their levitation positions. In one implementation images of the levitating cells are acquired with a microscope, but here we also introduce a cell phone-based device that integrates the magnets, capillary, and a lens into a compact and portable unit that acquires images with the phone's camera. To demonstrate the effectiveness of magnetic levitation in cell density analysis we carried out levitation experiments using red blood cells with artificially altered densities, and also levitated those from donors. We observed that we can distinguish red blood cells of an anemic donor from those that are healthy. Since a plethora of disease states are characterized by changes in cell density magnetic cell levitation promises to be an effective tool in identifying and analyzing pathologic states. Furthermore, the low cost, portability, and ease of use of the cell phone-based system may potentially lead to its deployment in low-resource environments. PMID:27431921

  15. Effect of different magnetic nanoparticle coatings on the efficiency of stem cell labeling

    NASA Astrophysics Data System (ADS)

    Horák, Daniel; Babič, Michal; Jendelová, Pavla; Herynek, Vít; Trchová, Miroslava; Likavčanová, Katarina; Kapcalová, Miroslava; Hájek, Milan; Syková, Eva

    2009-05-01

    Maghemite nanoparticles with various coatings were prepared by the coprecipitation method and characterized by transmission electron microscopy, dynamic light scattering and IR in terms of morphology, size, polydispersity and surface coating. The labeling efficiency and the viability of both rat and human mesenchymal stem cells labeled with Endorem ®, poly( L-lysine) (PLL)-modified Endorem ®, uncoated γ-Fe 2O 3, D-mannose-, PLL- or poly( N,N-dimethylacrylamide) (PDMAAm)-coated γ-Fe 2O 3 nanoparticles were compared. Coated γ-Fe 2O 3 nanoparticles labeled cells better than did Endorem ®. High relaxation rates and in vitro magnetic resonance imaging of cells labeled with coated nanoparticles showed clearly visible contrast compared with unlabeled cells or cells labeled with Endorem ®.

  16. Manipulation of red blood cells with electric field

    NASA Astrophysics Data System (ADS)

    Saboonchi, Hossain; Esmaeeli, Asghar

    2009-11-01

    Manipulation of bioparticles and macromolecules is the central task in many biological and biotechnological processes. The current methods for physical manipulation takes advantage of different forces such as acoustic, centrifugal, magnetic, electromagnetic, and electric forces, as well as using optical tweezers or filtration. Among all these methods, however, the electrical forces are particularly attractive because of their favorable scale up with the system size which makes them well-suited for miniaturization. Currently the electric field is used for transportation, poration, fusion, rotation, and separation of biological cells. The aim of the current research is to gain fundamental understanding of the effect of electric field on the human red blood cells (RBCs) using direct numerical simulation. A front tracking/finite difference technique is used to solve the fluid flow and electric field equations, where the fluid in the cell and the blood (plasma) is modeled as Newtonian and incompressible, and the interface separating the two is treated as an elastic membrane. The behavior of RBCs is investigated as a function of the controlling parameters of the problem such as the strength of the electric field.

  17. Of macrophages and red blood cells; a complex love story.

    PubMed

    de Back, Djuna Z; Kostova, Elena B; van Kraaij, Marian; van den Berg, Timo K; van Bruggen, Robin

    2014-01-01

    Macrophages tightly control the production and clearance of red blood cells (RBC). During steady state hematopoiesis, approximately 10(10) RBC are produced per hour within erythroblastic islands in humans. In these erythroblastic islands, resident bone marrow macrophages provide erythroblasts with interactions that are essential for erythroid development. New evidence suggests that not only under homeostasis but also under stress conditions, macrophages play an important role in promoting erythropoiesis. Once RBC have matured, these cells remain in circulation for about 120 days. At the end of their life span, RBC are cleared by macrophages residing in the spleen and the liver. Current theories about the removal of senescent RBC and the essential role of macrophages will be discussed as well as the role of macrophages in facilitating the removal of damaged cellular content from the RBC. In this review we will provide an overview on the role of macrophages in the regulation of RBC production, maintenance and clearance. In addition, we will discuss the interactions between these two cell types during transfer of immune complexes and pathogens from RBC to macrophages. PMID:24523696

  18. Fibrinogen and red blood cells in venous thrombosis

    PubMed Central

    Aleman, Maria M.; Walton, Bethany L.; Byrnes, James R.; Wolberg, Alisa S.

    2014-01-01

    Deep vein thrombosis and pulmonary embolism, collectively termed venous thromboembolism (VTE), affect over 1 million Americans each year. VTE is triggered by inflammation and blood stasis leading to the formation of thrombi rich in fibrin and red blood cells (RBCs). However, little is known about mechanisms regulating fibrin and RBC incorporation into venous thrombi, or how these components mediate thrombus size or resolution. Both elevated circulating fibrinogen (hyperfibrinogenemia) and abnormal fibrin(ogen) structure and function, including increased fibrin network density and resistance to fibrinolysis, have been observed in plasmas from patients with VTE. Abnormalities in RBC number and/or function have also been associated with VTE risk. RBC contributions to VTE are thought to stem from their effects on blood viscosity and margination of platelets to the vessel wall. More recent studies suggest RBCs also express phosphatidylserine, support thrombin generation, and decrease fibrinolysis. RBC interactions with fibrin(ogen) and cells, including platelets and endothelial cells, may also promote thrombus formation. The contributions of fibrin(ogen) and RBCs to the pathophysiology of VTE warrants further investigation. PMID:24759140

  19. Cytoskeletal control of the red-blood cell membrane

    NASA Astrophysics Data System (ADS)

    Gov, Nir; Safran, Sam

    2004-03-01

    We have shown (Physical Review Letters, 90, 228101 (2003)) that the thermal fluctuations of red blood cells can be accounted for by a model of a nearly-free, but confined bilayer membrane with a finite tension; both the confinement and tension arise from the coupling of the membrane with the cytoskeleton. Recently, we have shown that these relatively gentle effects of the cytoskeleton-membrane couplings on the membrane fluctuations are due to the dilute nature of the coupling molecules. To quantify this, we predict the fluctuation amplitude for a microscopic model of the inhomogeneous coupling of a fluid membrane and a fixed cytoskeleton. The coupling is modeled as periodic and harmonic, and we consider the linear response of the membrane. We find that there is indeed, an effective surface tension and confinement of such a membrane, in accord with our phenomenological model, and relate these quantities to the strength and periodicity of the microscopic coupling. We also find, surprisingly, that the membrane can develop a spontaneous breaking of the cytoskeleton symmetry, at low confinements. Finally we address the role of ATP activity on the cytoskeleton-driven fluctuations and the equilibrium shape of the cell. We examine in detail the role of spectrin disconnections as the main ATP-activated network defects on the global cell shape and membrane fluctuations.

  20. Loss of deformability of malaria-infected red blood cells

    NASA Astrophysics Data System (ADS)

    Hosseini, S. Majid; Feng, James

    2012-11-01

    The pathogenesis of malaria is largely due to stiffening of the infected red blood cells (RBCs). Contemporary understanding ascribes the loss of RBC deformability to a 10-fold increase in membrane stiffness caused by extra cross-linking in the spectrin network. Local measurements by micropipette aspiration, however, have reported only an increase of 3-fold in the shear modulus. We believe the discrepancy stems from the rigid parasite particles inside infected cells, and have carried out numerical simulations to demonstrate this mechanism. The cell membrane is represented by a set of discrete particles connected by linearly elastic springs. The cytosol is modeled as a homogeneous Newtonian fluid, and discretized by particles as in standard smoothed particle hydrodynamics. The malaria parasite is modeled as an aggregate of particles constrained to rigid-body motion. We simulate RBC stretching tests by optical tweezers in three dimensions. The results demonstrate that the presence of a sizeable parasite greatly reduces the ability of RBCs to deform under stretching. With the solid inclusion, the observed loss of deformability can be predicted quantitatively using the local membrane elasticity measured by micropipettes.

  1. Red blood cell as an adaptive optofluidic microlens

    NASA Astrophysics Data System (ADS)

    Miccio, L.; Memmolo, P.; Merola, F.; Netti, P. A.; Ferraro, P.

    2015-03-01

    The perspective of using live cells as lenses could open new revolutionary and intriguing scenarios in the future of biophotonics and biomedical sciences for endoscopic vision, local laser treatments via optical fibres and diagnostics. Here we show that a suspended red blood cell (RBC) behaves as an adaptive liquid-lens at microscale, thus demonstrating its imaging capability and tunable focal length. In fact, thanks to the intrinsic elastic properties, the RBC can swell up from disk volume of 90 fl up to a sphere reaching 150 fl, varying focal length from negative to positive values. These live optofluidic lenses can be fully controlled by triggering the liquid buffer’s chemistry. Real-time accurate measurement of tunable focus capability of RBCs is reported through dynamic wavefront characterization, showing agreement with numerical modelling. Moreover, in analogy to adaptive optics testing, blood diagnosis is demonstrated by screening abnormal cells through focal-spot analysis applied to an RBC ensemble as a microlens array.

  2. Labeling of human mesenchymal stem cell: Comparison between paramagnetic and superparamagnetic agents

    NASA Astrophysics Data System (ADS)

    Yang, Chung-Yi; Tai, Ming-Fong; Chen, Shin-Tai; Wang, Yi-Ting; Chen, Ya-Fang; Hsiao, Jong-Kai; Wang, Jaw-Lin; Liu, Hon-Man

    2009-04-01

    Paramagnetic and superparamagnetic substances are used to trace stem cell in living organisms under magnetic resonance imaging (MRI). We compared paramagnetic and superparamagnetic substance for their labeling efficiency by using clinically widely used gadolinium chelates and iron oxide nanoparticles. Without the aid of transfection agent, human mesenchymal stem cells were labeled with each agent separately in different concentration and the optimized concentration was determined by maintaining same cell viability as unlabeled cells. Iron oxide nanoparticle labeling has a detecting threshold of 12 500 cells in vitro, while gadolinium chelates labeling could be detected for at least 50 000 cells. In life animal study, we found there is an eightfold sensitivity in cells labeled with iron oxide superparamagnetic nanoparticles; however, the magnetic susceptibility artifact would obscure the detail of adjacent anatomical structures. We conclude that labeling stem cells with superparamagnetic substance is more efficacious. However, the cells labeled by superparamagnetic nanoparticles might interfere with the interpretation of anatomical structure. These findings would be beneficial to applications of magnetic substances toward stem cell biology and tissue engineering.

  3. Red blood cells radial dispersion in blood flowing through microchannels: The role of temperature.

    PubMed

    Pinho, Diana; Rodrigues, Raquel O; Faustino, Vera; Yaginuma, Tomoko; Exposto, José; Lima, Rui

    2016-07-26

    The behavior of suspensions of individual blood cells, such as red blood cells (RBCs), flowing through microvessels and microfluidic systems depend strongly on the hematocrit (Hct), microvessel topology and cell properties. Although it is well known that blood rheological properties are temperature dependent, to the best of our knowledge no work has studied the role of the temperature on the RBCs dispersion. A powerful way to investigate this latter effect is through a high-speed video microscopy system, which provides detailed flow measurements of each individual RBC. Hence, the effect of temperature on the RBCs dispersion flowing through a 100μm glass capillary was examined by means of a confocal micro-PTV system. Hundreds of labeled RBCs were tracked at moderate Hct (12%) and at four different temperatures, i.e., 25°C, 32°C, 37°C and 42°C. The results yielded an enhancement of the RBCs diffusion as the temperature increases. Hence, our findings show that RBCs radial dispersion is temperature dependent and as a result the temperature should not be ignored in future blood flow studies. We believe that this finding is important for a better understanding of blood mass transport mechanisms under both physiological and pathological conditions. PMID:26671221

  4. Red Blood Cell Susceptibility to Pneumolysin: CORRELATION WITH MEMBRANE BIOCHEMICAL AND PHYSICAL PROPERTIES.

    PubMed

    Bokori-Brown, Monika; Petrov, Peter G; Khafaji, Mawya A; Mughal, Muhammad K; Naylor, Claire E; Shore, Angela C; Gooding, Kim M; Casanova, Francesco; Mitchell, Tim J; Titball, Richard W; Winlove, C Peter

    2016-05-01

    This study investigated the effect of the biochemical and biophysical properties of the plasma membrane as well as membrane morphology on the susceptibility of human red blood cells to the cholesterol-dependent cytolysin pneumolysin, a key virulence factor of Streptococcus pneumoniae, using single cell studies. We show a correlation between the physical properties of the membrane (bending rigidity and surface and dipole electrostatic potentials) and the susceptibility of red blood cells to pneumolysin-induced hemolysis. We demonstrate that biochemical modifications of the membrane induced by oxidative stress, lipid scrambling, and artificial cell aging modulate the cell response to the toxin. We provide evidence that the diversity of response to pneumolysin in diabetic red blood cells correlates with levels of glycated hemoglobin and that the mechanical properties of the red blood cell plasma membrane are altered in diabetes. Finally, we show that diabetic red blood cells are more resistant to pneumolysin and the related toxin perfringolysin O relative to healthy red blood cells. Taken together, these studies indicate that the diversity of cell response to pneumolysin within a population of human red blood cells is influenced by the biophysical and biochemical status of the plasma membrane and the chemical and/or oxidative stress pre-history of the cell. PMID:26984406

  5. Snap-, CLIP- and Halo-Tag Labelling of Budding Yeast Cells

    PubMed Central

    Stagge, Franziska; Mitronova, Gyuzel Y.; Belov, Vladimir N.; Wurm, Christian A.; Jakobs, Stefan

    2013-01-01

    Fluorescence microscopy of the localization and the spatial and temporal dynamics of specifically labelled proteins is an indispensable tool in cell biology. Besides fluorescent proteins as tags, tag-mediated labelling utilizing self-labelling proteins as the SNAP-, CLIP-, or the Halo-tag are widely used, flexible labelling systems relying on exogenously supplied fluorophores. Unfortunately, labelling of live budding yeast cells proved to be challenging with these approaches because of the limited accessibility of the cell interior to the dyes. In this study we developed a fast and reliable electroporation-based labelling protocol for living budding yeast cells expressing SNAP-, CLIP-, or Halo-tagged fusion proteins. For the Halo-tag, we demonstrate that it is crucial to use the 6′-carboxy isomers and not the 5′-carboxy isomers of important dyes to ensure cell viability. We report on a simple rule for the analysis of 1H NMR spectra to discriminate between 6′- and 5′-carboxy isomers of fluorescein and rhodamine derivatives. We demonstrate the usability of the labelling protocol by imaging yeast cells with STED super-resolution microscopy and dual colour live cell microscopy. The large number of available fluorophores for these self-labelling proteins and the simplicity of the protocol described here expands the available toolbox for the model organism Saccharomyces cerevisiae. PMID:24205303

  6. Macrophages and iron trafficking at the birth and death of red cells

    PubMed Central

    Korolnek, Tamara

    2015-01-01

    Macrophages play a critical role in iron homeostasis via their intimate association with developing and dying red cells. Central nurse macrophages promote erythropoiesis in the erythroblastic island niche. These macrophages make physical contact with erythroblasts, enabling signaling and the transfer of growth factors and possibly nutrients to the cells in their care. Human mature red cells have a lifespan of 120 days before they become senescent and again come into contact with macrophages. Phagocytosis of red blood cells is the main source of iron flux in the body, because heme must be recycled from approximately 270 billion hemoglobin molecules in each red cell, and roughly 2 million senescent red cells are recycled each second. Here we will review pathways for iron trafficking found at the macrophage-erythroid axis, with a focus on possible roles for the transport of heme in toto. PMID:25778532

  7. The influence of nanodiamond on the oxygenation states and micro rheological properties of human red blood cells in vitro

    NASA Astrophysics Data System (ADS)

    Lin, Yu-Chung; Tsai, Lin-Wei; Perevedentseva, Elena; Chang, Hsin-Hou; Lin, Ching-Hui; Sun, Der-Shan; Lugovtsov, Andrei E.; Priezzhev, Alexander; Mona, Jani; Cheng, Chia-Liang

    2012-10-01

    Nanodiamond has been proven to be biocompatible and proposed for various biomedical applications. Recently, nanometer-sized diamonds have been demonstrated as an effective Raman/fluorescence probe for bio-labeling, as well as, for drug delivery. Bio-labeling/drug delivery can be extended to the human blood system, provided one understands the interaction between nanodiamonds and the blood system. Here, the interaction of nanodiamonds (5 and 100 nm) with human red blood cells (RBC) in vitro is discussed. Measurements have been facilitated using Raman spectroscopy, laser scanning fluorescence spectroscopy, and laser diffractometry (ektacytometry). Data on cell viability and hemolytic analysis are also presented. Results indicate that the nanodiamonds in the studied condition do not cause hemolysis, and the cell viability is not affected. Importantly, the oxygenation/deoxygenation process was not found to be altered when nanodiamonds interacted with the RBC. However, the nanodiamond can affect some RBC properties such as deformability and aggregation in a concentration dependent manner. These results suggest that the nanodiamond can be used as an effective bio-labeling and drug delivery tool in ambient conditions, without complicating the blood's physiological conditions. However, controlling the blood properties including deformability of RBCs and rheological properties of blood is necessary during treatment.

  8. Cell Labeling and Tracking Method without Distorted Signals by Phagocytosis of Macrophages

    PubMed Central

    Kang, Sun-Woong; Lee, Sangmin; Na, Jin Hee; Yoon, Hwa In; Lee, Dong-Eun; Koo, Heebeom; Cho, Yong Woo; Kim, Sun Hwa; Jeong, Seo Young; Kwon, Ick Chan; Choi, Kuiwon; Kim, Kwangmeyung

    2014-01-01

    Cell labeling and tracking are important processes in understanding biologic mechanisms and the therapeutic effect of inoculated cells in vivo. Numerous attempts have been made to label and track inoculated cells in vivo; however, these methods have limitations as a result of their biological effects, including secondary phagocytosis of macrophages and genetic modification. Here, we investigated a new cell labeling and tracking strategy based on metabolic glycoengineering and bioorthogonal click chemistry. We first treated cells with tetra-acetylated N-azidoacetyl-D-mannosamine to generate unnatural sialic acids with azide groups on the surface of the target cells. The azide-labeled cells were then transplanted to mouse liver, and dibenzyl cyclooctyne-conjugated Cy5 (DBCO-Cy5) was intravenously injected into mice to chemically bind with the azide groups on the surface of the target cells in vivo for target cell visualization. Unnatural sialic acids with azide groups could be artificially induced on the surface of target cells by glycoengineering. We then tracked the azide groups on the surface of the cells by DBCO-Cy5 in vivo using bioorthogonal click chemistry. Importantly, labeling efficacy was enhanced and false signals by phagocytosis of macrophages were reduced. This strategy will be highly useful for cell labeling and tracking. PMID:24578725

  9. Electrostatically Stabilized Magnetic Nanoparticles - An Optimized Protocol to Label Murine T Cells for in vivo MRI.

    PubMed

    Wuerfel, Eva; Smyth, Maureen; Millward, Jason M; Schellenberger, Eyk; Glumm, Jana; Prozorovski, Timour; Aktas, Orhan; Schulze-Topphoff, Ulf; Schnorr, Jörg; Wagner, Susanne; Taupitz, Matthias; Infante-Duarte, Carmen; Wuerfel, Jens

    2011-01-01

    We present a novel highly efficient protocol to magnetically label T cells applying electrostatically stabilized very small superparamagnetic iron oxide particles (VSOP). Our long-term aim is to use magnetic resonance imaging (MRI) to investigate T cell dynamics in vivo during the course of neuroinflammatory disorders such as experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. Encephalitogenic T cells were co-incubated with VSOP, or with protamine-complexed VSOP (VProt), respectively, at different conditions, optimizing concentrations and incubation times. Labeling efficacy was determined by atomic absorption spectrometry as well as histologically, and evaluated on a 7 T MR system. Furthermore, we investigated possible alterations of T cell physiology caused by the labeling procedure. T cell co-incubation with VSOP resulted in an efficient cellular iron uptake. T2 times of labeled cells dropped significantly, resulting in prominent hypointensity on T2*-weighted scans. Optimal labeling efficacy was achieved by VProt (1 mM Fe/ml, 8 h incubation; T2 time shortening of ∼80% compared to untreated cells). Although VSOP promoted T cell proliferation and altered the ratio of T cell subpopulations toward a CD4(+) phenotype, no effects on CD4 T cell proliferation or phenotypic stability were observed by labeling in vitro differentiated Th17 cells with VProt. Yet, high concentrations of intracellular iron oxide might induce alterations in T cell function, which should be considered in cell tagging studies. Moreover, we demonstrated that labeling of encephalitogenic T cells did not affect pathogenicity; labeled T cells were still capable of inducing EAE in susceptible recipient mice. PMID:22203815

  10. Red Ginseng Extract Reduced Metastasis of Colon Cancer Cells In Vitro and In Vivo

    PubMed Central

    Seo, Eun Young; Kim, Woo Kyoung

    2011-01-01

    This study investigated the effect of red ginseng extract on metastasis of colon cancer cells in vitro and in vivo. Wound healing migration, cell motility, invasion, and activity, protein expression, and mRNA expression of matrix metalloproteinases (MMPs) were examined in SW480 human colon cancer cells. SW480 cells were cultured with or without 100 μg/L PMA in the absence or presence of various concentrations (100, 200, or 300 μg/mL) of red ginseng extract. Red ginseng extract treatment caused significant suppression of cell motility and invasion (p<0.05) in SW480 cells. Red ginseng extract inhibited MMP-2 and MMP-9 activity and their protein and mRNA expression in a dose-dependent manner (p<0.05) in SW480 cells. For experimental metastasis, BALB/c mice were injected intravenously with CT-26 mouse colon cancer cells in the tail vein, and were orally administered various concentrations (0, 75, 150, or 300 mg/kg body weight) of red ginseng extract for 3 weeks. Numbers of pulmonary nodules were significantly decreased in mice that were fed red ginseng extract (p<0.05). Plasma MMP-2 and MMP-9 activity significantly decreased in response to treatment with red ginseng extract in mice (p<0.05). These data suggest that red ginseng extract may be useful for prevention of cancer invasion and metastasis through inhibition of MMP-2 and MMP-9 pathways. PMID:23717075

  11. Modulation of ligand-mediated human red cell agglutinability by prostaglandins

    SciTech Connect

    McLawhon, R.W.; Marikovsky, Y.; Weinstein, R.S.

    1986-03-01

    Ethanol induces the transformation of human red cells from bioconcave discs to echinocytes in vitro. In addition, they have observed that ethanol can enhance the agglutination of red cells by the plant lectin wheat germ agglutinin or poly-L-lysine. Incubation of washed human red cells with 5 and 10% ethanol (v/v) in phosphate buffered saline, pH 7.3 at 25/sup 0/C produced a 30% increase in ligand-mediated agglutinability within 12 min. Simultaneous addition of ethanol and one of the following prostaglandin derivatives, PGE/sub 1/, pge/sub 2/, pgf/sub 2/-alpha, or PGl/sub 2/ (10/sup -9/ to 5 x 10/sup -7/ M) prevented the shape-associated increases in red cell agglutinability. Thromboxane-B/sub 2/ had no effect on agglutinability. Prostaglandins did not prevent ethanol-induced red cell shape transformations per se under identical experimental conditions. As intragastric administration of 100% ethanol results in the formation of spiculated red cell thrombi in postcapillary venules of rat gastric mucosa, they postulate that the cytoprotective role of prostanoids in preventing mucosal ulceration may be due in part to their capacity to inhibit intravascular ligand mediated red cell agglutination, hemostasis, and their sequelae, epithelial necrosis. Moreover, the data suggest that ethanol-induced red cell shape transformations and ligand-mediated agglutination represent two distinct and independent biological phenomena.

  12. Antibody-mediated red blood cell agglutination resulting in spontaneous echocardiographic contrast.

    PubMed

    Miller, M R; Thompson, W R; Casella, J F; Spevak, P J

    1999-01-01

    Spontaneous echocardiographic contrast is well reported in states of low flow and low shear stress, and the primary blood component involved has been reported as red blood cells via rouleaux formation. This report describes the occurrence of spontaneous echocardiographic contrast from a unique mechanism of IgM-mediated red blood cell agglutination and describes the clinical sequelae. PMID:10368455

  13. Mach-Zehnder interferometer for separation of platelets from red blood cells using dielectrophoretics

    NASA Astrophysics Data System (ADS)

    Shwetha, M.; Narayan, K.

    2016-03-01

    In this work, separation of platelets from red blood cells using Mach-Zehnder interferometer is shown using Dielectrophoretics (DEP). The proposed model demonstrates continuous separation of platelets from red blood cells. Mach-Zehnder Interferometer (MZI) has two arms, in which sensing arm will sense according to the applied voltage and separate the platelets from mixed blood cells. The platelets and the red blood cells will flow in two outlets of MZI. Microfluidic device is used to separate the RBC's and the platelets from the mixed blood cells.

  14. Mechanical properties of stored red blood cells using optical tweezers

    NASA Astrophysics Data System (ADS)

    Fontes, Adriana; Alexandre de Thomaz, Andre; de Ysasa Pozzo, Liliana; de Lourdes Barjas-Castro, Maria; Brandao, Marcelo M.; Saad, Sara T. O.; Barbosa, Luiz Carlos; Cesar, Carlos Lenz

    2005-08-01

    We have developed a method for measuring the red blood cell (RBC) membrane overall elasticity μ by measuring the deformation of the cells when dragged at a constant velocity through a plasma fluid by an optical tweezers. The deformability of erythrocytes is a critical determinant of blood flow in the microcirculation. We tested our method and hydrodynamic models, which included the presence of two walls, by measuring the RBC deformation as a function of drag velocity and of the distance to the walls. The capability and sensitivity of this method can be evaluated by its application to a variety of studies, such as, the measurement of RBC elasticity of sickle cell anemia patients comparing homozygous (HbSS), including patients taking hydroxyrea (HU) and heterozygous (HbAS) with normal donors and the RBC elasticity measurement of gamma irradiated stored blood for transfusion to immunosupressed patients as a function of time and dose. These studies show that the technique has the sensitivity to discriminate heterozygous and homozygous sickle cell anemia patients from normal donors and even follow the course of HU treatment of Homozygous patients. The gamma irradiation studies show that there is no significant change in RBC elasticity over time for up to 14 days of storage, regardless of whether the unit was irradiated or not, but there was a huge change in the measured elasticity for the RBC units stored for more than 21 days after irradiation. These finds are important for the assessment of stored irradiated RBC viability for transfusion purposes because the present protocol consider 28 storage days after irradiation as the limit for the RBC usage.

  15. Targeted erythropoietin selectively stimulates red blood cell expansion in vivo.

    PubMed

    Burrill, Devin R; Vernet, Andyna; Collins, James J; Silver, Pamela A; Way, Jeffrey C

    2016-05-10

    The design of cell-targeted protein therapeutics can be informed by natural protein-protein interactions that use cooperative physical contacts to achieve cell type specificity. Here we applied this approach in vivo to the anemia drug erythropoietin (EPO), to direct its activity to EPO receptors (EPO-Rs) on red blood cell (RBC) precursors and prevent interaction with EPO-Rs on nonerythroid cells, such as platelets. Our engineered EPO molecule was mutated to weaken its affinity for EPO-R, but its avidity for RBC precursors was rescued via tethering to an antibody fragment that specifically binds the human RBC marker glycophorin A (huGYPA). We systematically tested the impact of these engineering steps on in vivo markers of efficacy, side effects, and pharmacokinetics. huGYPA transgenic mice dosed with targeted EPO exhibited elevated RBC levels, with only minimal platelet effects. This in vivo selectivity depended on the weakening EPO mutation, fusion to the RBC-specific antibody, and expression of huGYPA. The terminal plasma half-life of targeted EPO was ∼28.3 h in transgenic mice vs. ∼15.5 h in nontransgenic mice, indicating that huGYPA on mature RBCs acted as a significant drug sink but did not inhibit efficacy. In a therapeutic context, our targeting approach may allow higher restorative doses of EPO without platelet-mediated side effects, and also may improve drug pharmacokinetics. These results demonstrate how rational drug design can improve in vivo specificity, with potential application to diverse protein therapeutics. PMID:27114509

  16. Effect of Hydroperoxides on Red Blood Cell Membrane Mechanical Properties

    PubMed Central

    Hale, John P.; Winlove, C. Peter; Petrov, Peter G.

    2011-01-01

    We investigate the effect of oxidative stress on red blood cell membrane mechanical properties in vitro using detailed analysis of the membrane thermal fluctuation spectrum. Two different oxidants, the cytosol-soluble hydrogen peroxide and the membrane-soluble cumene hydroperoxide, are used, and their effects on the membrane bending elastic modulus, surface tension, strength of confinement due to the membrane skeleton, and 2D shear elastic modulus are measured. We find that both oxidants alter significantly the membrane elastic properties, but their effects differ qualitatively and quantitatively. While hydrogen peroxide mainly affects the elasticity of the membrane protein skeleton (increasing the membrane shear modulus), cumene hydroperoxide has an impact on both membrane skeleton and lipid bilayer mechanical properties, as can be seen from the increased values of the shear and bending elastic moduli. The biologically important implication of these results is that the effects of oxidative stress on the biophysical properties, and hence the physiological functions, of the cell membrane depend on the nature of the oxidative agent. Thermal fluctuation spectroscopy provides a means of characterizing these different effects, potentially in a clinical milieu. PMID:22004746

  17. Reduction of prion infectivity in packed red blood cells

    SciTech Connect

    Morales, Rodrigo; Buytaert-Hoefen, Kimberley A.; Gonzalez-Romero, Dennisse; Castilla, Joaquin; Hansen, Eric T.; Hlavinka, Dennis; Goodrich, Raymond P.; Soto, Claudio

    2008-12-12

    The link between a new variant form of Creutzfeldt-Jakob disease (vCJD) and the consumption of prion contaminated cattle meat as well as recent findings showing that vCJD can be transmitted by blood transfusion have raised public health concerns. Currently, a reliable test to identify prions in blood samples is not available. The purpose of this study was to evaluate the possibility to remove scrapie prion protein (PrP{sup Sc}) and infectivity from red blood cell (RBC) suspensions by a simple washing procedure using a cell separation and washing device. The extent of prion removal was assessed by Western blot, PMCA and infectivity bioassays. Our results revealed a substantial removal of infectious prions ({>=}3 logs of infectivity) by all techniques used. These data suggest that a significant amount of infectivity present in RBC preparations can be removed by a simple washing procedure. This technology may lead to increased safety of blood products and reduce the risk of further propagation of prion diseases.

  18. Kinematics of red cell aspiration by fluorescence-imaged microdeformation.

    PubMed

    Discher, D E; Mohandas, N

    1996-10-01

    Maps of fluorescing red cell membrane components on a pipette-aspirated projection are quantitated in an effort to elucidate and unify the heterogeneous kinematics of deformation. Transient gradients of diffusing fluorescent lipid first demonstrate the fluidity of an otherwise uniform-density bilayer and corroborate a "universal" calibration scale for relative surface density. A steep but smooth and stable gradient in the densities of the skeleton components spectrin, actin, and protein 4.1 is used to estimate large elastic strains along the aspirated skeleton. The deformation fields are argued to be an unhindered response to loading in the surface normal direction. Density maps intermediate to those of the compressible skeleton and fluid bilayer are exhibited by particular transmembrane proteins (e.g., Band 3) and yield estimates for the skeleton-connected fractions. Such connected proteins appear to occupy a significant proportion of the undeformed membrane surface and can lead to steric exclusion of unconnected integral membrane proteins from regions of network condensation. Consistent with membrane repatterning kinematics in reversible deformation, final vesiculation of the projection tip produces a cell fragment concentrated in freely diffusing proteins but depleted of skeleton. PMID:8889146

  19. Kinematics of red cell aspiration by fluorescence-imaged microdeformation.

    PubMed Central

    Discher, D E; Mohandas, N

    1996-01-01

    Maps of fluorescing red cell membrane components on a pipette-aspirated projection are quantitated in an effort to elucidate and unify the heterogeneous kinematics of deformation. Transient gradients of diffusing fluorescent lipid first demonstrate the fluidity of an otherwise uniform-density bilayer and corroborate a "universal" calibration scale for relative surface density. A steep but smooth and stable gradient in the densities of the skeleton components spectrin, actin, and protein 4.1 is used to estimate large elastic strains along the aspirated skeleton. The deformation fields are argued to be an unhindered response to loading in the surface normal direction. Density maps intermediate to those of the compressible skeleton and fluid bilayer are exhibited by particular transmembrane proteins (e.g., Band 3) and yield estimates for the skeleton-connected fractions. Such connected proteins appear to occupy a significant proportion of the undeformed membrane surface and can lead to steric exclusion of unconnected integral membrane proteins from regions of network condensation. Consistent with membrane repatterning kinematics in reversible deformation, final vesiculation of the projection tip produces a cell fragment concentrated in freely diffusing proteins but depleted of skeleton. Images FIGURE 1 FIGURE 2 FIGURE 4 FIGURE 5 FIGURE 7 FIGURE 8 FIGURE 9 FIGURE 10 FIGURE 11 PMID:8889146

  20. Mechanical response of red blood cells entering a constriction.

    PubMed

    Zeng, Nancy F; Ristenpart, William D

    2014-11-01

    Most work on the dynamic response of red blood cells (RBCs) to hydrodynamic stress has focused on linear velocity profiles. Relatively little experimental work has examined how individual RBCs respond to pressure driven flow in more complex geometries, such as the flow at the entrance of a capillary. Here, we establish the mechanical behaviors of healthy RBCs undergoing a sudden increase in shear stress at the entrance of a narrow constriction. We pumped RBCs through a constriction in a microfluidic device and used high speed video to visualize and track the flow behavior of more than 4400 RBCs. We show that approximately 85% of RBCs undergo one of four distinct modes of motion: stretching, twisting, tumbling, or rolling. Intriguingly, a plurality of cells (∼30%) exhibited twisting (rotation around the major axis parallel to the flow direction), a mechanical behavior that is not typically observed in linear velocity profiles. We present detailed statistical analyses on the dynamics of each motion and demonstrate that the behavior is highly sensitive to the location of the RBC within the channel. We further demonstrate that the observed tumbling, twisting, and rolling rotations can be rationalized qualitatively in terms of rigid body mechanics. The detailed experimental statistics presented here should serve as a useful resource for modeling of RBC behavior under physiologically important flow conditions. PMID:25553197

  1. Twisting of Red Blood Cells Entering a Constriction

    NASA Astrophysics Data System (ADS)

    Zeng, Nancy; Ristenpart, William

    2014-11-01

    Most work on the dynamic response of red blood cells (RBCs) to hydrodynamic stress has focused on linear velocity profiles. Relatively little experimental work has examined how individual RBCs respond to pressure driven flow in more complex geometries, such as the flow at the entrance of a capillary. Here, we establish the mechanical behaviors of healthy RBCs undergoing a sudden increase in shear stress at the entrance of a narrow constriction. We pumped RBCs through a constriction in an ex vivo microfluidic device and used high speed video to visualize and track the flow behavior of more than 4,400 RBCs. We show that approximately 85% of RBCs undergo one of four distinct modes of motion: stretching, twisting, tumbling, or rolling. Intriguingly, a plurality of cells (~30%) exhibited twisting (rotation around the major axis parallel to the flow direction), a mechanical behavior that is not typically observed in linear velocity profiles. We examine the mechanical origin of twisting using, as a limiting case, the equations of motion for rigid ellipsoids, and we demonstrate that the observed rotation is qualitatively consistent with rigid body theory.

  2. Enhancement of red blood cell aggregation by plasma triglycerides.

    PubMed

    Cicha, I; Suzuki, Y; Tateishi, N; Maeda, N

    2001-01-01

    The effects of plasma triglycerides level on human red blood cells (RBCs) indices, hematological parameters, RBCs aggregation velocity and whole blood viscosity were studied at 2 hours after high-fat or low-fat meal. Proteins, triglycerides and cholesterol levels of plasma were analysed. The RBCs rouleaux formation rate was measured in 70% autologous plasma (with 30% phosphate-buffered saline, PBS) or 1 g/dl dextran T70 solution (with 4 g/dl bovine serum albumin) in PBS, using a low-shear rheoscope. The results were grouped according to triglycerides content in plasma. No significant difference in whole blood viscosity, hematological parameters, RBC indices, protein and cholesterol content was observed between high-fat and low-fat blood samples. There was a significant increase in rouleaux formation rate of samples with high triglyceride levels, when measured in 70% autologous plasma, but it was not significant in dextran T70 containing medium. In conclusion, the results obtained suggest that alteration of plasma lipid levels as well as possible changes in the cell membrane lipid composition lead to enhanced RBC aggregation. PMID:11564913

  3. Small-molecule labeling of live cell surfaces for three-dimensional super-resolution microscopy.

    PubMed

    Lee, Marissa K; Rai, Prabin; Williams, Jarrod; Twieg, Robert J; Moerner, W E

    2014-10-01

    Precise imaging of the cell surface of fluorescently labeled bacteria requires super-resolution methods because the size-scale of these cells is on the order of the diffraction limit. In this work, we present a photocontrollable small-molecule rhodamine spirolactam emitter suitable for non-toxic and specific labeling of the outer surface of cells for three-dimensional (3D) super-resolution (SR) imaging. Conventional rhodamine spirolactams photoswitch to the emitting form with UV light; however, these wavelengths can damage cells. We extended photoswitching to visible wavelengths >400 nm by iterative synthesis and spectroscopic characterization to optimize the substitution on the spirolactam. Further, an N-hydroxysuccinimide-functionalized derivative enabled covalent labeling of amines on the surface of live Caulobacter crescentus cells. Resulting 3D SR reconstructions of the labeled cell surface reveal uniform and specific sampling with thousands of localizations per cell and excellent localization precision in x, y, and z. The distribution of cell stalk lengths (a sub-diffraction-sized cellular structure) was quantified for a mixed population of cells. Pulse-chase experiments identified sites of cell surface growth. Covalent labeling with the optimized rhodamine spirolactam label provides a general strategy to study the surfaces of living cells with high specificity and resolution down to 10-20 nm. PMID:25222297

  4. Small-Molecule Labeling of Live Cell Surfaces for Three-Dimensional Super-Resolution Microscopy

    PubMed Central

    2015-01-01

    Precise imaging of the cell surface of fluorescently labeled bacteria requires super-resolution methods because the size-scale of these cells is on the order of the diffraction limit. In this work, we present a photocontrollable small-molecule rhodamine spirolactam emitter suitable for non-toxic and specific labeling of the outer surface of cells for three-dimensional (3D) super-resolution (SR) imaging. Conventional rhodamine spirolactams photoswitch to the emitting form with UV light; however, these wavelengths can damage cells. We extended photoswitching to visible wavelengths >400 nm by iterative synthesis and spectroscopic characterization to optimize the substitution on the spirolactam. Further, an N-hydroxysuccinimide-functionalized derivative enabled covalent labeling of amines on the surface of live Caulobacter crescentus cells. Resulting 3D SR reconstructions of the labeled cell surface reveal uniform and specific sampling with thousands of localizations per cell and excellent localization precision in x, y, and z. The distribution of cell stalk lengths (a sub-diffraction-sized cellular structure) was quantified for a mixed population of cells. Pulse-chase experiments identified sites of cell surface growth. Covalent labeling with the optimized rhodamine spirolactam label provides a general strategy to study the surfaces of living cells with high specificity and resolution down to 10–20 nm. PMID:25222297

  5. Labeling of mesenchymal stem cells for MRI with single-cell sensitivity.

    PubMed

    Ariza de Schellenberger, Angela; Kratz, Harald; Farr, Tracy D; Löwa, Norbert; Hauptmann, Ralf; Wagner, Susanne; Taupitz, Matthias; Schnorr, Jörg; Schellenberger, Eyk A

    2016-01-01

    Sensitive cell detection by magnetic resonance imaging (MRI) is an important tool for the development of cell therapies. However, clinically approved contrast agents that allow single-cell detection are currently not available. Therefore, we compared very small iron oxide nanoparticles (VSOP) and new multicore carboxymethyl dextran-coated iron oxide nanoparticles (multicore particles, MCP) designed by our department for magnetic particle imaging (MPI) with discontinued Resovist(®) regarding their suitability for detection of single mesenchymal stem cells (MSC) by MRI. We achieved an average intracellular nanoparticle (NP) load of >10 pg Fe per cell without the use of transfection agents. NP loading did not lead to significantly different results in proliferation, colony formation, and multilineage in vitro differentiation assays in comparison to controls. MRI allowed single-cell detection using VSOP, MCP, and Resovist(®) in conjunction with high-resolution T2*-weighted imaging at 7 T with postprocessing of phase images in agarose cell phantoms and in vivo after delivery of 2,000 NP-labeled MSC into mouse brains via the left carotid artery. With optimized labeling conditions, a detection rate of ~45% was achieved; however, the experiments were limited by nonhomogeneous NP loading of the MSC population. Attempts should be made to achieve better cell separation for homogeneous NP loading and to thus improve NP-uptake-dependent biocompatibility studies and cell detection by MRI and future MPI. Additionally, using a 7 T MR imager equipped with a cryocoil resulted in approximately two times higher detection. In conclusion, we established labeling conditions for new high-relaxivity MCP, VSOP, and Resovist(®) for improved MRI of MSC with single-cell sensitivity. PMID:27110112

  6. Labeling of mesenchymal stem cells for MRI with single-cell sensitivity

    PubMed Central

    Ariza de Schellenberger, Angela; Kratz, Harald; Farr, Tracy D; Löwa, Norbert; Hauptmann, Ralf; Wagner, Susanne; Taupitz, Matthias; Schnorr, Jörg; Schellenberger, Eyk A

    2016-01-01

    Sensitive cell detection by magnetic resonance imaging (MRI) is an important tool for the development of cell therapies. However, clinically approved contrast agents that allow single-cell detection are currently not available. Therefore, we compared very small iron oxide nanoparticles (VSOP) and new multicore carboxymethyl dextran-coated iron oxide nanoparticles (multicore particles, MCP) designed by our department for magnetic particle imaging (MPI) with discontinued Resovist® regarding their suitability for detection of single mesenchymal stem cells (MSC) by MRI. We achieved an average intracellular nanoparticle (NP) load of >10 pg Fe per cell without the use of transfection agents. NP loading did not lead to significantly different results in proliferation, colony formation, and multilineage in vitro differentiation assays in comparison to controls. MRI allowed single-cell detection using VSOP, MCP, and Resovist® in conjunction with high-resolution T2*-weighted imaging at 7 T with postprocessing of phase images in agarose cell phantoms and in vivo after delivery of 2,000 NP-labeled MSC into mouse brains via the left carotid artery. With optimized labeling conditions, a detection rate of ~45% was achieved; however, the experiments were limited by nonhomogeneous NP loading of the MSC population. Attempts should be made to achieve better cell separation for homogeneous NP loading and to thus improve NP-uptake-dependent biocompatibility studies and cell detection by MRI and future MPI. Additionally, using a 7 T MR imager equipped with a cryocoil resulted in approximately two times higher detection. In conclusion, we established labeling conditions for new high-relaxivity MCP, VSOP, and Resovist® for improved MRI of MSC with single-cell sensitivity. PMID:27110112

  7. MR Imaging Features of Gadofluorine-Labeled Matrix-Associated Stem Cell Implants in Cartilage Defects

    PubMed Central

    Do, Thuy; Sutton, Elizabeth J.; Baehner, Frederick; Horvai, Andrew; Sennino, Barbara; McDonald, Donald; Meier, Reinhard; Misselwitz, Bernd; Link, Thomas M.; Daldrup-Link, Heike E.

    2012-01-01

    Objectives The purpose of our study was to assess the chondrogenic potential and the MR signal effects of GadofluorineM-Cy labeled matrix associated stem cell implants (MASI) in pig knee specimen. Materials and Methods Human mesenchymal stem cells (hMSCs) were labeled with the micelle-based contrast agent GadofluorineM-Cy. Ferucarbotran-labeled hMSCs, non-labeled hMSCs and scaffold only served as controls. Chondrogenic differentiation was induced and gene expression and histologic evaluation were performed. The proportions of spindle-shaped vs. round cells of chondrogenic pellets were compared between experimental groups using the Fisher's exact test. Labeled and unlabeled hMSCs and chondrocytes in scaffolds were implanted into cartilage defects of porcine femoral condyles and underwent MR imaging with T1- and T2-weighted SE and GE sequences. Contrast-to-noise ratios (CNR) between implants and adjacent cartilage were determined and analyzed for significant differences between different experimental groups using the Kruskal-Wallis test. Significance was assigned for p<0.017, considering a Bonferroni correction for multiple comparisons. Results Collagen type II gene expression levels were not significantly different between different groups (p>0.017). However, hMSC differentiation into chondrocytes was superior for unlabeled and GadofluorineM-Cy-labeled cells compared with Ferucarbotran-labeled cells, as evidenced by a significantly higher proportion of spindle cells in chondrogenic pellets (p<0.05). GadofluorineM-Cy-labeled hMSCs and chondrocytes showed a positive signal effect on T1-weighted images and a negative signal effect on T2-weighted images while Ferucarbotran-labeled cells provided a negative signal effect on all sequences. CNR data for both GadofluorineM-Cy-labeled and Ferucarbotran-labeled hMSCs were significantly different compared to unlabeled control cells on T1-weighted SE and T2*-weighted MR images (p<0.017). Conclusion hMSCs can be labeled by

  8. Modified procedure for labelling target cells in a europium release assay of natural killer cell activity.

    PubMed

    Pacifici, R; Di Carlo, S; Bacosi, A; Altieri, I; Pichini, S; Zuccaro, P

    1993-05-01

    Lanthanide europium chelated to diethylenetriaminopentaacetate (EuDTPA) can be used to label target cells such as tumor cells and lymphocytes (Blomberg et al., 1986a,b; Granberg et al., 1988). This procedure has permitted the development of new non-radioactive methods for the detection of target cell cytolysis by natural killer (NK) cells (Blomberg et al., 1986a,b), cytotoxic T lymphocytes (CTL) (Granberg et al., 1988) or complement-mediated cytolysis (Cui et al., 1992). However, we had no success with this method because of a lack of comparability between human NK cell activity simultaneously measured by a classical 51Cr release assay (Seaman et al., 1981) and EuDTPA release assay (Blomberg et al., 1986a). Furthermore, cell division and cell viability were significantly impaired by the suggested concentrations of EuCl3. In this paper, we present a modified non-cytotoxic method for target cell labelling with EuDTPA while cells are growing in culture medium. PMID:8486925

  9. Electrophoretic characterization of aldehyde-fixed red blood cells, kidney cells, lynphocytes and chamber coatings

    NASA Technical Reports Server (NTRS)

    1976-01-01

    Ground-based electrokinetic data on the electrophoresis flight experiment to be flown on the Apollo-Soyuz Test Project experiment MA-011 are stipulated. Aldehyde-fixed red blood cells, embryonic kidney cells and lymphocytes were evaluated by analytical particle electrophoresis. The results which aided in the interpretation of the final analysis of the MA-011 experiment are documented. The electrophoresis chamber surface modifications, the buffer, and the material used in the column system are also discussed.

  10. Comparison of the labeling efficiency of BrdU, DiI and FISH labeling techniques in bone marrow stromal cells.

    PubMed

    Li, Na; Yang, Hui; Lu, Lingling; Duan, Chunli; Zhao, Chunli; Zhao, Huanying

    2008-06-18

    Cells are generally labeled during in vivo implantation studies enabling the cells to be traced. The relationship between the labeling efficiency and cellular proliferation after transplantation is critical for the interpretation of data obtained by detection of the signals on tissue sections. Here, we compare cellular labeling methods of rat marrow stromal cells that were labeled with 5-bromo-2-deoxyuridine (BrdU), 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) and fluorescence in situ hybridization (FISH). Our data show that (i) BrdU uniformly labeled the nuclei, (ii) DiI-labeled cells had many dots or stained clear and uniform when a longer exposure time was used during detection and (iii) FISH labeled the cells with dots along the edges of the nuclei. The labeling efficiency was 94.1+/-8.6%, 97.6+/-3.4% and 90.5+/-3.0%, in BrdU, DiI- and FISH-labeled cells, respectively. After sub-culturing of labeled cells, the percentage of BrdU-positive cells was found to be 71.9+/-18.0% and 18.4+/-6.9%, after the first and second passages, respectively. The percentage of DiI-labeled cells detected depended on the exposure time: a long exposure time (>10 s) resulted in identification of 95.1+/-4.0% and 94.5+/-3.9% DiI-positive cells after the first and second sub-cultures, respectively. The percentage of FISH-positive cells was found to be 87.0+/-3.0% and 89.1+/-9.7%. The BrdU labeling signal quickly decreased over time. Thus, BrdU should only be used to temporarily label dividing cells. In contrast, our data indicate that DiI and FISH labeling may be used to steadily trace cells during in vivo experiments. To our knowledge, this is the first time that the effects of different labeling methods over time have been examined during a cell transplantation study. PMID:18468584

  11. Influence of red cell concentration on filtration of blood cell suspensions.

    PubMed

    Schmalzer, E A; Skalak, R; Usami, S; Vayo, M; Chien, S

    1983-01-01

    Pressure-time curves obtained by passing suspensions of blood cells in Ringer solution through a 5 microns polycarbonate filter at constant flow (1.6 ml/min) were evaluated for their ability to reflect the deformability of the erythrocytes. The initial pressure reading (Pi) obtained in a quasi-steady state during the first 1-2 sec of pumping was found to be reproducible for hematocrit values between 10 and 30 percent. This Pi value was normalized by the pressure generated by the cell-free suspending medium (PO) at the same flow rate. The ratio Pi/PO was found to be linearly proportional to hematocrit up to 30 percent but independent of leukocyte concentration up to 12,000/mm3. Later portions of the curve did vary with leukocyte count. By using the equations developed from theoretical modeling of cells passing through a filter, the experimentally determined relation of Pi/PO to hematocrit, and the known geometry of the filter pores, we were able to calculate parameters reflecting the deformability of red cells. These include beta, the ratio of resistance in a pore containing a red cell to that in a pore containing only the suspending medium, and alpha, the proportion of pores filled by erythrocytes in transit. The application of theoretical analysis to experimental data has provided quantitative insights into the behavior of red cells during filtration tests in normal and disease states. PMID:6871424

  12. Time Dependent Assessment of Morphological Changes: Leukodepleted Packed Red Blood Cells Stored in SAGM

    PubMed Central

    2016-01-01

    Usually packed red blood cells (pRBCs) require specific conditions in storage procedures to ensure the maximum shelf life of up to 42 days in 2–6°C. However, molecular and biochemical consequences can affect the stored blood cells; these changes are collectively labeled as storage lesions. In this study, the effect of prolonged storage was assessed through investigating morphological changes and evaluating oxidative stress. Samples from leukodepleted pRBC in SAGM stored at 4°C for 42 days were withdrawn aseptically on day 0, day 14, day 28, and day 42. Morphological changes were observed using scanning electron microscopy and correlated with osmotic fragility and hematocrit. Oxidative injury was studied through assessing MDA level as a marker for lipid peroxidation. Osmotic fragility test showed that extended storage time caused increase in the osmotic fragility. The hematocrit increased by 6.6% from day 0 to day 42. The last 2 weeks show alteration in the morphology with the appearance of echinocytes and spherocytes. Storage lesions and morphological alterations appeared to affect RBCs during the storage period. Further studies should be performed to develop strategies that will aid in the improvement of stored pRBC quality and efficacy. PMID:26904677

  13. Indolic Uremic Solutes Enhance Procoagulant Activity of Red Blood Cells through Phosphatidylserine Exposure and Microparticle Release

    PubMed Central

    Gao, Chunyan; Ji, Shuting; Dong, Weijun; Qi, Yushan; Song, Wen; Cui, Debin; Shi, Jialan

    2015-01-01

    Increased accumulation of indolic uremic solutes in the blood of uremic patients contributes to the risk of thrombotic events. Red blood cells (RBCs), the most abundant blood cells in circulation, may be a privileged target of these solutes. However, the effect of uremic solutes indoxyl sulfate (IS) and indole-3-acetic acid (IAA) on procoagulant activity (PCA) of erythrocyte is unclear. Here, RBCs from healthy adults were treated with IS and IAA (mean and maximal concentrations reported in uremic patients). Phosphatidylserine (PS) exposure of RBCs and their microparticles (MPs) release were labeled with Alexa Fluor 488-lactadherin and detected by flow cytometer. Cytosolic Ca2+ ([Ca2+]) with Fluo 3/AM was analyzed by flow cytometer. PCA was assessed by clotting time and purified coagulation complex assays. We found that PS exposure, MPs generation, and consequent PCA of RBCs at mean concentrations of IS and IAA enhanced and peaked in maximal uremic concentrations. Moreover, 128 nM lactadherin, a PS inhibitor, inhibited over 90% PCA of RBCs and RMPs. Eryptosis or damage, by indolic uremic solutes was due to, at least partially, the increase of cytosolic [Ca2+]. Our results suggest that RBC eryptosis in uremic solutes IS and IAA plays an important role in thrombus formation through releasing RMPs and exposing PS. Lactadherin acts as an efficient anticoagulant in this process. PMID:26516916

  14. Indolic uremic solutes enhance procoagulant activity of red blood cells through phosphatidylserine exposure and microparticle release.

    PubMed

    Gao, Chunyan; Ji, Shuting; Dong, Weijun; Qi, Yushan; Song, Wen; Cui, Debin; Shi, Jialan

    2015-11-01

    Increased accumulation of indolic uremic solutes in the blood of uremic patients contributes to the risk of thrombotic events. Red blood cells (RBCs), the most abundant blood cells in circulation, may be a privileged target of these solutes. However, the effect of uremic solutes indoxyl sulfate (IS) and indole-3-acetic acid (IAA) on procoagulant activity (PCA) of erythrocyte is unclear. Here, RBCs from healthy adults were treated with IS and IAA (mean and maximal concentrations reported in uremic patients). Phosphatidylserine (PS) exposure of RBCs and their microparticles (MPs) release were labeled with Alexa Fluor 488-lactadherin and detected by flow cytometer. Cytosolic Ca(2+) ([Ca(2+)]) with Fluo 3/AM was analyzed by flow cytometer. PCA was assessed by clotting time and purified coagulation complex assays. We found that PS exposure, MPs generation, and consequent PCA of RBCs at mean concentrations of IS and IAA enhanced and peaked in maximal uremic concentrations. Moreover, 128 nM lactadherin, a PS inhibitor, inhibited over 90% PCA of RBCs and RMPs. Eryptosis or damage, by indolic uremic solutes was due to, at least partially, the increase of cytosolic [Ca(2+)]. Our results suggest that RBC eryptosis in uremic solutes IS and IAA plays an important role in thrombus formation through releasing RMPs and exposing PS. Lactadherin acts as an efficient anticoagulant in this process. PMID:26516916

  15. Simultaneous measurement of NK cell cytotoxicity against two target cell lines labelled with fluorescent lanthanide chelates.

    PubMed

    Lövgren, J; Blomberg, K

    1994-07-12

    We describe a cytotoxicity assay which permits the simultaneous measurement of natural killer cell activity against two different cell lines. The target cell lines are labelled either with a fluorescent europium chelate or with a fluorescent terbium chelate and cell death is quantified by measuring the chelate release. K-562, Molt4 and Daudi cell lines have been used as targets. The release of the two chelates from the target cells can be detected with the help of time resolved fluorometry. As the measurements are made after background fluorescence has decayed no additional steps are needed to correct for the background from the medium. The assay procedure used for measurement of cytotoxicity against two target cell lines is very similar to the widely used 51Cr release assay. PMID:8034979

  16. Colloidal Properties of Nanoerythrosomes Derived from Bovine Red Blood Cells.

    PubMed

    Kuo, Yuan-Chia; Wu, Hsuan-Chen; Hoang, Dao; Bentley, William E; D'Souza, Warren D; Raghavan, Srinivasa R

    2016-01-12

    Liposomes are nanoscale containers that are typically synthesized from lipids using a high-shear process such as extrusion or sonication. While liposomes are extensively used in drug delivery, they do suffer from certain problems including limited colloidal stability and short circulation times in the body. As an alternative to liposomes, we explore a class of container structures derived from erythrocytes (red blood cells). The procedure involves emptying the inner contents of these cells (specifically hemoglobin) and resuspending the empty structures in buffer, followed by sonication. The resulting structures are termed nanoerythrosomes (NERs), i.e., they are membrane-covered nanoscale containers, much like liposomes. Cryo-transmission electron microscopy (cryo-TEM) and small-angle neutron scattering (SANS) are employed for the first time to study these NERs. The results reveal that the NERs are discrete spheres (∼110 nm diameter) with a unilamellar membrane of thickness ∼4.5 nm. Remarkably, the biconcave disc-like shape of erythrocytes is also exhibited by the NERs under hypertonic conditions. Moreover, unlike typical liposomes, NERs show excellent colloidal stability in both buffer as well as in serum at room temperature, and are also able to withstand freeze-thaw cycling. We have explored the potential for using NERs as colloidal vehicles for targeted delivery. Much like conventional liposomes, NER membranes can be decorated with fluorescent or other markers, solutes can be encapsulated in the cores of the NERs, and NERs can be targeted to specifically bind to mammalian cells. Our study shows that NERs are a promising and versatile class of nanostructures. NERs that are harvested from a patient's own blood and reconfigured for nanomedicine can potentially offer several benefits including biocompatibility, minimization of immune response, and extended circulation time in the body. PMID:26684218

  17. Antigen site distribution among weak A' red cell populations. A study of A3, Ax and Aend variants.

    PubMed Central

    Cartron, J P; Reyes, F; Gourdin, M F; Garretta, M; Salmon, C H

    1977-01-01

    The distribution of the A receptors was studied among 'agglutinated' and 'free' populations of A variant RBC (A3, AX, Aend) known to be either partially or weakly agglutinated by human anti-A reagents. Following separation of the red cell populations and disaggregation of the clumps by mild treatment with soluble blood group substances, it was shown after appropriate controls, that among A3 ARBC, the 'agglutinated' RBC have at least five times as 'free' RBC, these latter however being strongly A positive. The differences between the A antigenic content of the AX RBC were less pronounced. The most striking result was obtained with the Aend RBC, where two populations are clearly demonstrated; the first, including 5-10 per cent of the RBC, strongly agglutinates with anti-A and contains erythrocytes of high antigenic content (140,000 A receptors per cell). The second, including the majority of RBC could not be differentiated from the control O RBC. A wide heterogeneity of antibody binding capacity of the various populations of A3, AX and Aend red cells, was also demonstrated following ultrastructural examination by immunoelectron microscopy with peroxidase-conjugated antibodies. Such study reveals furthermore an heterogeneity of labelling from one cell to another in the same population of red blood cells. Comparison of 'week A' RBC and O RBC enzymatically converted into A RBC, demonstrates a similar pattern of reactivity between these cells, and supports the general relationship between antigen site density and red cell agglutination. It is concluded that the typical pattern of agglutinability of A3 and AX RBC arises both from their heterogeneous antigenic content and from the occurrence of an antigenic threshold below which red cells become non-agglutinable. The typical mixed-field agglutination pattern of Aend RBC merely reflects the occurrence of a probably true dual population of RBC. Finally, the mechanisms of inheritance of such well-known Mendelian characters

  18. Differentiation of cytotoxicity using target cells labelled with europium and samarium by electroporation.

    PubMed

    Bohlen, H; Manzke, O; Engert, A; Hertel, M; Hippler-Altenburg, R; Diehl, V; Tesch, H

    1994-07-12

    We report the simultaneous use of europium-DTPA (Eu-DTPA) and samarium-DTPA (Sm-DTPA) in cytotoxicity experiments to analyze simultaneously LAK and NK cell lysis and to differentiate between specific target lysis and bystander killing. The target cells were either labelled with Eu-DTPA or Sm-DTPA chelates by electroporation, which permits the use of target cell lines or primary leukemic B cells (B-CLL) that cannot be labelled by the conventional dextran-sulphate method. The release of europium and samarium reaches a maximum at comparable time intervals (2-3 h). Due to the shorter counting interval within the samarium window the labelling efficiency is about ten times less efficient compared to europium. Using europium as label for the LAK target Daudi and samarium as label for the NK sensitive cell line K562 the differentiation of LAK versus NK activity can be performed in a single culture assay. Also, the killing of B cells and bystander cells by cytotoxic T cells was analyzed in a system where T cells were redirected to B cells through CD3 x CD19 bispecific antibodies. In fact, no bystander killing was noted when bispecific antibodies were used to bridge cytotoxic T cells to the B cells. This approach provides a simple non-radioactive method for evaluating cytotoxicity against two different cells in a single culture well. PMID:8034986

  19. Solvatochromic Nile Red probes with FRET quencher reveal lipid order heterogeneity in living and apoptotic cells.

    PubMed

    Kreder, Rémy; Pyrshev, Kyrylo A; Darwich, Zeinab; Kucherak, Oleksandr A; Mély, Yves; Klymchenko, Andrey S

    2015-06-19

    Detecting and imaging lipid microdomains (rafts) in cell membranes remain a challenge despite intensive research in the field. Two types of fluorescent probes are used for this purpose: one specifically labels a given phase (liquid ordered, Lo, or liquid disordered, Ld), while the other, being environment-sensitive (solvatochromic), stains the two phases in different emission colors. Here, we combined the two approaches by designing a phase-sensitive probe of the Ld phase and a quencher of the Ld phase. The former is an analogue of the recently developed Nile Red-based probe NR12S, bearing a bulky hydrophobic chain (bNR10S), while the latter is based on Black Hole Quencher-2 designed as bNR10S (bQ10S). Fluorescence spectroscopy of large unilamellar vesicles and microscopy of giant vesicles showed that the bNR10S probe can partition specifically into the Ld phase, while bQ10S can specifically quench the NR12S probe in the Ld phase so that only its fraction in the Lo phase remains fluorescent. Thus, the toolkit of two probes with quencher can specifically target Ld and Lo phases and identify their lipid order from the emission color. Application of this toolkit in living cells (HeLa, CHO, and 293T cell lines) revealed heterogeneity in the cell plasma membranes, observed as distinct probe environments close to the Lo and Ld phases of model membranes. In HeLa cells undergoing apoptosis, our toolkit showed the formation of separate domains of the Ld-like phase in the form of blebs. The developed tools open new possibilities in lipid raft research. PMID:25710589

  20. Theoretical models for near forward light scattering by a Plasmodium falciparum infected red blood cell

    NASA Astrophysics Data System (ADS)

    Sharma, S. K.

    2012-12-01

    A number of experimental elastic light scattering studies have been performed in the past few years with the aim of developing automated in vivo tools for differentiating a healthy red blood cell from a Plasmodium falciparum infected cell. This paper examines some theoretical aspects of the problem. An attempt has been made to simulate the scattering patterns of healthy as well as infected individual red blood cells. Two models, namely, a homogeneous sphere model and a coated sphere model have been considered. The scattering patterns predicted by these models are examined. A possible method for discriminating infected red blood cells from healthy ones has been suggested.

  1. Mn-doped Zinc Sulphide nanocrystals for immunofluorescent labeling of epidermal growth factor receptors on cells and clinical tumor tissues

    NASA Astrophysics Data System (ADS)

    J, Aswathy; V, Seethalekshmy N.; R, Hiran K.; R, Bindhu M.; K, Manzoor; Nair, Shantikumar V.; Menon, Deepthy

    2014-11-01

    The field of molecular detection and targeted imaging has evolved considerably with the introduction of fluorescent semiconductor nanocrystals. Manganese-doped zinc sulphide nanocrystals (ZnS:Mn NCs), which are widely used in electroluminescent displays, have been explored for the first time for direct immunofluorescent (IF) labeling of clinical tumor tissues. ZnS:Mn NCs developed through a facile wet chemistry route were capped using amino acid cysteine, conjugated to streptavidin and thereafter coupled to biotinylated epidermal growth factor receptor (EGFR) antibody utilizing the streptavidin-biotin linkage. The overall conjugation yielded stable EGFR antibody conjugated ZnS:Mn NCs (EGFR ZnS:Mn NCs) with a hydrodynamic diameter of 65 ± 15 nm, and having an intense orange-red fluorescence emission at 598 nm. Specific labeling of EGF receptors on EGFR+ve A431 cells in a co-culture with EGFR-ve NIH3T3 cells was demonstrated using these nanoprobes. The primary antibody conjugated fluorescent NCs could also clearly delineate EGFR over-expressing cells on clinical tumor tissues processed by formalin fixation as well as cryopreservation with a specificity of 86% and accuracy of 88%, in comparison to immunohistochemistry. Tumor tissues labeled with EGFR ZnS:Mn NCs showed good fluorescence emission when imaged after storage even at 15 months. Thus, ZnS nanobioconjugates with dopant-dependent and stable fluorescence emission show promise as an efficient, target-specific fluorophore that would enable long term IF labeling of any antigen of interest on clinical tissues.

  2. 111Indium labeling of hepatocytes for analysis of short-term biodistribution of transplanted cells.

    PubMed

    Gupta, S; Lee, C D; Vemuru, R P; Bhargava, K K

    1994-03-01

    Hepatocyte transplantation is useful for ex vivo gene therapy and liver repopulation. Methods for hepatic reconstitution have recently been developed but optimization of hepatocyte transplantation systems is necessary. To develop systems for noninvasive assessment of the biodistribution of transplanted cells, we labeled hepatocytes with 111indium-oxine. Our initial studies showed that hepatocytes incorporated 111indium-oxine with an efficiency of approximately 20%. After labeling, cell viability was unchanged and 111indium was present in hepatocytes after overnight culture, as well as after intrasplenic transplantation. Transplanted cells were successfully localized by means of scintigraphic imaging. The scintigraphic patterns of cell distribution were different when hepatocytes were transplanted by means of either spleen or internal jugular vein, which deposit cells into separate vascular beds. Quantitative analysis of the biodistribution of 111indium-labeled hepatocytes indicated that within 2 hr of intrasplenic transplantation, cells were predominantly localized in liver and spleen, and occasionally in lungs. To determine whether the rate of intrasplenic cell injection influenced translocation of hepatocytes, we transplanted cells in normal rats. Despite intrasplenic cell injection at a variety of rates, organ-specific distribution of 111indium-labeled hepatocytes remained unchanged. Labeling with 111indium did not affect long-term survival of transplanted hepatocytes. These results indicate that 111indium-labeling of hepatocytes should greatly assist noninvasive analysis in the short-term of the biodistribution of transplanted hepatocytes. PMID:8119703

  3. GABAergic and glycinergic pathways to goldfish retinal ganglion cells: an ultrastructural double label study

    SciTech Connect

    Muller, J.F.

    1987-01-01

    An ultrastructural double label has been employed to compare GABAergic and glycinergic systems in the inner plexiform layer (IPL) of the goldfish retina. Electron microscope autoradiography of /sup 3/H-GABA and /sup 3/H-glycine uptake was combined with retrograde HRP-labeling of ganglion cells. When surveyed for distribution, GABAergic and glycinergic synapses were found onto labeled ganglion cells throughout the IPL. This reinforces previous physiological work that described GABAergic and glycinergic influences on a variety of ganglion cells in goldfish and carp; These physiological effects often reflect direct inputs.

  4. Cell Surface Proteome of Dental Pulp Stem Cells Identified by Label-Free Mass Spectrometry.

    PubMed

    Niehage, Christian; Karbanová, Jana; Steenblock, Charlotte; Corbeil, Denis; Hoflack, Bernard

    2016-01-01

    Multipotent mesenchymal stromal cells (MSCs) are promising tools for regenerative medicine. They can be isolated from different sources based on their plastic-adherence property. The identification of reliable cell surface markers thus becomes the Holy Grail for their prospective isolation. Here, we determine the cell surface proteomes of human dental pulp-derived MSCs isolated from single donors after culture expansion in low (2%) or high (10%) serum-containing media. Cell surface proteins were tagged on intact cells using cell impermeable, cleavable sulfo-NHS-SS-biotin, which allows their enrichment by streptavidin pull-down. For the proteomic analyses, we first compared label-free methods to analyze cell surface proteomes i.e. composition, enrichment and proteomic differences, and we developed a new mathematical model to determine cell surface protein enrichment using a combinatorial gene ontology query. Using this workflow, we identified 101 cluster of differentiation (CD) markers and 286 non-CD cell surface proteins. Based on this proteome profiling, we identified 14 cell surface proteins, which varied consistently in abundance when cells were cultured under low or high serum conditions. Collectively, our analytical methods provide a basis for identifying the cell surface proteome of dental pulp stem cells isolated from single donors and its evolution during culture or differentiation. Our data provide a comprehensive cell surface proteome for the precise identification of dental pulp-derived MSC populations and their isolation for potential therapeutic intervention. PMID:27490675

  5. Cell Surface Proteome of Dental Pulp Stem Cells Identified by Label-Free Mass Spectrometry

    PubMed Central

    Niehage, Christian; Karbanová, Jana; Steenblock, Charlotte

    2016-01-01

    Multipotent mesenchymal stromal cells (MSCs) are promising tools for regenerative medicine. They can be isolated from different sources based on their plastic-adherence property. The identification of reliable cell surface markers thus becomes the Holy Grail for their prospective isolation. Here, we determine the cell surface proteomes of human dental pulp-derived MSCs isolated from single donors after culture expansion in low (2%) or high (10%) serum-containing media. Cell surface proteins were tagged on intact cells using cell impermeable, cleavable sulfo-NHS-SS-biotin, which allows their enrichment by streptavidin pull-down. For the proteomic analyses, we first compared label-free methods to analyze cell surface proteomes i.e. composition, enrichment and proteomic differences, and we developed a new mathematical model to determine cell surface protein enrichment using a combinatorial gene ontology query. Using this workflow, we identified 101 cluster of differentiation (CD) markers and 286 non-CD cell surface proteins. Based on this proteome profiling, we identified 14 cell surface proteins, which varied consistently in abundance when cells were cultured under low or high serum conditions. Collectively, our analytical methods provide a basis for identifying the cell surface proteome of dental pulp stem cells isolated from single donors and its evolution during culture or differentiation. Our data provide a comprehensive cell surface proteome for the precise identification of dental pulp-derived MSC populations and their isolation for potential therapeutic intervention. PMID:27490675

  6. Photosensitized labeling of a functional multidrug transporter in living drug-resistant tumor cells

    SciTech Connect

    Raviv, Y.; Pollard, H.B.; Bruggemann, E.P.; Pastan, I.; Gottesman, M.M. )

    1990-03-05

    A 170,000-Da glycoprotein (P170 multidrug transporter) becomes specifically labeled in multidrug-resistant human KB carcinoma cells by the photolabile lipophilic membrane probe 5-(125I)iodonaphthalene-1-azide ((125I)INA) when photoactivation of the probe is triggered by energy transfer from intracellular doxorubicin or rhodamine 123. In contrast, in drug-sensitive cells, drug-induced specific labeling of membrane proteins with (125I)INA was not observed. Instead, multiple membrane proteins became labeled in a nonspecific manner. This phenomenon of drug-induced specific labeling of P170 by (125I)INA is observed only in living cells, but not in purified membrane vesicles or lysed cells. It is generated by doxorubicin and rhodamine 123, drugs that are chromophores and to which the cells exhibit resistance; but it is not observed with other drugs or dyes. Verapamil, a calcium channel blocker which reverses resistance to doxorubicin, also abolishes doxorubicin-induced specific (125I)INA labeling of P170. These results reveal that a specific interaction between P170 and doxorubicin takes place in living cells and demonstrate that P170 is directly involved in the mechanism of drug resistance in vivo. They also provide a possible means to label functional domains in the multidrug transporter. The results demonstrate that photosensitized (125I)INA labeling is a technique which provides sufficient spatial and time resolution to detect specific intracellular interactions between chromophores and proteins in vivo.

  7. Neonatal nucleated red blood cells in G6PD deficiency.

    PubMed

    Yeruchimovich, Mark; Shapira, Boris; Mimouni, Francis B; Dollberg, Shaul

    2002-05-01

    The objective of this study is to study the absolute number of nucleated red blood cells (RBC) at birth, an index of active fetal erythropoiesis, in infants with G6PD deficiency and in controls. We tested the hypothesis that hematocrit and hemoglobin would be lower, and absolute nucleated RBC counts higher, in the G6PD deficient and that these changes would be more prominent in infants exposed passively to fava bean through maternal diet. Thirty-two term infants with G6PD deficiency were compared with 30 term controls. Complete blood counts with manual differential counts were obtained within 12 hours of life. Absolute nucleated RBC and corrected leukocyte counts were computed from the Coulter results and the differential count. G6PD deficient patients did not differ from controls in terms of gestational age, birth weight, or Apgar scores or in any of the hematologic parameters studied, whether or not the mother reported fava beans consumption in the days prior to delivery. Although intrauterine hemolysis is possible in G6PD deficient fetuses exposed passively to fava beans, our study supports that such events must be very rare. PMID:12012283

  8. Characterization of Red Blood Cells with Multiwavelength Transmission Spectroscopy

    PubMed Central

    Serebrennikova, Yulia M.; Huffman, Debra E.; Garcia-Rubio, Luis H.

    2015-01-01

    Multiwavelength transmission (MWT) spectroscopy was applied to the investigation of the morphological parameters and composition of red blood cells (RBCs). The MWT spectra were quantitatively analyzed with a Mie theory based interpretation model modified to incorporate the effects of the nonsphericity and orientation of RBCs. The MWT spectra of the healthy and anemic samples were investigated for the RBC indices in open and blinded studies. When MWT performance was evaluated against a standard reference system, very good agreement between two methods, with R2 > 0.85 for all indices studied, was demonstrated. The RBC morphological parameters were used to characterize three types of anemia and to draw an association between RBC morphology and anemia severity. The MWT spectra of RBCs infected with malaria parasite Plasmodium falciparum at different life cycle stages were analyzed for RBC morphological parameters. The changes in the RBC volume, surface area, aspect ratio, and hemoglobin composition were used to trace the morphological and compositional alterations in the infected RBCs occurring with parasites' development and to provide insights into parasite-host interactions. The MWT method was shown to be reliable for determination of the RBC morphological parameters and to be valuable for identification of the RBC pathologic changes and disease states. PMID:25654099

  9. Characterization of red blood cells with multiwavelength transmission spectroscopy.

    PubMed

    Serebrennikova, Yulia M; Huffman, Debra E; Garcia-Rubio, Luis H

    2015-01-01

    Multiwavelength transmission (MWT) spectroscopy was applied to the investigation of the morphological parameters and composition of red blood cells (RBCs). The MWT spectra were quantitatively analyzed with a Mie theory based interpretation model modified to incorporate the effects of the nonsphericity and orientation of RBCs. The MWT spectra of the healthy and anemic samples were investigated for the RBC indices in open and blinded studies. When MWT performance was evaluated against a standard reference system, very good agreement between two methods, with R (2) > 0.85 for all indices studied, was demonstrated. The RBC morphological parameters were used to characterize three types of anemia and to draw an association between RBC morphology and anemia severity. The MWT spectra of RBCs infected with malaria parasite Plasmodium falciparum at different life cycle stages were analyzed for RBC morphological parameters. The changes in the RBC volume, surface area, aspect ratio, and hemoglobin composition were used to trace the morphological and compositional alterations in the infected RBCs occurring with parasites' development and to provide insights into parasite-host interactions. The MWT method was shown to be reliable for determination of the RBC morphological parameters and to be valuable for identification of the RBC pathologic changes and disease states. PMID:25654099

  10. Ratio of Spleen Diameter to Red Blood Cell Distribution Width

    PubMed Central

    Balaban, Daniel Vasile; Popp, Alina; Lungu, Andrei Marian; Costache, Raluca Simona; Anca, Ioana Alina; Jinga, Mariana

    2015-01-01

    Abstract Celiac disease (CD) is currently considerably underdiagnosed, setting the need for developing tools to select patients with probability of CD, who warrant further testing. Red blood cell distribution width (RDW) has been shown in previous studies to be a sensitive predictor for CD, but it lacks specificity. Splenic hypotrophy is also noted frequently in celiac patients. Our aim was to evaluate if spleen diameter to RDW ratio can be used as an indicator for CD. We evaluated 15 newly diagnosed CD patients, 52 patients with inflammatory bowel disease, and 35 patients with irritable bowel syndrome (IBS). We evaluated the differences in spleen diameter, RDW, and their ratio among the four groups. Two-thirds of the CD patients had elevated RDW, compared to 9% in the IBS group. A small spleen was seen in 80% of the celiacs, compared to 21.9% in the ulcerative colitis group, 10% in the Crohn disease group, and 9% in the IBS group. A spleen diameter to RDW ratio under 6 had a sensitivity of 73.3% and specificity of 88.5% in predicting CD, with an AUROC of 0.737. Spleen diameter to RDW ratio is a simple, widely available score, which can be used to select adult patients with probability of CD. PMID:25881851

  11. Alteration of red blood cell aggregation during blood storage

    NASA Astrophysics Data System (ADS)

    Lim, Hyun-Jung; Nam, Jeong-Hun; Lee, Byoung-Kwon; Suh, Jang-Soo; Shin, Sehyun

    2011-06-01

    Even though the trade-off between the benefits and risks of blood transfusion has been discussed for the last several decades, it requires further understanding of the rheological changes in stored blood that include the alteration of red blood cell (RBC) aggregation. The RBC aggregation of stored blood in its autologous plasma was monitored through the storage period (35 days). The critical shear stress, as a measure of RBC aggregation, was determined by using a microfluidic aggregometer. Blood was processed into a blood bag containing the anticoagulant CPDA1 and stored at 4°C. It was subjected to assays after zero, seven, 14, and 35 days. The critical shear stress for stored blood did not change up to 14 days of storage but exhibited a significant decrease after 35 days of storage. These results were identical to those of the conventional aggregation index (AI). Also, in the alteration of RBC aggregation for blood storage, the effect of the plasma factor was slightly stronger than that of the cellular factor. Through the present study, the critical shear stress as a new measure of RBC aggregation may help to monitor and control the quality of blood storage.

  12. Harmless Pregnancy-Induced Warm Autoantibodies to Red Blood Cells

    PubMed Central

    Sürücü, Gülüstan; Mayer, Beate; Märzacker, Anneliese; Yürek, Salih; Salama, Abdulgabar

    2015-01-01

    Summary Background There is little information concerning the development and significance of autoantibodies (aab) to red blood cells (RBCs) during pregnancy. Methods Unselected pregnant women were routinely screened for the presence of unexpected antibodies to RBCs using standard techniques. Results Between 2009 and 2013, 153,612 pregnant women were tested. The antibody screening test was positive in 1,721 women (1.12%). In 1,602 (1.04%) cases, immune and/or non-immune alloantibodies and cold-reactive aab were detected, whereas warm-reactive aab were found in 119 women (0.08%). In almost all cases, warm-reactive aab belonged to the IgG class. No evidence of the presence of significant haemolysis in affected women was observed. Conclusion Pregnant women may rarely develop aab to RBCs, which do not appear to cause haemolytic anaemia. Further clarification is required on the reasons behind the development of these aab and their clinical insignificance. PMID:26696801

  13. Continuum modeling of deformation and aggregation of red blood cells.

    PubMed

    Yoon, Daegeun; You, Donghyun

    2016-07-26

    In order to gain better understanding for rheology of an isolated red blood cell (RBC) and a group of multiple RBCs, new continuum models for describing mechanical properties of cellular structures of an RBC and inter-cellular interactions among multiple RBCs are developed. The viscous property of an RBC membrane, which characterizes dynamic behaviors of an RBC under stress loading and unloading processes, is determined using a generalized Maxwell model. The present model is capable of predicting stress relaxation and stress-strain hysteresis, of which prediction is not possible using the commonly used Kelvin-Voigt model. Nonlinear elasticity of an RBC is determined using the Yeoh hyperelastic material model in a framework of continuum mechanics using finite-element approximation. A novel method to model inter-cellular interactions among multiple adjacent RBCs is also developed. Unlike the previous modeling approaches for aggregation of RBCs, where interaction energy for aggregation is curve-fitted using a Morse-type potential function, the interaction energy is analytically determined. The present aggregation model, therefore, allows us to predict various effects of physical parameters such as the osmotic pressure, the thickness of a glycocalyx layer, the penetration depth, and the permittivity, on the depletion and electrostatic energy among RBCs. Simulations for elongation and recovery deformation of an RBC and for aggregation of multiple RBCs are conducted to evaluate the efficacy of the present continuum modeling methods. PMID:26706720

  14. Interactions between earthworm hemolysins and sheep red blood cell membranes.

    PubMed

    Roch, P; Canicatti, C; Valembois, P

    1989-08-01

    The hemolytic activity exhibited by the coelomic fluid of the Annelid Eisenia fetida andrei is mediated by two lipoproteins of mass 40 and 45 kDa, each of them capable of hemolysis. Such an activity is not inhibited by zymosan, inulin or lipopolysaccharide (LPS), nor by hydrazine or methylamine, suggesting that earthworm hemolysins are not related to C3 or C3b complement components. Among the membrane lipids tested (phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, sphingomyelin and cholesterol) only sphingomyelin inhibited hemolysis. The analysis of E.f. andrei proteins bound to sphingomyelin microvesicles, as well as to sheep red blood cell (SRBC) membranes, revealed a polymerization of E.f. andrei 40 kDa and/or 45 kDa hemolysins. Consequently, sphingomyelin appears a likely candidate for hemolytic complex receptor. Electron microscopy observations suggested that the polymerization causes an open channel through the lipid bilayer. As demonstrated using metal ions, heparin, chondroitin sulfate, poly(L-lysine) and protamine chloride, the mode of action of earthworm hemolytic complex is not analogous to that of C9 or perforine. PMID:2758056

  15. Perioperative Red Blood Cell Transfusion: What We Do Not Know

    PubMed Central

    Lei, Chong; Xiong, Li-Ze

    2015-01-01

    Objective: Blood transfusion saves lives but may also increase the risk of injury. The objective of this review was to evaluate the possible adverse effects related to transfusion of red blood cell (RBC) concentrates stored for prolonged periods. Data Sources: The data used in this review were mainly from PubMed articles published in English up to February 2015. Study Selection: Clinical and basic research articles were selected according to their relevance to this topic. Results: The ex vivo changes to RBC that occur during storage are collectively called storage lesion. It is still inconclusive if transfusion of RBC with storage lesion has clinical relevance. Multiple ongoing prospective randomized controlled trials are aimed to clarify this clinical issue. It was observed that the adverse events related to stored RBC transfusion were prominent in certain patient populations, including trauma, critical care, pediatric, and cardiac surgery patients, which leads to the investigation of underlying mechanisms. It is demonstrated that free hemoglobin toxicity, decreasing of nitric oxide bioavailability, and free iron-induced increasing of inflammation may play an important role in this process. Conclusion: It is still unclear whether transfusion of older RBC has adverse effects, and if so, which factors determine such clinical effects. However, considering the magnitude of transfusion and the widespread medical significance, potential preventive strategies should be considered, especially for the susceptible recipients. PMID:26315088

  16. Red blood cell deformability in diabetes mellitus: effect of phytomenadione.

    PubMed

    Sabo, A; Jakovljević, V; Stanulović, M; Lepsanović, L; Pejin, D

    1993-01-01

    Decreased deformability of red blood cells (RBC) in diabetes mellitus (DM) is considered to be linked to microcirculatory complications in this condition. As we found that phytomenadione increased RBC deformability in experimental animals, the question was raised, whether phytomenadione had the same effect on the RBC of diabetic patients. The study was performed in 10 patients with insulin-dependent diabetes mellitus, where the erythrocyte deformability was impaired. Patients received 10 mg/day phytomenadione i.m. for five days. Deformability was measured with policarbonate membranes (Nucleopore) with pore diameter 5 microns, under gravity. The results were expressed as the ratio (r) between the flow of 1.5 ml (r1) and 2 ml (r2) of RBC suspension and 1.5 ml of buffer. Phytomenadione increased the erythrocyte deformability in patients with diabetes mellitus, lowering the value r1 from 3.54 +/- 0.84 to 2.32 +/- 0.61 (p 0.02) and r2 from 7.80 +/- 2.41 to 4.65 +/- 1.07 (p 0.01). The values after treatment reached the range of healthy controls (r1 3.11 +/- 0.98, r2 6.52 +/- 3.04). The whole blood viscosity was significantly lowered after phytomenadione (5.28 +/- 0.58 mPas before, 4.64 +/- 0.74 mPas after, p < 0.02) with unchanged plasma viscosity, but significantly lowered internal viscosity of erythrocytes. PMID:8444511

  17. flowCL: ontology-based cell population labelling in flow cytometry

    PubMed Central

    Courtot, Mélanie; Meskas, Justin; Diehl, Alexander D.; Droumeva, Radina; Gottardo, Raphael; Jalali, Adrin; Taghiyar, Mohammad Jafar; Maecker, Holden T.; McCoy, J. Philip; Ruttenberg, Alan; Scheuermann, Richard H.; Brinkman, Ryan R.

    2015-01-01

    Motivation: Finding one or more cell populations of interest, such as those correlating to a specific disease, is critical when analysing flow cytometry data. However, labelling of cell populations is not well defined, making it difficult to integrate the output of algorithms to external knowledge sources. Results: We developed flowCL, a software package that performs semantic labelling of cell populations based on their surface markers and applied it to labelling of the Federation of Clinical Immunology Societies Human Immunology Project Consortium lyoplate populations as a use case. Conclusion: By providing automated labelling of cell populations based on their immunophenotype, flowCL allows for unambiguous and reproducible identification of standardized cell types. Availability and implementation: Code, R script and documentation are available under the Artistic 2.0 license through Bioconductor (http://www.bioconductor.org/packages/devel/bioc/html/flowCL.html). Contact: rbrinkman@bccrc.ca Supplementary information: Supplementary data are available at Bioinformatics online. PMID:25481008

  18. Tumor-initiating label-retaining cancer cells in human gastrointestinal cancers undergo asymmetric cell division.

    PubMed

    Xin, Hong-Wu; Hari, Danielle M; Mullinax, John E; Ambe, Chenwi M; Koizumi, Tomotake; Ray, Satyajit; Anderson, Andrew J; Wiegand, Gordon W; Garfield, Susan H; Thorgeirsson, Snorri S; Avital, Itzhak

    2012-04-01

    Label-retaining cells (LRCs) have been proposed to represent adult tissue stem cells. LRCs are hypothesized to result from either slow cycling or asymmetric cell division (ACD). However, the stem cell nature and whether LRC undergo ACD remain controversial. Here, we demonstrate label-retaining cancer cells (LRCCs) in several gastrointestinal (GI) cancers including fresh surgical specimens. Using a novel method for isolation of live LRCC, we demonstrate that a subpopulation of LRCC is actively dividing and exhibits stem cells and pluripotency gene expression profiles. Using real-time confocal microscopic cinematography, we show live LRCC undergoing asymmetric nonrandom chromosomal cosegregation LRC division. Importantly, LRCCs have greater tumor-initiating capacity than non-LRCCs. Based on our data and that cancers develop in tissues that harbor normal-LRC, we propose that LRCC might represent a novel population of GI stem-like cancer cells. LRCC may provide novel mechanistic insights into the biology of cancer and regenerative medicine and present novel targets for cancer treatment. PMID:22331764

  19. Labeling pluripotent stem cell-derived neural progenitors with iron oxide particles for magnetic resonance imaging.

    PubMed

    Sart, Sébastien; Bejarano, Fabian Calixto; Yan, Yuanwei; Grant, Samuel C; Li, Yan

    2015-01-01

    Due to the unlimited proliferation capacity and the unique differentiation ability of pluripotent stem cells (PSCs), including both embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), large numbers of PSC-derived cell products are in demand for applications in drug screening, disease modeling, and especially cell therapy. In stem cell-based therapy, tracking transplanted cells with magnetic resonance imaging (MRI) has emerged as a powerful technique to reveal cell survival and distribution. This chapter illustrated the basic steps of labeling PSC-derived neural progenitors (NPs) with micron-sized particles of iron oxide (MPIO, 0.86 μm) for MRI analysis. The protocol described PSC expansion and differentiation into NPs, and the labeling of the derived cells either after replating on adherent surface or in suspension. The labeled cells can be analyzed using in vitro MRI analysis. The methods presented here can be easily adapted for cell labeling in cell processing facilities under current Good Manufacturing Practices (cGMP). The iron oxide-labeled NPs can be used for cellular monitoring of in vitro cultures and in vivo transplantation. PMID:25304204

  20. Studies with nonradioisotopic sodium chromate. II. Single- and double-label sup 52 Cr/ sup 51 Cr posttransfusion recovery estimations

    SciTech Connect

    Heaton, W.A.; Keegan, T.; Hanbury, C.M.; Holme, S.; Pleban, P. )

    1989-10-01

    A recently developed nonradioisotopic 52Cr technique was used to measure either red cell volume or posttransfusion recovery of stored red cells. The experimental method uses Zeeman electrothermal atomic absorption spectrophotometry to measure red cell chromium. Results from the 52Cr method were compared with those from 51Cr single-label and 125I-albumin/51Cr double-label procedures using 49-day AS-1 red cell concentrates drawn and prepared according to standard procedures. In the first group of five donors, red cell volume was estimated concurrently with both 52Cr-labeled fresh red cells and 125I-albumin. The latter measured plasma volume from which red cell volume was estimated on the basis of the hematocrit (125I red cell volume). 51Cr-labeled stored red cells were transfused to measure posttransfusion recoveries. The correlation between 52Cr and 125I red cell volumes was significant (r = 0.68, p less than 0.01), and, in this group, the differences were not significant (p less than 0.05). Twenty-four-hour posttransfusion recoveries of 51Cr-labeled stored red cells averaged 66 +/- 5 percent when measured with the 125I/51Cr technique and 69 +/- 8 percent when measured with the 52Cr/51Cr method. In the second group of five donors, red cell volume was estimated by the 125I-albumin technique, and the posttransfusion recovery of stored red cells was quantitated by 51Cr- and 52Cr-labeled stored cells simultaneously. In this group, posttransfusion recoveries with 125I/51Cr averaged 73 +/- 7 percent; with 125I/52Cr, they averaged 75 +/- 10 percent. Using the single-label method of calculation, recoveries averaged 76 +/- 7 and 75 +/- 10 percent for the 51Cr and 52Cr methods, respectively.

  1. Scattering pulse of label free fine structure cells to determine the size scale of scattering structures

    NASA Astrophysics Data System (ADS)

    Zhang, Lu; Chen, Xingyu; Zhang, Zhenxi; Chen, Wei; Zhao, Hong; Zhao, Xin; Li, Kaixing; Yuan, Li

    2016-04-01

    Scattering pulse is sensitive to the morphology and components of each single label-free cell. The most direct detection result, label free cell's scattering pulse is studied in this paper as a novel trait to recognize large malignant cells from small normal cells. A set of intrinsic scattering pulse calculation method is figured out, which combines both hydraulic focusing theory and small particle's scattering principle. Based on the scattering detection angle ranges of widely used flow cytometry, the scattering pulses formed by cell scattering energy in forward scattering angle 2°-5° and side scattering angle 80°-110° are discussed. Combining the analysis of cell's illuminating light energy, the peak, area, and full width at half maximum (FWHM) of label free cells' scattering pulses for fine structure cells with diameter 1-20 μm are studied to extract the interrelations of scattering pulse's features and cell's morphology. The theoretical and experimental results show that cell's diameter and FWHM of its scattering pulse agree with approximate linear distribution; the peak and area of scattering pulse do not always increase with cell's diameter becoming larger, but when cell's diameter is less than about 16 μm the monotone increasing relation of scattering pulse peak or area with cell's diameter can be obtained. This relationship between the features of scattering pulse and cell's size is potentially a useful but very simple criterion to distinguishing malignant and normal cells by their sizes and morphologies in label free cells clinical examinations.

  2. Procoagulant activity in stored units of red blood cells.

    PubMed

    Aleshnick, Maya; Foley, Jonathan H; Keating, Friederike K; Butenas, Saulius

    2016-06-10

    The procoagulant activity (PA) of stored units of red blood cells (RBC) increases over time, which is related to the expression/exposure of tissue factor (TF). However, there is a discrepancy between the TF measured and changes in PA observed, suggesting that other blood components contribute to this activity. Our goal was to evaluate changes in PA of stored RBCs and to determine possible contributors to it. RBC units from 4 healthy donors were prepared and stored at 4 °C. On selected days, RBC aliquots were reconstituted with autologous plasma and tested in the thromboelastography assay. Corresponding supernatants were tested in a clotting assay. For all donors, the clotting time (CT) of reconstituted RBC units decreased from ∼3000-4000s on day 1 to ∼1000-1600s on day 30, with the most dramatic changes occurring between days 1 and 5. Anti-TF antibody slightly prolonged the CT. The concentration of TF did not change significantly over time and was within the range of 0.3-2.3 pM. Bovine lactadherin (LTD) prolonged the CT of the RBC (by 2.4-3.4-fold in days 3-5 and by 1.3-1.8-fold at day 30). Anti-TF antibody together with LTD had a cumulative effect on the CT prolongation. CT of supernatants responded to both anti-TF and anti-FXIa antibodies. Three contributors to the PA of stored RBC were identified, i.e. FXIa in solution and phosphatidylserine and TF exposed on blood cells and microparticles. Failure of LTD and antibodies to completely eliminate PA suggests that other components of blood could contribute to it. PMID:27150627

  3. Measuring red blood cell aggregation forces using double optical tweezers.

    PubMed

    Fernandes, Heloise P; Fontes, Adriana; Thomaz, André; Castro, Vagner; Cesar, Carlos L; Barjas-Castro, Maria L

    2013-04-01

    Classic immunohematology approaches, based on agglutination techniques, have been used in manual and automated immunohematology laboratory routines. Red blood cell (RBC) agglutination depends on intermolecular attractive forces (hydrophobic bonds, Van der Walls, electrostatic forces and hydrogen bonds) and repulsive interactions (zeta potential). The aim of this study was to measure the force involved in RBC aggregation using double optical tweezers, in normal serum, in the presence of erythrocyte antibodies and associated to agglutination potentiator solutions (Dextran, low ionic strength solution [LISS] and enzymes). The optical tweezers consisted of a neodymium:yattrium aluminium garnet (Nd:YAG) laser beam focused through a microscope equipped with a minicam, which registered the trapped cell image in a computer where they could be analyzed using a software. For measuring RBC aggregation, a silica bead attached to RBCs was trapped and the force needed to slide one RBC over the other, as a function of the velocities, was determined. The median of the RBC aggregation force measured in normal serum (control) was 1 × 10(-3) (0.1-2.5) poise.cm. The samples analyzed with anti-D showed 2 × 10(-3) (1.0-4.0) poise.cm (p < 0.001). RBC diluted in potentiator solutions (Dextran 0.15%, Bromelain and LISS) in the absence of erythrocyte antibodies, did not present agglutination. High adherence was observed when RBCs were treated with papain. Results are in agreement with the imunohematological routine, in which non-specific results are not observed when using LISS, Dextran and Bromelain. Nevertheless, false positive results are frequently observed in manual and automated microplate analyzer using papain enzyme. The methodology proposed is simple and could provide specific information with the possibility of meansuration regarding RBC interaction. PMID:23402665

  4. Algal autolysate medium to label proteins for NMR in mammalian cells.

    PubMed

    Fuccio, Carmelo; Luchinat, Enrico; Barbieri, Letizia; Neri, Sara; Fragai, Marco

    2016-04-01

    In-cell NMR provides structural and functional information on proteins directly inside living cells. At present, the high costs of the labeled media for mammalian cells represent a limiting factor for the development of this methodology. Here we report a protocol to prepare a homemade growth medium from Spirulina platensis autolysate, suitable to express uniformly labeled proteins inside mammalian cells at a reduced cost-per-sample. The human proteins SOD1 and Mia40 were overexpressed in human cells grown in (15)N-enriched S. platensis algal-derived medium, and high quality in-cell NMR spectra were obtained. PMID:27106902

  5. Detection of Bacteria in Red Blood Cell Concentrates by the Scansystem Method

    PubMed Central

    Ribault, S.; Faucon, A.; Grave, L.; Nannini, P.; Faure, I. Besson

    2005-01-01

    Bacterial contamination remains one of the major risks associated with blood product transfusion. The kinetics of bacterial growth in red blood cell concentrates (RBCC) is different than otherwise due to storage at 4°C, conditions in which most bacteria do not survive. Psychrophilic bacteria such as Yersinia enterocolitica, however, can proliferate from a very low level of contamination to clinically significant levels at 4°C and are known to cause severe transfusion-related infections. A screening method allowing the early detection of very low levels of bacteria in RBCC would improve transfusion safety. The Scansystem method has been previously described for detection of bacteria in platelet concentrates. We present here a modification of the system for detection of low levels of bacteria in RBCC. The Scansystem RBC kit protocol requires three steps, i.e., the agglutination and selective removal of RBCs, a labeling stage during which bacteria are labeled with a DNA-specific fluorophore, and finally recovery of bacteria on the surface of a black membrane for analysis using the Scansystem. The entire procedure from sampling to result can be completed in 90 min. Both gram-negative and gram-positive bacteria in RBCC are detected with a higher sensitivity than with currently available culture-based methods. The Scansystem RBC kit is shown to be sensitive enough to identify low-level bacterial contamination in a single unit tested in a pool of up to 20 RBCC samples (detection limit of between 1 and 10 CFU/ml depending on the bacterial strain). The method therefore lends itself to incorporation into high-sample-throughput screening programs. PMID:15872251

  6. Detection of bacteria in red blood cell concentrates by the Scansystem method.

    PubMed

    Ribault, S; Faucon, A; Grave, L; Nannini, P; Faure, I Besson

    2005-05-01

    Bacterial contamination remains one of the major risks associated with blood product transfusion. The kinetics of bacterial growth in red blood cell concentrates (RBCC) is different than otherwise due to storage at 4 degrees C, conditions in which most bacteria do not survive. Psychrophilic bacteria such as Yersinia enterocolitica, however, can proliferate from a very low level of contamination to clinically significant levels at 4 degrees C and are known to cause severe transfusion-related infections. A screening method allowing the early detection of very low levels of bacteria in RBCC would improve transfusion safety. The Scansystem method has been previously described for detection of bacteria in platelet concentrates. We present here a modification of the system for detection of low levels of bacteria in RBCC. The Scansystem RBC kit protocol requires three steps, i.e., the agglutination and selective removal of RBCs, a labeling stage during which bacteria are labeled with a DNA-specific fluorophore, and finally recovery of bacteria on the surface of a black membrane for analysis using the Scansystem. The entire procedure from sampling to result can be completed in 90 min. Both gram-negative and gram-positive bacteria in RBCC are detected with a higher sensitivity than with currently available culture-based methods. The Scansystem RBC kit is shown to be sensitive enough to identify low-level bacterial contamination in a single unit tested in a pool of up to 20 RBCC samples (detection limit of between 1 and 10 CFU/ml depending on the bacterial strain). The method therefore lends itself to incorporation into high-sample-throughput screening programs. PMID:15872251

  7. Seasonal variation in cell proliferation and cell migration in the brain of adult red-sided garter snakes (Thamnophis sirtalis parietalis).

    PubMed

    Maine, Ashley R; Powers, Sean D; Lutterschmidt, Deborah I

    2014-01-01

    Plasticity in the adult central nervous system has been described in all vertebrate classes as well as in some invertebrate groups. However, the limited taxonomic diversity represented in the current neurogenesis literature limits our ability to assess the functional significance of adult neurogenesis for natural behaviors as well as the evolution of its regulatory mechanisms. In the present study, we used free-ranging red-sided garter snakes (Thamnophis sirtalis parietalis) to test the hypothesis that seasonal shifts in physiology and behavior are associated with seasonal variation in postembryonic neurogenesis. Specifically, we used the thymidine analog 5-bromo-2'-deoxyuridine (BrdU) to determine if the rates of cell proliferation in the adult brain vary between male snakes collected during spring and fall at 1, 5, and 10 days post-BrdU treatment. To assess rates of cell migration within the brain, we further categorized BrdU-labeled cells according to their location within the ventricular zone or parenchymal region. BrdU-labeled cells were localized mainly within the lateral, dorsal, and medial cortex, septal nucleus, nucleus sphericus, preoptic area, and hypothalamus. In all regions, the number of BrdU-labeled cells in the ventricular zone was higher in the fall compared to spring. In the parenchymal region, a significantly higher number of labeled cells was also observed during the fall, but only within the nucleus sphericus and the combined preoptic area/hypothalamus. The immunoreactive cell number did not vary significantly with days post-BrdU treatment in either season or in any brain region. While it is possible that the higher rates of cell proliferation in the fall simply reflect increased growth of all body tissues, including the brain, our data show that seasonal changes in cell migration into the parenchyma are region specific. In red-sided garter snakes and other reptiles, the dorsal and medial cortex is important for spatial navigation and memory

  8. Perturbation of red blood cell flow in small tubes by white blood cells.

    PubMed

    Thompson, T N; La Celle, P L; Cokelet, G R

    1989-02-01

    The flow of blood in the microcirculation is facilitated by the dynamic reduction in viscosity (Fahraeus-Lindquist effect) resulting from the axial flow of deforming erythrocytes (RBCs) and from the decrease in the ratio of cell to vessel diameter. RBC velocity exceeds that of average fluid velocity; however the slower moving white blood cells (WBC) perturb flow velocity and the ratio of cell to vessel diameter by obstructing red cell flow through formation of "trains" of red cells collecting behind the white cell. This effect of white cells was studied quantitatively in a model in vitro tubes less than 10 microns in diameter with the demonstration that flow resistance increases linearly with white cell numbers up to 1,000 WBC/mm3 at tube hematocrit of 17.7%. The increase in resistance exceeds the flow resistance of WBC and appears to relate directly to train formation. A mechanical model of train formation developed to predict WBC influence in flow resistance over the range of WBC studied reasonably fits observed WBC effects. PMID:2928089

  9. Integrating Cell Phone Imaging with Magnetic Levitation (i-LEV) for Label-Free Blood Analysis at the Point-of-Living.

    PubMed

    Baday, Murat; Calamak, Semih; Durmus, Naside Gozde; Davis, Ronald W; Steinmetz, Lars M; Demirci, Utkan

    2016-03-01

    There is an emerging need for portable, robust, inexpensive, and easy-to-use disease diagnosis and prognosis monitoring platforms to share health information at the point-of-living, including clinical and home settings. Recent advances in digital health technologies have improved early diagnosis, drug treatment, and personalized medicine. Smartphones with high-resolution cameras and high data processing power enable intriguing biomedical applications when integrated with diagnostic devices. Further, these devices have immense potential to contribute to public health in resource-limited settings where there is a particular need for portable, rapid, label-free, easy-to-use, and affordable biomedical devices to diagnose and continuously monitor patients for precision medicine, especially those suffering from rare diseases, such as sickle cell anemia, thalassemia, and chronic fatigue syndrome. Here, a magnetic levitation-based diagnosis system is presented in which different cell types (i.e., white and red blood cells) are levitated in a magnetic gradient and separated due to their unique densities. Moreover, an easy-to-use, smartphone incorporated levitation system for cell analysis is introduced. Using our portable imaging magnetic levitation (i-LEV) system, it is shown that white and red blood cells can be identified and cell numbers can be quantified without using any labels. In addition, cells levitated in i-LEV can be distinguished at single-cell resolution, potentially enabling diagnosis and monitoring, as well as clinical and research applications. PMID:26523938

  10. Effects of Poloxamer 188 on red blood cell membrane properties in sickle cell anaemia.

    PubMed

    Sandor, Barbara; Marin, Mickaël; Lapoumeroulie, Claudine; Rabaï, Miklos; Lefevre, Sophie D; Lemonne, Nathalie; El Nemer, Wassim; Mozar, Anaïs; Français, Olivier; Le Pioufle, Bruno; Connes, Philippe; Le Van Kim, Caroline

    2016-04-01

    Vaso-occlusive crisis (VOC) is the main acute complication in sickle cell anaemia (SS) and several clinical trials are investigating different drugs to improve the clinical severity of SS patients. A phase III study is currently exploring the profit of Velopoloxamer in SS during VOCs. We analysed, in-vitro, the effect of poloxamer (P188) on red blood cell (RBC) properties by investigating haemorheology, mechanical and adhesion functions using ektacytometry, microfluidics and dynamic adhesion approaches, respectively. We show that poloxamer significantly reduces blood viscosity, RBC aggregation and adhesion to endothelial cells, supporting the beneficial use of this molecule in SS therapy. PMID:26846309

  11. Membrane characteristics and osmotic fragility of red cells, fractionated with anglehead centrifugation and counterflow centrifugation.

    PubMed

    van der Vegt, S G; Ruben, A M; Werre, J M; de Gier, J; Staal, G E

    1985-11-01

    Red cell populations were separated on the basis of differences in density using anglehead centrifugation and on the basis of differences in mean cell volume using counterflow centrifugation. In the different fractions, mean surface area was calculated, phospholipid and cholesterol content determined as well as the osmotic behaviour in hypotonic salt solutions. Older red cells appeared to be more resistant to hypotonic salt solutions, due to favourable surface area to volume ratio. PMID:4063204

  12. Deoxygenation-induced and Ca(2+) dependent phosphatidylserine externalisation in red blood cells from normal individuals and sickle cell patients.

    PubMed

    Weiss, Erwin; Cytlak, Urszula M; Rees, David C; Osei, Anna; Gibson, John S

    2012-01-01

    Phosphatidylserine (PS) is usually confined to the inner leaflet of the red blood cell (RBC) membrane. It may become externalised in various conditions, however, notably in RBCs from patients with sickle cell disease (SCD) where exposed PS may contribute to anaemic and ischaemic complications. PS externalisation requires both inhibition of the aminophospholipid translocase (or flippase) and activation of the scramblase. Both may follow from elevation of intracellular Ca(2+). Flippase inhibition occurs at low [Ca(2+)](i), about 1μM, but [Ca(2+)](i) required for scrambling is reported to be much higher (around 100μM). In this work, FITC-labelled lactadherin and FACS were used to measure externalised PS, with [Ca(2+)](i) altered using bromo-A23187 and EGTA/Ca(2+) mixtures. Two components of Ca(2+)-induced scrambling were apparent, of high (EC(50) 1.8±0.3μM) and low (306±123μM) affinity, in RBCs from normal individuals and the commonest SCD genotypes, HbSS and HbSC. The high affinity component was lost in the presence of unphysiologically high [Mg(2+)] but was unaffected by high K(+) (90mM) or vanadate (1mM). The high affinity component accounted for PS scrambling in ≥2/3rd RBCs. It is likely to be most significant in vivo and may be involved in the pathophysiology of SCD or other conditions involving eryptosis. PMID:22197026

  13. Lentivirus-mediated bifunctional cell labeling for in vivo melanoma study

    PubMed Central

    Day, Chi-Ping; Carter, John; Bonomi, Carrie; Esposito, Dominic; Crise, Bruce; Ortiz-Conde, Betty; Hollingshead, Melinda; Merlino, Glenn

    2009-01-01

    SUMMARY Lentiviral vectors (LVs) are capable of labeling a broad spectrum of cell types, achieving stable expression of transgenes. However, for in vivo studies, the duration of marker gene expression has been highly variable. We have developed a series of LVs harboring different promoters for expressing reporter gene in mouse cells. Long-term culture and colony formation of several LV-labeled mouse melanoma cells showed that promoters derived from mammalian house-keeping genes, especially those encoding RNA polymerase II (Pol2) and ferritin (FerH), provided the highest consistency for reporter expression. For in vivo studies, primary B16BL6 mouse melanoma were infected with LVs whose luciferase-GFP fusion gene (Luc/GFP) was driven by either Pol2 or FerH promoters. When transplanted into syngeneic C57BL/6 mice, Luc/GFP-labeled B16BL6 mouse melanoma cells can be monitored by bioluminescence imaging in vivo, and GFP-positive cells can be isolated from the tumors by FACS. Pol2-Luc/GFP labeling, while lower in activity, was more sustainable than FerH-Luc/GFP labeling in B16BL6 over consecutive passages into mice. We conclude that Pol-2-Luc/GFP labeling allows long-term in vivo monitoring and tumor cell isolation in immunocompetent mouse melanoma models. SIGNIFICANCE In this study we have developed and identified lentiviral vectors that allow labeled mouse melanoma cells to maintain long-term and consistent expression of a bifunctional luciferase-GFP marker gene, even in syngeneic mice with an intact immune function. This cell-labeling system can be used to build immunocompetent mouse melanoma models that permit both tumor monitoring and FACS-based tumor cell isolation from tissues, greatly facilitating the in vivo study of melanoma. PMID:19175523

  14. A study of membrane protein defects and alpha hemoglobin chains of red blood cells in human beta thalassemia

    SciTech Connect

    Rouyer-Fessard, P.; Garel, M.C.; Domenget, C.; Guetarni, D.; Bachir, D.; Colonna, P.; Beuzard, Y. )

    1989-11-15

    The soluble pool of alpha hemoglobin chains present in blood or bone marrow cells was measured with a new affinity method using a specific probe, beta A hemoglobin chain labeled with ({sup 3}H)N-ethylmaleimide. This pool of soluble alpha chains was 0.067 {plus minus} 0.017% of hemoglobin in blood of normal adult, 0.11 {plus minus} 0.03% in heterozygous beta thalassemia and ranged from 0.26 to 1.30% in homozygous beta thalassemia intermedia. This elevated pool of soluble alpha chains observed in human beta thalassemia intermedia decreased 33-fold from a value of 10% of total hemoglobin in bone marrow cells to 0.3% in the most dense red blood cells. The amount of insoluble alpha chains was measured by using the polyacrylamide gel electrophoresis in urea and Triton X-100. In beta thalassemia intermedia the amount of insoluble alpha chains was correlated with the decreased spectrin content of red cell membrane and was associated with a decrease in ankyrin and with other abnormalities of the electrophoretic pattern of membrane proteins. The loss and topology of the reactive thiol groups of membrane proteins was determined by using ({sup 3}H)N-ethylmaleimide added to membrane ghosts prior to urea and Triton X-100 electrophoresis. Spectrin and ankyrin were the major proteins with the most important decrease of thiol groups.

  15. A Novel Antifouling Defense Strategy from Red Seaweed: Exocytosis and Deposition of Fatty Acid Derivatives at the Cell Wall Surface.

    PubMed

    Paradas, Wladimir Costa; Tavares Salgado, Leonardo; Pereira, Renato Crespo; Hellio, Claire; Atella, Georgia Correa; de Lima Moreira, Davyson; do Carmo, Ana Paula Barbosa; Soares, Angélica Ribeiro; Menezes Amado-Filho, Gilberto

    2016-05-01

    We investigated the organelles involved in the biosynthesis of fatty acid (FA) derivatives in the cortical cells of Laurencia translucida (Rhodophyta) and the effect of these compounds as antifouling (AF) agents. A bluish autofluorescence (with emission at 500 nm) within L. translucida cortical cells was observed above the thallus surface via laser scanning confocal microscopy (LSCM). A hexanic extract (HE) from L. translucida was split into two isolated fractions called hydrocarbon (HC) and lipid (LI), which were subjected to HPLC coupled to a fluorescence detector, and the same autofluorescence pattern as observed by LSCM analyses (emission at 500 nm) was revealed in the LI fraction. These fractions were analyzed by gas chromatography-mass spectrometry (GC-MS), which revealed that docosane is the primary constituent of HC, and hexadecanoic acid and cholesterol trimethylsilyl ether are the primary components of LI. Nile red (NR) labeling (lipid fluorochrome) presented a similar cellular localization to that of the autofluorescent molecules. Transmission and scanning electron microscopy (TEM and SEM) revealed vesicle transport processes involving small electron-lucent vesicles, from vacuoles to the inner cell wall. Both fractions (HC and LI) inhibited micro-fouling [HC, lower minimum inhibitory concentration (MIC) values of 0.1 µg ml(-1); LI, lower MIC value of 10 µg ml(-1)]. The results suggested that L. translucida cortical cells can produce FA derivatives (e.g. HCs and FAs) and secrete them to the thallus surface, providing a unique and novel protective mechanism against microfouling colonization in red algae. PMID:26936789

  16. Marked increase of calcium uptake in the ATP-depleted red cells of patients with iron deficiency

    SciTech Connect

    Shimoda, M.; Yawata, Y.

    1985-05-01

    Calcium (Ca) uptake was markedly increased in ATP-depleted red cells of patients with iron deficiency anemia (IDA) compared to ATP- depleted normal red cells. The extent of increased Ca uptake was related to the severity of iron deficiency as judged by decreased mean cell volume. Moreover, the increased Ca uptake returned to normal levels after oral iron supplementation therapy. The net calcium content of fresh red cells from iron-deficient individuals was the same as in red cells from normal subjects. Sodium influx and ferric ion uptake appeared to be virtually unaffected in the iron deficient red cells.

  17. Lentivirus-mediated bifunctional cell labeling for in vivo melanoma study.

    PubMed

    Day, Chi-Ping; Carter, John; Bonomi, Carrie; Esposito, Dominic; Crise, Bruce; Ortiz-Conde, Betty; Hollingshead, Melinda; Merlino, Glenn

    2009-06-01

    Lentiviral vectors (LVs) are capable of labeling a broad spectrum of cell types, achieving stable expression of transgenes. However, for in vivo studies, the duration of marker gene expression has been highly variable. We have developed a series of LVs harboring different promoters for expressing reporter gene in mouse cells. Long-term culture and colony formation of several LV-labeled mouse melanoma cells showed that promoters derived from mammalian house-keeping genes, especially those encoding RNA polymerase II (Pol2) and ferritin (FerH), provided the highest consistency for reporter expression. For in vivo studies, primary B16BL6 mouse melanoma were infected with LVs whose luciferase-green fluorescence protein fusion gene (Luc/GFP) was driven by either Pol2 or FerH promoters. When transplanted into syngeneic C57BL/6 mice, Luc/GFP-labeled B16BL6 mouse melanoma cells can be monitored by bioluminescence imaging in vivo, and GFP-positive cells can be isolated from the tumors by fluorescence-activated cell sorter. Pol2-Luc/GFP labeling, while lower in activity, was more sustainable than FerH-Luc/GFP labeling in B16BL6 over consecutive passages into mice. We conclude that Pol-2-Luc/GFP labeling allows long-term in vivo monitoring and tumor cell isolation in immunocompetent mouse melanoma models. PMID:19175523

  18. Rheological properties of RBC in the microcirculation of mammalian skeletal muscle. [red blood cells

    NASA Technical Reports Server (NTRS)

    Ehrenberg, M. H.

    1974-01-01

    In the investigation the established technique of direct microscopic viewing was combined with the use of a closed circuit television system and cinematography. The red cell flow patterns in all capillaries were found to be oscillatory with characteristic cycle frequencies and amplitudes for all concentrations of inspired oxygen greater than 8%. Generally, there was a transient decrease in mean flow rate with increasing severity of hypoxia, with a gradual return toward control values. Red cell flow patterns are discussed along with questions of red cell configuration.

  19. The acute effects of nifedipine on red cell deformability in angina pectoris.

    PubMed Central

    Waller, D G; Nicholson, H P; Roath, S

    1984-01-01

    In a randomised double-blind study, the effects on red cell deformability of a single sublingual dose of nifedipine were compared with placebo in eight patients with stable angina pectoris. Red cell deformability, measured by filtration and centrifugation techniques, was significantly increased at rest in all eight patients 1 h after nifedipine, while no change occurred after placebo. The improvement in deformability after nifedipine was maintained at the end of a period of exercise and unchanged from resting values after placebo. The results suggest that the increased deformability of red cells after nifedipine could contribute to the therapeutic effects of the drug in myocardial ischaemia. PMID:6704282

  20. The energy-less red blood cell is lost: erythrocyte enzyme abnormalities of glycolysis.

    PubMed

    van Wijk, Richard; van Solinge, Wouter W

    2005-12-15

    The red blood cell depends solely on the anaerobic conversion of glucose by the Embden-Meyerhof pathway for the generation and storage of high-energy phosphates, which is necessary for the maintenance of a number of vital functions. Many red blood cell enzymopathies have been described that disturb the erythrocyte's integrity, shorten its cellular survival, and result in hemolytic anemia. By far the majority of these enzymopathies are hereditary in nature. In this review, we summarize the current knowledge regarding the genetic, biochemical, and structural features of clinically relevant red blood cell enzymopathies involved in the Embden-Meyerhof pathway and the Rapoport-Luebering shunt. PMID:16051738

  1. Detection of IgG sensitization of red cells with /sup 125/I staphylococcal protein A

    SciTech Connect

    Yam, P.; Petz, L.D.; Spath, P.

    1982-06-01

    Most cases of immune hemolytic anemia are associated with a positive direct antiglobulin test. However, in some cases, the antiglobulin test is not sensitive enough to detect low levels of red-cell bound antibodies. This report describes a method using radiolabelled purified staphylococcal protein A which is capable of detecting IgG sensitization of red cells beyond the threshold of serologic techniques. It is less cumbersome than previously described methods and does not require antibody purification procedures. Its effectiveness was demonstrated for the detection of red-cell alloantibodies and in evaluation of patients with acquired hemolytic anemias associated with a negative direct antiglobulin test.

  2. Iodine-131 labeled anti-CEA antibodies uptake by Huerthle cell carcinoma

    SciTech Connect

    Abdel-Nabi, H.; Hinkle, G.H.; Falko, J.M.; Kelly, D.; Olsen, J.O.; Martin, E.W. Jr.

    1985-10-01

    Localization of Huerthle cell cancer deposits in the lung with I-131 labeled anti-carcinoembryonic antigen (CEA) monoclonal antibody is described. This technique may prove useful if conventional scanning with I-131 sodium iodide for distant metastases is negative.

  3. Erythropoietic Protoporphyria: Lipid Peroxidation and Red Cell Membrane Damage Associated with Photohemolysis

    PubMed Central

    Goldstein, Bernard D.; Harber, Leonard C.

    1972-01-01

    The mechanism by which long wavelength ultraviolet light hemolyzes red cells obtained from patients with erythropoietic protoporphyria (EPP) was investigated. Previous studies had suggested that irradiation of these red cells with wavelengths of light capable of eliciting dermatological manifestations led to oxygen-dependent colloid osmotic hemolysis through the formation of peroxides. In the present report, lipid peroxidation during in vitro irradiation of EPP red cells with long ultraviolet light was demonstrated by: (a) the formation of 2-thiobarbituric acid reactants; (b) the presence of conjugated diene bonds in red cell lipid; and (c) the selective loss of unsaturated fatty acids proportional to the number of carbon-carbon double bonds in each. Irradiation of EPP red cells was also shown to result in the formation of hydrogen peroxide. Before photohemolysis there was a decline in cell membrane sulfhydryl groups and a loss in activity of the cell membrane enzyme acetylcholinesterase. These parameters provide further evidence suggesting that the cell membrane is a primary site of the photohemolytic effect of long ultraviolet light in EPP red cells. Further evaluation of the radiation-induced inactivation of EPP red cell acetylcholinesterase was performed by radiating mixtures containing bovine erythrocyte acetylcholinesterase and protoporphyrin IX. These studies revealed that the rate of decline in enzyme activity is accelerated by the addition of linoleic acid, an unsaturated fatty acid, but not by palmitic acid, a saturated fatty acid. Partial protection against both photohemolysis and acetylcholinesterase decline is provided by alpha-to-copherol. This lipid antioxidant loses its activity during the irradiation of EPP red cells suggesting that it is utilized in this process. PMID:5014616

  4. Safe extension of red blood cell storage life at 4{degree}C

    SciTech Connect

    Bitensky, M.; Yoshida, Tatsuro

    1996-04-01

    The project sought to develop methods to extend the storage life of red blood cells. Extended storage would allow donor to self or autologous transfusion, expand and stabilize the blood supply, reduce the cost of medical care and eliminate the risk of transfusion related infections, including a spectrum of hepatitides (A, B and C) and HIV. The putative cause of red blood cell spoilage at 4 C has been identified as oxidative membrane damage resulting from deoxyhemoglobin and its denaturation products including hemichrome, hemin and Fe{sup 3+}. Trials with carbon monoxide, which is a stabilizer of hemoglobin, have produced striking improvement of red blood cell diagnostics for cells stored at 4 C. Carbonmonoxy hemoglobin is readily converted to oxyhemoglobin by light in the presence of oxygen. These findings have generated a working model and an approach to identify the best protocols for optimal red cell storage and hemoglobin regeneration.

  5. Multicolor protein labeling in living cells using mutant β-lactamase-tag technology.

    PubMed

    Watanabe, Shuji; Mizukami, Shin; Hori, Yuichiro; Kikuchi, Kazuya

    2010-12-15

    Protein labeling techniques using small molecule probes have become important as practical alternatives to the use of fluorescent proteins (FPs) in live cell imaging. These labeling techniques can be applied to more sophisticated fluorescence imaging studies such as pulse-chase imaging. Previously, we reported a novel protein labeling system based on the combination of a mutant β-lactamase (BL-tag) with coumarin-derivatized probes and its application to specific protein labeling on cell membranes. In this paper, we demonstrated the broad applicability of our BL-tag technology to live cell imaging by the development of a series of fluorescence labeling probes for this technology, and the examination of the functions of target proteins. These new probes have a fluorescein or rhodamine chromophore, each of which provides enhanced photophysical properties relative to coumarins for the purpose of cellular imaging. These probes were used to specifically label the BL-tag protein and could be used with other small molecule fluorescent probes. Simultaneous labeling using our new probes with another protein labeling technology was found to be effective. In addition, it was also confirmed that this technology has a low interference with respect to the functions of target proteins in comparison to GFP. Highly specific and fast covalent labeling properties of this labeling technology is expected to provide robust tools for investigating protein functions in living cells, and future applications can be improved by combining the BL-tag technology with conventional imaging techniques. The combination of probe synthesis and molecular biology techniques provides the advantages of both techniques and can enable the design of experiments that cannot currently be performed using existing tools. PMID:20961132

  6. Stable isotope labeling by amino acids in cell culture (SILAC) for quantitative proteomics.

    PubMed

    Hoedt, Esthelle; Zhang, Guoan; Neubert, Thomas A

    2014-01-01

    Stable isotope labeling by amino acids in cell culture (SILAC) is a powerful approach for high-throughput quantitative proteomics. SILAC allows highly accurate protein quantitation through metabolic encoding of whole cell proteomes using stable isotope labeled amino acids. Since its introduction in 2002, SILAC has become increasingly popular. In this chapter we review the methodology and application of SILAC, with an emphasis on three research areas: dynamics of posttranslational modifications, protein-protein interactions, and protein turnover. PMID:24952180

  7. Sickle cell disease biochip: a functional red blood cell adhesion assay for monitoring sickle cell disease.

    PubMed

    Alapan, Yunus; Kim, Ceonne; Adhikari, Anima; Gray, Kayla E; Gurkan-Cavusoglu, Evren; Little, Jane A; Gurkan, Umut A

    2016-07-01

    Sickle cell disease (SCD) afflicts millions of people worldwide and is associated with considerable morbidity and mortality. Chronic and acute vaso-occlusion are the clinical hallmarks of SCD and can result in pain crisis, widespread organ damage, and early movtality. Even though the molecular underpinnings of SCD were identified more than 60 years ago, there are no molecular or biophysical markers of disease severity that are feasibly measured in the clinic. Abnormal cellular adhesion to vascular endothelium is at the root of vaso-occlusion. However, cellular adhesion is not currently evaluated clinically. Here, we present a clinically applicable microfluidic device (SCD biochip) that allows serial quantitative evaluation of red blood cell (RBC) adhesion to endothelium-associated protein-immobilized microchannels, in a closed and preprocessing-free system. With the SCD biochip, we have analyzed blood samples from more than 100 subjects and have shown associations between the measured RBC adhesion to endothelium-associated proteins (fibronectin and laminin) and individual RBC characteristics, including hemoglobin content, fetal hemoglobin concentration, plasma lactate dehydrogenase level, and reticulocyte count. The SCD biochip is a functional adhesion assay, reflecting quantitative evaluation of RBC adhesion, which could be used at baseline, during crises, relative to various long-term complications, and before and after therapeutic interventions. PMID:27063958

  8. Sickle cell disease biochip: a functional red blood cell adhesion assay for monitoring sickle cell disease

    PubMed Central

    ALAPAN, YUNUS; KIM, CEONNE; ADHIKARI, ANIMA; GRAY, KAYLA E.; GURKAN-CAVUSOGLU, EVREN; LITTLE, JANE A.; GURKAN, UMUT A.

    2016-01-01

    Sickle cell disease (SCD) afflicts millions of people worldwide and is associated with considerable morbidity and mortality. Chronic and acute vaso-occlusion are the clinical hallmarks of SCD and can result in pain crisis, widespread organ damage, and early movtality. Even though the molecular underpinnings of SCD were identified more than 60 years ago, there are no molecular or biophysical markers of disease severity that are feasibly measured in the clinic. Abnormal cellular adhesion to vascular endothelium is at the root of vaso-occlusion. However, cellular adhesion is not currently evaluated clinically. Here, we present a clinically applicable microfluidic device (SCD biochip) that allows serial quantitative evaluation of red blood cell (RBC) adhesion to endothelium-associated protein-immobilized microchannels, in a closed and preprocessing-free system. With the SCD biochip, we have analyzed blood samples from more than 100 subjects and have shown associations between the measured RBC adhesion to endothelium-associated proteins (fibronectin and laminin) and individual RBC characteristics, including hemoglobin content, fetal hemoglobin concentration, plasma lactate dehydrogenase level, and reticulocyte count. The SCD biochip is a functional adhesion assay, reflecting quantitative evaluation of RBC adhesion, which could be used at baseline, during crises, relative to various long-term complications, and before and after therapeutic interventions. PMID:27063958

  9. Reconciling Estimates of Cell Proliferation from Stable Isotope Labeling Experiments.

    PubMed

    Ahmed, Raya; Westera, Liset; Drylewicz, Julia; Elemans, Marjet; Zhang, Yan; Kelly, Elizabeth; Reljic, Rajko; Tesselaar, Kiki; de Boer, Rob J; Macallan, Derek C; Borghans, José A M; Asquith, Becca

    2015-10-01

    Stable isotope labeling is the state of the art technique for in vivo quantification of lymphocyte kinetics in humans. It has been central to a number of seminal studies, particularly in the context of HIV-1 and leukemia. However, there is a significant discrepancy between lymphocyte proliferation rates estimated in different studies. Notably, deuterated (2)H2-glucose (D2-glucose) labeling studies consistently yield higher estimates of proliferation than deuterated water (D2O) labeling studies. This hampers our understanding of immune function and undermines our confidence in this important technique. Whether these differences are caused by fundamental biochemical differences between the two compounds and/or by methodological differences in the studies is unknown. D2-glucose and D2O labeling experiments have never been performed by the same group under the same experimental conditions; consequently a direct comparison of these two techniques has not been possible. We sought to address this problem. We performed both in vitro and murine in vivo labeling experiments using identical protocols with both D2-glucose and D2O. This showed that intrinsic differences between the two compounds do not cause differences in the proliferation rate estimates, but that estimates made using D2-glucose in vivo were susceptible to difficulties in normalization due to highly variable blood glucose enrichment. Analysis of three published human studies made using D2-glucose and D2O confirmed this problem, particularly in the case of short term D2-glucose labeling. Correcting for these inaccuracies in normalization decreased proliferation rate estimates made using D2-glucose and slightly increased estimates made using D2O; thus bringing the estimates from the two methods significantly closer and highlighting the importance of reliable normalization when using this technique. PMID:26437372

  10. Study on the best method of SPIO labeling on the cell line ECV304

    NASA Astrophysics Data System (ADS)

    Yang, FuYuan; Yu, MingXi; Chen, WenLi; Zhou, Quan

    2010-02-01

    Studies the superparamagnetic iron oxide (SPIO) label ECV304 method and test its effect on cell physiological activity .We use different concentration PLL-SPIO incubate ECV304 cells, Prussian blue dye was taken to examine SPIO mark efficiency, DCF dyeing and PI & Annexin the VFITC double dyeing was carried out to analyze cell apoptosis. The result shows that 25μg/ml gave the most efficient labeling concentration, there is also no toxic effect on cells in this concentration. This outcome can be used in MRI of living body cell transplant tracking.

  11. Isoelectric focusing of red blood cells in a density gradient stabilized column

    NASA Technical Reports Server (NTRS)

    Smolka, A. J. K.; Miller, T. Y.

    1980-01-01

    The effects of Ficoll and cell application pH on red blood cell electrophoretic mobility and focusing pH were investigated by focusing cells in a density gradient stabilized column. Sample loading, cell dispersion, column conductivity, resolution of separation, and the effect of Ampholines were examined.

  12. Evaluation of Fluorophores to Label SNAP-Tag Fused Proteins for Multicolor Single-Molecule Tracking Microscopy in Live Cells

    PubMed Central

    Bosch, Peter J.; Corrêa, Ivan R.; Sonntag, Michael H.; Ibach, Jenny; Brunsveld, Luc; Kanger, Johannes S.; Subramaniam, Vinod

    2014-01-01

    Single-molecule tracking has become a widely used technique for studying protein dynamics and their organization in the complex environment of the cell. In particular, the spatiotemporal distribution of membrane receptors is an active field of study due to its putative role in the regulation of signal transduction. The SNAP-tag is an intrinsically monovalent and highly specific genetic tag for attaching a fluorescent label to a protein of interest. Little information is currently available on the choice of optimal fluorescent dyes for single-molecule microscopy utilizing the SNAP-tag labeling system. We surveyed 6 green and 16 red excitable dyes for their suitability in single-molecule microscopy of SNAP-tag fusion proteins in live cells. We determined the nonspecific binding levels and photostability of these dye conjugates when bound to a SNAP-tag fused membrane protein in live cells. We found that only a limited subset of the dyes tested is suitable for single-molecule tracking microscopy. The results show that a careful choice of the dye to conjugate to the SNAP-substrate to label SNAP-tag fusion proteins is very important, as many dyes suffer from either rapid photobleaching or high nonspecific staining. These characteristics appear to be unpredictable, which motivated the need to perform the systematic survey presented here. We have developed a protocol for evaluating the best dyes, and for the conditions that we evaluated, we find that Dy 549 and CF 640 are the best choices tested for single-molecule tracking. Using an optimal dye pair, we also demonstrate the possibility of dual-color single-molecule imaging of SNAP-tag fusion proteins. This survey provides an overview of the photophysical and imaging properties of a range of SNAP-tag fluorescent substrates, enabling the selection of optimal dyes and conditions for single-molecule imaging of SNAP-tagged fusion proteins in eukaryotic cell lines. PMID:25140415

  13. Red Cell Properties after Different Modes of Blood Transportation

    PubMed Central

    Makhro, Asya; Huisjes, Rick; Verhagen, Liesbeth P.; Mañú-Pereira, María del Mar; Llaudet-Planas, Esther; Petkova-Kirova, Polina; Wang, Jue; Eichler, Hermann; Bogdanova, Anna; van Wijk, Richard; Vives-Corrons, Joan-Lluís; Kaestner, Lars

    2016-01-01

    Transportation of blood samples is unavoidable for assessment of specific parameters in blood of patients with rare anemias, blood doping testing, or for research purposes. Despite the awareness that shipment may substantially alter multiple parameters, no study of that extent has been performed to assess these changes and optimize shipment conditions to reduce transportation-related artifacts. Here we investigate the changes in multiple parameters in blood of healthy donors over 72 h of simulated shipment conditions. Three different anticoagulants (K3EDTA, Sodium Heparin, and citrate-based CPDA) for two temperatures (4°C and room temperature) were tested to define the optimal transportation conditions. Parameters measured cover common cytology and biochemistry parameters (complete blood count, hematocrit, morphological examination), red blood cell (RBC) volume, ion content and density, membrane properties and stability (hemolysis, osmotic fragility, membrane heat stability, patch-clamp investigations, and formation of micro vesicles), Ca2+ handling, RBC metabolism, activity of numerous enzymes, and O2 transport capacity. Our findings indicate that individual sets of parameters may require different shipment settings (anticoagulants, temperature). Most of the parameters except for ion (Na+, K+, Ca2+) handling and, possibly, reticulocytes counts, tend to favor transportation at 4°C. Whereas plasma and intraerythrocytic Ca2+ cannot be accurately measured in the presence of chelators such as citrate and EDTA, the majority of Ca2+-dependent parameters are stabilized in CPDA samples. Even in blood samples from healthy donors transported using an optimized shipment protocol, the majority of parameters were stable within 24 h, a condition that may not hold for the samples of patients with rare anemias. This implies for as short as possible shipping using fast courier services to the closest expert laboratory at reach. Mobile laboratories or the travel of the patients to

  14. Vesiculation of healthy and defective red blood cells

    NASA Astrophysics Data System (ADS)

    Li, He; Lykotrafitis, George

    2015-07-01

    Vesiculation of mature red blood cells (RBCs) contributes to removal of defective patches of the erythrocyte membrane. In blood disorders, which are related to defects in proteins of the RBC membrane, vesiculation of the plasma membrane is intensified. Several hypotheses have been proposed to explain RBC vesiculation but the exact underlying mechanisms and what determines the sizes of the vesicles are still not completely understood. In this work, we apply a two-component coarse-grained molecular dynamics RBC membrane model to study how RBC vesiculation is controlled by the membrane spontaneous curvature and by lateral compression of the membrane. Our simulation results show that the formation of small homogeneous vesicles with a diameter less than 40 nm can be attributed to a large spontaneous curvature of membrane domains. On the other hand, compression on the membrane can cause the formation of vesicles with heterogeneous composition and with sizes comparable with the size of the cytoskeleton corral. When spontaneous curvature and lateral compression are simultaneously considered, the compression on the membrane tends to facilitate formation of vesicles originating from curved membrane domains. We also simulate vesiculation of RBCs with membrane defects connected to hereditary elliptocytosis (HE) and to hereditary spherocytosis (HS). When the vertical connectivity between the lipid bilayer and the membrane skeleton is elevated, as in normal RBCs, multiple vesicles are shed from the compressed membrane with diameters similar to the cytoskeleton corral size. In HS RBCs, where the connectivity between the lipid bilayer and the cytoskeleton is reduced, larger-size vesicles are released under the same compression ratio as in normal RBCs. Lastly, we find that vesicles released from HE RBCs can contain cytoskeletal filaments due to fragmentation of the membrane skeleton while vesicles released from the HS RBCs are depleted of cytoskeletal filaments.

  15. Red Cell Properties after Different Modes of Blood Transportation.

    PubMed

    Makhro, Asya; Huisjes, Rick; Verhagen, Liesbeth P; Mañú-Pereira, María Del Mar; Llaudet-Planas, Esther; Petkova-Kirova, Polina; Wang, Jue; Eichler, Hermann; Bogdanova, Anna; van Wijk, Richard; Vives-Corrons, Joan-Lluís; Kaestner, Lars

    2016-01-01

    Transportation of blood samples is unavoidable for assessment of specific parameters in blood of patients with rare anemias, blood doping testing, or for research purposes. Despite the awareness that shipment may substantially alter multiple parameters, no study of that extent has been performed to assess these changes and optimize shipment conditions to reduce transportation-related artifacts. Here we investigate the changes in multiple parameters in blood of healthy donors over 72 h of simulated shipment conditions. Three different anticoagulants (K3EDTA, Sodium Heparin, and citrate-based CPDA) for two temperatures (4°C and room temperature) were tested to define the optimal transportation conditions. Parameters measured cover common cytology and biochemistry parameters (complete blood count, hematocrit, morphological examination), red blood cell (RBC) volume, ion content and density, membrane properties and stability (hemolysis, osmotic fragility, membrane heat stability, patch-clamp investigations, and formation of micro vesicles), Ca(2+) handling, RBC metabolism, activity of numerous enzymes, and O2 transport capacity. Our findings indicate that individual sets of parameters may require different shipment settings (anticoagulants, temperature). Most of the parameters except for ion (Na(+), K(+), Ca(2+)) handling and, possibly, reticulocytes counts, tend to favor transportation at 4°C. Whereas plasma and intraerythrocytic Ca(2+) cannot be accurately measured in the presence of chelators such as citrate and EDTA, the majority of Ca(2+)-dependent parameters are stabilized in CPDA samples. Even in blood samples from healthy donors transported using an optimized shipment protocol, the majority of parameters were stable within 24 h, a condition that may not hold for the samples of patients with rare anemias. This implies for as short as possible shipping using fast courier services to the closest expert laboratory at reach. Mobile laboratories or the travel of the

  16. Effect of Irradiation on Microparticles in Red Blood Cell Concentrates.

    PubMed

    Cho, Chi Hyun; Yun, Seung Gyu; Koh, Young Eun; Lim, Chae Seung

    2016-07-01

    Changes in microparticles (MP) from red blood cell (RBC) concentrates in the context of irradiation have not been investigated. The aim of this study was to evaluate how irradiation affects the number of MPs within transfusion components. Twenty RBC concentrates, within 14 days after donation, were exposed to gamma rays (dose rate: 25 cGy) from a cesium-137 irradiator. Flow cytometry was used to determine the numbers of MPs derived from RBC concentrates before and 24 hr after irradiation. The mean number of MPs (±standard deviation) in RBC concentrates was 21.9×10⁹/L (±22.7×10⁹/L), and the total number of MPs ranged from 2.6×10⁹/L to 96.9×10⁹/L. The mean number of MPs increased to 22.6×10⁹/L (±31.6×10⁹/L) after irradiation. Before irradiation, the CD41-positive and CD235a-positive MPs constituted 9.5% (1.0×10⁹/L) and 2.2% (263×10⁶/L) of total MPs, respectively. After irradiation, CD41-positive MPs increased to 12.1% (1.5×10⁹/L) (P=0.014), but the CD235a-positive MPs decreased to 2.0% (214×10⁶/L) of the total MPs (P=0.369). Irradiation increases the number of CD41-positive MPs within RBC concentrates, suggesting the irradiation of RBC concentrates could be associated with thrombotic risk of circulating blood through the numerical change. PMID:27139610

  17. Red blood cell sodium transport in patients with cirrhosis.

    PubMed

    Henriksen, Ulrik Lütken; Kiszka-Kanowitz, Marianne; Bendtsen, Flemming; Henriksen, Jens H

    2016-09-01

    Patients with advanced cirrhosis have abnormal sodium homoeostasis. The study was undertaken to quantify the sodium transport across the plasma membrane of red blood cells (RBC) in patients with cirrhosis. RBC efflux and influx of sodium were studied in vitro with tracer (22) Na(+) according to linear kinetics in 24 patients with cirrhosis and 14 healthy controls. The sodium efflux was modified by ouabain (O), furosemide (F) and a combination of O and F (O + F). RBC sodium was significantly decreased (4·6 versus control 6·3 mmol l(-1) , P<0·001) and directly related to serum sodium (r = 0·57, P<0·05). The RBC fractional sodium efflux was higher in patients with cirrhosis (+46%, P<0·01) compared to controls. Inhibition in both high (145 mmol l(-1) )- and low (120 mmol l(-1) )-sodium buffers showed that the F-insensitive sodium efflux was twice as high in cirrhosis as in controls (P = 0·03-0·007), especially the O-sensitive, F-insensitive efflux was increased (+ 225%, P = 0·01-0·006). Fractional F-sensitive transport was normal in cirrhosis. RBC sodium influx was largely normal in cirrhosis. In conclusion, RBC sodium content is reduced in patients with cirrhosis with a direct relation to serum sodium. Increased RBC sodium efflux is especially related to ouabain-sensitive, furosemide-insensitive transport and thus most likely due to upregulated activity of the sodium-potassium pump. The study gives no evidence to an altered intracellular/extracellular sodium ratio or to a reduced fractional furosemide-sensitive sodium transport in cirrhosis. PMID:26016736

  18. Effect of Irradiation on Microparticles in Red Blood Cell Concentrates

    PubMed Central

    Cho, Chi Hyun; Yun, Seung Gyu; Koh, Young Eun

    2016-01-01

    Changes in microparticles (MP) from red blood cell (RBC) concentrates in the context of irradiation have not been investigated. The aim of this study was to evaluate how irradiation affects the number of MPs within transfusion components. Twenty RBC concentrates, within 14 days after donation, were exposed to gamma rays (dose rate: 25 cGy) from a cesium-137 irradiator. Flow cytometry was used to determine the numbers of MPs derived from RBC concentrates before and 24 hr after irradiation. The mean number of MPs (±standard deviation) in RBC concentrates was 21.9×109/L (±22.7×109/L), and the total number of MPs ranged from 2.6×109/L to 96.9×109/L. The mean number of MPs increased to 22.6×109/L (±31.6×109/L) after irradiation. Before irradiation, the CD41-positive and CD235a-positive MPs constituted 9.5% (1.0×109/L) and 2.2% (263×106/L) of total MPs, respectively. After irradiation, CD41-positive MPs increased to 12.1% (1.5×109/L) (P=0.014), but the CD235a-positive MPs decreased to 2.0% (214×106/L) of the total MPs (P=0.369). Irradiation increases the number of CD41-positive MPs within RBC concentrates, suggesting the irradiation of RBC concentrates could be associated with thrombotic risk of circulating blood through the numerical change. PMID:27139610

  19. Effects of pegylated hamster red blood cells on microcirculation.

    PubMed

    Chen, Peter C Y; Huang, Wei; Stassinopoulos, Adonis; Cheung, Anthony T W

    2008-01-01

    The objective of this study was to examine the effects of polyethylene glycol (PEG) treated red blood cells (RBCs) on the microcirculation in a hamster back skin window chamber model. Donor hamster RBCs were PEGylated through an incubation with an activated PEG solution, washed, resuspended, and infused through a 10% volume top loading procedure into the carotid artery in an awake Syrian Golden hamster. Eight hamster groups were treated with activated PEG different sizes and concentrations: 0.05 mM-5 kDa PEG, 0.5 mM-5 kDa PEG, 1.1 mM-5 kDa PEG, 2.2 mM-5 kDa PEG, 22 mM-5 kDa PEG, 0.05 mM-20 kDa PEG, 0.5 mM-20 kDa PEG, and 5 mM-20 kDa PEG. Non-treated RBCs were used as control. The microvascular bed under observation was videotaped 30 min before the infusion and followed for 30 min post infusion. The diameter of individual blood vessels and blood flow velocities in selected vessels was measured. Hematocrit and hemoglobin concentration were recorded before infusion and at the end of experiment. Tissue pO(2) was also monitored. Results showed the hamsters tolerated the PEGylated RBCs without apparent ill effects. No significant changes were recorded for the hematocrit, the hemoglobin concentration, the blood vessel diameters, blood flow velocities, and the interstitial partial oxygen pressure (pO(2)) before, during, and after the injections of PEG-RBCs (P > 0.05). Unlike most hemoglobin-based oxygen carrying compounds, which can cause vasoconstriction, the PEGylated RBCs did not produce any measurable vasoactivity. Together with the absence of rouleaux formation and the fact that PEG molecules can mask the surface antigens on RBCs, PEGylation appeared promising as a circulation enhancement treatment. PMID:18649167

  20. Pure red-cell aplasia "epidemic"--mystery completely revealed?

    PubMed

    Locatelli, Francesco; Del Vecchio, Lucia; Pozzoni, Pietro

    2007-06-01

    Starting in 1998, the number of pure red-cell aplasia (PRCA) cases in patients treated with recombinant human erythropoietin (rHuEPO) increased dramatically. Most cases were observed in patients treated with epoetin alfa produced outside the United States. The peak was observed in 2002; since then, the PRCA incidence has declined. Many factors are likely to have contributed to this up-surge. The molecular structure of the various epoetins and patient characteristics do not seem to play a major role. The route of administration holds some importance, because most PRCA patients received rHuEPO subcutaneously. The peak of PRCA cases overlapped with the removal of human serum albumin from the Eprex formulation (Janssen-Pharmaceutica NV, Beerse, Belgium), for which polysorbate 80 and glycine were substituted. Polysorbate 80 may have increased the immunogenicity of Eprex by eliciting the formation of epoetin-containing micelles or by interacting with leachates released by the uncoated rubber stoppers of prefilled syringes. Compared with the previous formulation, the polysorbate 80 formulation has lower stability, making it more susceptible to stress conditions such as insufficient attention to the cold chain. This situation could facilitate protein denaturation or aggregate formation. Uncoated rubber stoppers were replaced with coated stoppers, and the cold chain was reinforced; the Eprex formulation has remained unchanged. Even though the incidence of PRCA returned to very low levels, discriminating the cause-effect relationship of a single action is difficult, given that all occurred with a similar chronology, and that PRCA develops after a relatively long exposure period. Careful observation of future trends of new PRCA cases is thus mandatory. PMID:17556324

  1. Increased phorbol 12,13-dibutyrate (PDBu) receptor function associated with sickle red cell membrane ghosts

    SciTech Connect

    Ramachandran, M.; Nair, C.N.; Abraham, E.C.

    1987-05-01

    The biological receptor for tumor-promoting phorbol esters has been identified as the CaS /phospholipid dependent enzyme, protein kinase C. In the red cell, this enzyme is mainly cytosolic but becomes translocated to the membrane if the cellular CaS is allowed to rise. Since cellular CaS in sickle red cells is high, it was reasoned that this enzyme may become more membrane-bound. In fact, the authors noticed a four-fold increase in the binding of TH-PDBu by membrane ghosts isolated from sickle red cells compared to normal red cells (pmoles PDBu bound/mg protein; normal = 0.3 vs sickle cell = 1.4). Attempts to assay the enzyme directly as phospholipid-activated TSP incorporation into the acid-precipitable membrane proteins also indicated a two-fold increase in the radiolabelling of sickle cell membrane ghosts. Autophosphorylation of membrane proteins and analysis of the phosphorylation profile by SDS-PAGE and autoradiography revealed phosphorylation predominantly of bands 3, 4.1 and 4.9 which are known protein kinase C substrates for the red cell enzyme. The increased membrane-associated protein kinase C in sickle red cells may have a bearing on the altered membrane properties reported in this condition.

  2. Active Targeting to Osteosarcoma Cells and Apoptotic Cell Death Induction by the Novel Lectin Eucheuma serra Agglutinin Isolated from a Marine Red Alga

    PubMed Central

    Hayashi, Keita; Walde, Peter; Miyazaki, Tatsuhiko; Sakayama, Kenshi; Nakamura, Atsushi; Kameda, Kenji; Masuda, Seizo; Umakoshi, Hiroshi; Kato, Keiichi

    2012-01-01

    Previously, we demonstrated that the novel lectin Eucheuma serra agglutinin from a marine red alga (ESA) induces apoptotic cell death in carcinoma. We now find that ESA induces apoptosis also in the case of sarcoma cells. First, propidium iodide assays with OST cells and LM8 cells showed a decrease in cell viability after addition of ESA. With 50 μg/ml ESA, the viabilities after 24 hours decreased to 54.7 ± 11.4% in the case of OST cells and to 41.7 ± 12.3% for LM8 cells. Second, using fluorescently labeled ESA and flow cytometric and fluorescence microscopic measurements, it could be shown that ESA does not bind to cells that were treated with glycosidases, indicating importance of the carbohydrate chains on the surface of the cells for efficient ESA-cell interactions. Third, Span 80 vesicles with surface-bound ESA as active targeting ligand were shown to display sarcoma cell binding activity, leading to apoptosis and complete OST cell death after 48 hours at 2 μg/ml ESA. The findings indicate that Span 80 vesicles with surface-bound ESA are a potentially useful drug delivery system not only for the treatment of carcinoma but also for the treatment of osteosarcoma. PMID:23346404

  3. Active Targeting to Osteosarcoma Cells and Apoptotic Cell Death Induction by the Novel Lectin Eucheuma serra Agglutinin Isolated from a Marine Red Alga.

    PubMed

    Hayashi, Keita; Walde, Peter; Miyazaki, Tatsuhiko; Sakayama, Kenshi; Nakamura, Atsushi; Kameda, Kenji; Masuda, Seizo; Umakoshi, Hiroshi; Kato, Keiichi

    2012-01-01

    Previously, we demonstrated that the novel lectin Eucheuma serra agglutinin from a marine red alga (ESA) induces apoptotic cell death in carcinoma. We now find that ESA induces apoptosis also in the case of sarcoma cells. First, propidium iodide assays with OST cells and LM8 cells showed a decrease in cell viability after addition of ESA. With 50 μg/ml ESA, the viabilities after 24 hours decreased to 54.7 ± 11.4% in the case of OST cells and to 41.7 ± 12.3% for LM8 cells. Second, using fluorescently labeled ESA and flow cytometric and fluorescence microscopic measurements, it could be shown that ESA does not bind to cells that were treated with glycosidases, indicating importance of the carbohydrate chains on the surface of the cells for efficient ESA-cell interactions. Third, Span 80 vesicles with surface-bound ESA as active targeting ligand were shown to display sarcoma cell binding activity, leading to apoptosis and complete OST cell death after 48 hours at 2 μg/ml ESA. The findings indicate that Span 80 vesicles with surface-bound ESA are a potentially useful drug delivery system not only for the treatment of carcinoma but also for the treatment of osteosarcoma. PMID:23346404

  4. Flow structures and red blood cell dynamics in arteriole of dilated or constricted cross section.

    PubMed

    Gambaruto, Alberto M

    2016-07-26

    Vessel with 'circular' or 'star-shaped' cross sections are studied, representing respectively dilated or constricted cases where endothelial cells smoothly line or bulge into the lumen. Computational haemodynamics simulations are carried out on idealised periodic arteriole-sized vessels, with red blood cell 'tube' hematocrit value=24%. A further simulation of a single red blood cell serves for comparison purposes. The bulk motion of the red blood cells reproduces well-known effects, including the presence of a cell-free layer and the apparent shear-thinning non-Newtonian rheology. The velocity flow field is analysed in a Lagrangian reference frame, relative to any given red blood cell, hence removing the bulk coaxial motion and highlighting instead the complex secondary flow patterns. An aggregate formation becomes apparent, continuously rearranging and dynamic, brought about by the inter-cellular fluid mechanics interactions and the deformability properties of the cells. The secondary flow field induces a vacillating radial migration of the red blood cells. At different radial locations, the red blood cells express different residence times, orientation and shape. The shear stresses exerted by the flow on the vessel wall are influenced by the motion of red blood cells, despite the presence of the cell-free layer. Spatial (and temporal) variations of wall shear stress patters are observed, especially for the 'circular' vessel. The 'star-shaped' vessel bears considerable stress at the protruding endothelial cell crests, where the stress vectors are coaxially aligned. The bulging endothelial cells hence regularise the transmission of stresses on the vessel wall. PMID:26822224

  5. Cell Labeling for 19F MRI: New and Improved Approach to Perfluorocarbon Nanoemulsion Design

    PubMed Central

    Patel, Sravan K.; Williams, Jonathan; Janjic, Jelena M.

    2013-01-01

    This report describes novel perfluorocarbon (PFC) nanoemulsions designed to improve ex vivo cell labeling for 19F magnetic resonance imaging (MRI). 19F MRI is a powerful non-invasive technique for monitoring cells of the immune system in vivo, where cells are labeled ex vivo with PFC nanoemulsions in cell culture. The quality of 19F MRI is directly affected by the quality of ex vivo PFC cell labeling. When co-cultured with cells for longer periods of time, nanoemulsions tend to settle due to high specific weight of PFC oils (1.5–2.0 g/mL). This in turn can decrease efficacy of excess nanoemulsion removal and reliability of the cell labeling in vitro. To solve this problem, novel PFC nanoemulsions are reported which demonstrate lack of sedimentation and high stability under cell labeling conditions. They are monodisperse, have small droplet size (~130 nm) and low polydispersity (<0.15), show a single peak in the 19F nuclear magnetic resonance spectrum at −71.4 ppm and possess high fluorine content. The droplet size and polydispersity remained unchanged after 160 days of follow up at three temperatures (4, 25 and 37 °C). Further, stressors such as elevated temperature in the presence of cells, and centrifugation, did not affect the nanoemulsion droplet size and polydispersity. Detailed synthetic methodology and in vitro testing for these new PFC nanoemulsions is presented. PMID:25586263

  6. Double labeling autoradiography. Cell kinetic studies with /sup 3/H- and /sup 14/C-thymidine

    SciTech Connect

    Schultze, B.

    1981-01-01

    Examples of the multiple applicability of the double labeling method with /sup 3/H- and /sup 14/C-TdR are demonstrated. Double labeling with /sup 3/H- and /sup 14/C-TdR makes it possible to determine the cycle and its phases with high precision by modifying the usual percent labeled mitoses method with a single injection of /sup 3/H-TdR. In addition, data is provided on the variances of the transit times through the cycle phases. For example, in the case of the jejunal crypt cells of the mouse, the transit times through successive cycle phases are uncorrelated. In the case of glial cells the double labeling method provides cell kinetic parameters despite the paucity of proliferating glial cells. In the adult untreated animal, glial cell mitoses are so rare that the percent labeled mitoses method can not be utilized. However, the S-phase duration can be measured by double labeling and the cycle time can be determined by the so-called method of labeled S phases. With the latter method the passage through the S phase of the /sup 3/H-TdR-labeled S phase cells can be registered by injecting /sup 14/C-TdR at different time intervals following /sup 3/H-TdR application. In this way an S-phase duration of about 10 hr and a cycle time of about 20 hr was found for glial cells in the adult untreated mouse. An exchange of glial cells between the growth fraction and the nongrowth fraction has also been shown by double labeling. A quite different application of the double labeling method with 3H- and /sup 14/C-TdR is the in vivo study of the cell cycle phase-specific effect of drugs used in chemotherapy of tumors. The effect of vincristine on these cells has been studied. Vincristine affects cells in S and G2 in such a manner that they are arrested during the next metaphase and subsequently become necrotic. It has no effect on G1 cells.

  7. Red light interferes in UVA-induced photoaging of human skin fibroblast cells.

    PubMed

    Niu, Tianhui; Tian, Yan; Ren, Qu; Wei, Lizhao; Li, Xiaoxin; Cai, Qing

    2014-01-01

    The possible regulation mechanism of red light was determined to discover how to retard UVA-induced skin photoaging. Human skin fibroblasts were cultured and irradiated with different doses of UVA, thus creating a photoaging model. Fibroblasts were also exposed to a subtoxic dose of UVA combined with a red light-emitting diode (LED) for five continuous days. Three groups were examined: control, UVA and UVA plus red light. Cumulative exposure doses of UVA were 25 J cm(-2), and the total doses of red light were 0.18 J cm(-2). Various indicators were measured before and after irradiation, including cell morphology, viability, β-galactosidase staining, apoptosis, cycle phase, the length of telomeres and the protein levels of photoaging-related genes. Red light irradiation retarded the cumulative low-dose UVA irradiation-induced skin photoaging, decreased the expression of senescence-associated β-galactosidase, upregulated SIRT1 expression, decreased matrix metalloproteinase MMP-1 and the acetylation of p53 expression, reduced the horizon of cell apoptosis and enhanced cell viability. Furthermore, the telomeres in UVA-treated cells were shortened compared to those of cells in the red light groups. These results suggest that red light plays a key role in the antiphotoaging of human skin fibroblasts by acting on different signaling transduction pathways. PMID:25039464

  8. Superparamagnetic iron oxide is suitable to label tendon stem cells and track them in vivo with MR imaging

    PubMed Central

    Yang, Yunfa; Zhang, Jianying; Qian, Yongxian; Dong, Shiwu; Huang, He; Boada, Fernando E; Fu, Freddie H.; Wang, James H-C.

    2013-01-01

    Tendon stem cells (TSCs) may be used to effectively repair or regenerate injured tendons. However, the fates of TSCs once implanted in vivo remain unclear. This study was aimed to determine the feasibility of labeling TSCs with super-paramagnetic iron oxide (SPIO) nano-particles to track TSCs in vivo using MRI. Rabbit TSCs were labeled by incubation with 50 μg/ml SPIO. Labeling efficiency, cell viability, and proliferation were then measured, and the stemness of TSCs was tested by quantitative real time RT-PCR (qRT-PCR) and immunocytochemistry. We found that the labeling efficiency of TSCs reached as high as 98%, and that labeling at 50 μg/ml SPIO concentrations did not alter cell viability and cell proliferation compared to non-labeled control cells. Moreover, the expression levels of stem cell markers (Nucleostemin, Nanog, and Oct-4) did not change in SPIO-labeled TSCs compared to non-labeled cells. Both labeled and non-labeled cells also exhibited similar differentiation potential. Finally, labeled TSCs could be detected by MRI both in vitro and in vivo. Taken together, the findings of this study show that labeling TSCs with SPIO particles is a feasible approach to track TSCs in vivo by MRI, which offers a noninvasive method to monitor repair of injured tendons. PMID:23549900

  9. A microfluidics-based technique for automated and rapid labeling of cells for flow cytometry

    NASA Astrophysics Data System (ADS)

    Patibandla, Phani K.; Estrada, Rosendo; Kannan, Manasaa; Sethu, Palaniappan

    2014-03-01

    Flow cytometry is a powerful technique capable of simultaneous multi-parametric analysis of heterogeneous cell populations for research and clinical applications. In recent years, the flow cytometer has been miniaturized and made portable for application in clinical- and resource-limited settings. The sample preparation procedure, i.e. labeling of cells with antibodies conjugated to fluorescent labels, is a time consuming (˜45 min) and labor-intensive procedure. Microfluidics provides enabling technologies to accomplish rapid and automated sample preparation. Using an integrated microfluidic device consisting of a labeling and washing module, we demonstrate a new protocol that can eliminate sample handling and accomplish sample and reagent metering, high-efficiency mixing, labeling and washing in rapid automated fashion. The labeling module consists of a long microfluidic channel with an integrated chaotic mixer. Samples and reagents are precisely metered into this device to accomplish rapid and high-efficiency mixing. The mixed sample and reagents are collected in a holding syringe and held for up to 8 min following which the mixture is introduced into an inertial washing module to obtain ‘analysis-ready’ samples. The washing module consists of a high aspect ratio channel capable of focusing cells to equilibrium positions close to the channel walls. By introducing the cells and labeling reagents in a narrow stream at the center of the channel flanked on both sides by a wash buffer, the elution of cells into the wash buffer away from the free unbound antibodies is accomplished. After initial calibration experiments to determine appropriate ‘holding time’ to allow antibody binding, both modules were used in conjunction to label MOLT-3 cells (T lymphoblast cell line) with three different antibodies simultaneously. Results confirm no significant difference in mean fluorescence intensity values for all three antibodies labels (p < 0.01) between the

  10. Clinical significance of anti-Yt(b). Report of a case using a 51-chromium red cell survival study

    SciTech Connect

    Levy, G.J.; Selset, G.; McQuiston, D.; Nance, S.J.; Garratty, G.; Smith, L.E.; Goldfinger, D.

    1988-05-01

    Several published reports have documented the variable survival of Yt(a+) red cells (RBC) in patients with anti-Yt(a) as measured by 51-Chromium (Cr)-labeled RBC survival studies. Similar studies with anti-Yt(b) have not been reported. A /sup 51/Cr-labeled RBC survival study was performed using Yt(b+) RBCs and a monocyte monolayer assay in a young hemodialysis patient who required chronic transfusion therapy and who had developed anti-Yt(b). The survival of the transfused RBCs was 100 and 93 percent at 1 and 24 hours, respectively, with a half life of 21 days at termination of the study (normal, 28 to 32 days). These results showed no evidence of rapid destruction of the Yt(b+) RBCs, indicating that this patient could be transfused safely with blood from Yt(b+) donors. Long-term survival of the /sup 51/Cr-labeled Yt(b+) RBCs was shortened moderately, however, a finding that correlated with a slightly abnormal monocyte monolayer assay test.

  11. Biosignatures of Kerala red rain cells: Implications in understanding their origin

    NASA Astrophysics Data System (ADS)

    Gangappa, R.; Thomas, M.; Hogg, S.

    2013-09-01

    The red rain that fell over Kerala, southern India (2001-2012) was characterised by the red pigmented particles. Earlier proposal claiming that these are known algal bloom blown from trees (Sampath et al, 2001; DiGregorio, 2007) has been studied by us and disproved. Also, further investigation reporting their extraordinary properties including a suggestion that they lack DNA (Louis and Kumar 2003; 2006; 2008) has been invalidated (Gangappa and Hogg, 2013). However, their claim regarding the growth and replication of these cells at 300ºC needs more investigation if it is to gain acceptance. Current study provide evidences regarding the biological properties of Kerala red rain cells to gain insights into environmental conditions from which they may have originated. Combined with various research strategies and high resolution instruments, we have demonstrated the following interesting properties of Kerala red rain cells: (1) unusually thick external envelope enclosing the central core; (2)stability of red pigment at temperatures about 100ºC and pH variations; (3) absence of eukaryotic ultrastructures; (4) possible replication at 121ºC with nanostructures (possible daughter cells) having similar morphological features inside the large mother cells at such high temperature. They contain high percentage of carbon, iron, silicon and aluminum and often enclosed in a silicon rich biofilms. Further investigation shows that the positive detection of DNA in these cells was possible only after the complete removal of red pigment, thereby providing an explanation for the negative outcome of earlier studies in this regard. Moreover, evidences are shown to support that these cells contain high amounts of UV absorbing compounds, porphyrin complexes and possible scytonemin. Kerala red rain cells may prove to be polyextermophiles belonging to prokaryotes and may have possibly originated from the environment containing above mentioned chemical elements, high energy UV exposure and

  12. Method for extending the useful shelf-life of refrigerated red blood cells by flushing with inert gas

    DOEpatents

    Bitensky, M.W.; Yoshida, Tatsuro

    1997-04-29

    A method is disclosed using oxygen removal for extending the useful shelf-life of refrigerated red blood cells. A cost-effective, 4 C storage procedure that preserves red cell quality and prolongs post-transfusion in vivo survival is described. Preservation of adenosine triphosphate levels and reduction in hemolysis and in membrane vesicle production of red blood cells stored at 4 C for prolonged periods of time is achieved by removing oxygen from the red blood cells at the time of storage; in particular, by flushing with an inert gas. Adenosine triphosphate levels of the stored red blood cells are boosted in some samples by addition of ammonium phosphate. 4 figs.

  13. Labeling and Imaging Mesenchymal Stem Cells with Quantum Dots

    EPA Science Inventory

    Mesenchymal stem cells (MSCs) are multipotent cells with the potential to differentiate into bone, cartilage, adipose and muscle cells. Adult derived MSCs are being actively investigated because of their potential to be utilized for therapeutic cell-based transplantation. Methods...

  14. Current issues relating to the transfusion of stored red blood cells.

    PubMed

    Zimrin, A B; Hess, J R

    2009-02-01

    The development of blood storage systems allowed donation and transfusion to be separated in time and space. This separation has permitted the regionalization of donor services with subsequent economies of scale and improvements in the quality and availability of blood products. However, the availability of storage raises the question of how long blood products can and should be stored and how long they are safe and effective. The efficacy of red blood cells was originally measured as the increment in haematocrit and safety began with typing and the effort to reduce the risk of bacterial contamination. Appreciation of a growing list of storage lesions of red blood cells has developed with our increasing understanding of red blood cell physiology and our experience with red blood cell transfusion. However, other than frank haemolysis, rare episodes of bacterial contamination and overgrowth, the reduction of oxygen-carrying capacity associated with the failure of some transfused cells to circulate, and the toxicity of lysophospholipids released from membrane breakdown, storage-induced lesions have not had obvious correlations with safety or efficacy. The safety of red blood cell storage has also been approached in retrospective epidemiologic studies of transfused patients, but the results are frequently biased by the fact that sicker patients are transfused more often and blood banks do not issue blood products in a random order. Several large prospective studies of the safety of stored red blood cells are planned. PMID:19152602

  15. Effects of 4000 rad irradiation on the in vitro storage properties of packed red cells

    SciTech Connect

    Moore, G.L.; Ledford, M.E.

    1985-11-01

    Immunosuppressed patients who require red cell transfusions receive irradiated (1500-3000 rad) packed red cells. These cells are irradiated immediately before infusion. If a large group of patients become immunosuppressed due to exposure to radiation or chemicals, the ability to supply large volumes of irradiated blood at the time of use might not be possible. An alternate solution to providing quantities of irradiated blood is to irradiate the units prior to storage. This study presents in vitro data comparing storage of paired packed red cell units either irradiated or not irradiated. Five units of fresh blood drawn into citrate-phosphate-dextrose-adenine (CPDA-1) were packed to a hematocrit of 75 +/- 1 percent, and then each unit was divided in two equal parts. One of each pair was irradiated (4000 rads), and both parts of each unit were stored for 35 days at 4 degrees C. Samples were analyzed every 7 days. Irradiation caused a slight drop in red cell adenosine triphosphate and 2,3 diphosphoglycerate and a slight increase in plasma hemoglobin compared to controls. Methemoglobin, pH, and glucose consumption were identical to the controls. The evidence indicates that irradiation did not cause biochemical or metabolic changes in the red cells that would lead us to suspect a difference between irradiated and nonirradiated stored red cells in function or viability. These negative findings require in vivo confirmation.

  16. Cell Proliferation Analysis Using EdU Labeling in Whole Plant and Histological Samples of Arabidopsis.

    PubMed

    Kazda, Anita; Akimcheva, Svetlana; Watson, J Matthew; Riha, Karel

    2016-01-01

    The ability to analyze cell division in both spatial and temporal dimensions within an organism is a key requirement in developmental biology. Specialized cell types within individual organs, such as those within shoot and root apical meristems, have often been identified by differences in their rates of proliferation prior to the characterization of distinguishing molecular markers. Replication-dependent labeling of DNA is a widely used method for assaying cell proliferation. The earliest approaches used radioactive labeling with tritiated thymidine, which were later followed by immunodetection of bromodeoxyuridine (BrdU). A major advance in DNA labeling came with the use of 5-ethynyl-2'deoxyuridine (EdU) which has proven to have multiple advantages over BrdU. Here we describe the methodology for analyzing EdU labeling and retention in whole plants and histological sections of Arabidopsis. PMID:26659962

  17. A covalent approach for site-specific RNA labeling in Mammalian cells.

    PubMed

    Li, Fahui; Dong, Jianshu; Hu, Xiaosong; Gong, Weimin; Li, Jiasong; Shen, Jing; Tian, Huifang; Wang, Jiangyun

    2015-04-01

    Advances in RNA research and RNA nanotechnology depend on the ability to manipulate and probe RNA with high precision through chemical approaches, both in vitro and in mammalian cells. However, covalent RNA labeling methods with scope and versatility comparable to those of current protein labeling strategies are underdeveloped. A method is reported for the site- and sequence-specific covalent labeling of RNAs in mammalian cells by using tRNA(Ile2) -agmatidine synthetase (Tias) and click chemistry. The crystal structure of Tias in complex with an azide-bearing agmatine analogue was solved to unravel the structural basis for Tias/substrate recognition. The unique RNA sequence specificity and plastic Tias/substrate recognition enable the site-specific transfer of azide/alkyne groups to an RNA molecule of interest in vitro and in mammalian cells. Subsequent click chemistry reactions facilitate the versatile labeling, functionalization, and visualization of target RNA. PMID:25694369

  18. New carboxysilane-coated iron oxide nanoparticles for nonspecific cell labelling.

    PubMed

    Bridot, Jean-Luc; Stanicki, Dimitri; Laurent, Sophie; Boutry, Sébastien; Gossuin, Yves; Leclère, Philippe; Lazzaroni, Roberto; Vander Elst, Luce; Muller, Robert N

    2013-01-01

    Magnetic resonance imaging (MRI) offers the possibility of tracking cells labelled with a contrast agent and evaluating the progress of cell therapies. This requires efficient cell labelling with contrast agents. A basic incubation of cells with iron oxide nanoparticles (NPs) is a common method. This study reports the synthesis at the gram scale of iron oxide nanoparticles as MRI T₂ contrast agents for cell labelling. These NPs are based on small iron oxide cores coated with a thin polysiloxane shell presenting carboxylic acid functions. The iron oxide cores produced have been characterized by transmission electron microscopy, X-ray diffraction, ζ-potential, infrared, photon correlation spectroscopy, atomic force microscopy, magnetometry and relaxometric measurements. These measurements confirmed the expected surface modification by carboxysilane. Carboxylic groups created electrostatic repulsion between NPs when they are deprotonated. Therefore, highly concentrated aqueous solutions of carboxysilane coated iron oxide NPs can be obtained, up to 70% (w/w). These NPs could be used for cell labelling owing to their aggregation and re-dispersion properties. NPs precipitated in Dulbecco's modified Eagle medium induced a rapid association with 3 T6 fibroblast cells and could easily be re-dispersed in phosphate buffer saline solution to obtain properly labelled cells. PMID:24375902

  19. In vitro red blood cell assay for oxidant toxicity of petroleum oil

    SciTech Connect

    Couillard, C.M.; Leighton, F.A. )

    1993-05-01

    Petroleum oil has caused hemolytic anemia in birds and mammals. In birds, an oxidant damage on circulating red cells has been identified as the primary toxic effect of ingested petroleum oils. An in vitro red blood cell assay was developed to discriminate among the oxidant activities of different petroleum oils. The assay used rabbit red blood cells with a rat liver enzyme system and formation of methemoglobin was measured as an indicator of oxidant damage to the red cells. The assay was applied to five different petroleum oils and to naphthalene, a petroleum hydrocarbon known to cause hemolytic anemia. Different petroleum oils differed in their capacity to induce methemoglobin formation. Methemoglobin levels varied from 2.9% with Arabian light crude oil to 6.2% with South Louisiana crude oil. Naphthalene induced formation of up to 37% methemoglobin. Naphthalene and the five petroleum oils generated methemoglobin only in the presence of liver enzymes.

  20. Fetal anaemia from red blood cell membrane defect and co-inherited haemoglobin Constant Spring.

    PubMed

    Srisupundit, Kasemsri; Charoenkwan, Pimlak; Traisrisilp, Kuntharee; Tongsong, Theera

    2015-01-01

    The case presented here is an example of hereditary red blood cell membrane defect with a co-inherited haemoglobin Constant Spring. This case is of an anaemic fetus that presented with isolated ascites at 18 weeks of gestation. Fetal blood analysis revealed abnormal shaped red blood cells. The same pattern of red blood cell morphology was also seen on paternal peripheral blood smear. Intrauterine blood transfusions were given twice to correct fetal anaemia. The fetus showed a good response to the transfusions and was delivered at term with mild anaemia and did not need blood transfusion after birth. This report describes a natural course of red blood cell membrane defect with co-inherited haemoglobin Constant Spring, indicating that the course of disease was more severe during fetal life. Intrauterine transfusion supported the transition of the fetus through the critical period in utero to a healthier life after birth. PMID:26216922