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Sample records for labelled red cell

  1. Technetium-99m-labeled red blood cell imaging

    SciTech Connect

    Front, D.; Israel, O.; Groshar, D.; Weininger, J.

    1984-07-01

    Red blood cells labeled with 99mTc constitute a suitable intravascular agent for imaging of vascular abnormalities. Hemangiomas are characterized by low perfusion and a high blood pool. This ''perfusion blood-pool mismatch,'' not encountered in other lesions, may help in the specific diagnosis of this tumor. This is particularly so in cavernous hemangiomas of the liver where three-phase 99mTc-labeled red blood cell scintigraphy should precede liver biopsy. Red cell scintigraphy also is useful for establishing the vascular nature of hemangiomas of the head and neck and the skin and for diagnosis of venous occlusion. Heat-damaged red blood cells provide a specific spleen imaging agent. This should be used when patients with suspected splenic pathology have equivocal colloid scintigraphy.

  2. Measurement of posttransfusion red cell survival with the biotin label.

    PubMed

    Mock, Donald M; Widness, John A; Veng-Pedersen, Peter; Strauss, Ronald G; Cancelas, Jose A; Cohen, Robert M; Lindsell, Christopher J; Franco, Robert S

    2014-07-01

    The goal of this review is to summarize and critically assess information concerning the biotin method to label red blood cells (RBC) for use in studies of RBC and transfusion biology-information that will prove useful to a broad audience of clinicians and scientists. A review of RBC biology, with emphasis on RBC senescence and in vivo survival, is included, followed by an analysis of the advantages and disadvantages of biotin-labeled RBC (BioRBC) for measuring circulating RBC volume, posttransfusion RBC recovery, RBC life span, and RBC age-dependent properties. The advantages of BioRBC over (51)Cr RBC labeling, the current reference method, are discussed. Because the biotin method is straightforward and robust, including the ability to follow the entire life spans of multiple RBC populations concurrently in the same subject, BioRBC offers distinct advantages for studying RBC biology and physiology, particularly RBC survival. The method for biotin labeling, validation of the method, and application of BioRBCs to studies of sickle cell disease, diabetes, and anemia of prematurity are reviewed. Studies documenting the safe use of BioRBC are reviewed; unanswered questions requiring future studies, remaining concerns, and regulatory barriers to broader application of BioRBC including adoption as a new reference method are also presented.

  3. 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.

  4. Radionuclide-labeled red blood cells: current status and future prospects

    SciTech Connect

    Srivastava, S.C.; Chervu, L.R.

    1984-04-01

    Radiolabeling of red cells and their clinical and research application in nuclear medicine constitute an area of continued interest and steady growth during the past two decades. Technetium-/sup 99/m-labeled red cells in particular have revolutionized the field of cardiovascular nuclear medicine by making possible the external evaluation of various heart parameters with minimum radiation dose or trauma to the patient. Among other areas of study that use /sup 99/mTc -RBC are blood pool imaging, detection of vascular malformations, red cell mass determination, detection of gastrointestinal bleeding, and of hemangiomas. Heat-damaged /sup 99/mTc -RBC find application in spleen imaging, accessory spleen localization, detection of GI bleeding, and in other areas. A critical evaluation is presented of the various in vitro and in vivo labeling techniques that are currently available for red cell labeling. Even though the presently used procedures provide satisfactory labeled preparations, ideal radioisotopic RBC labels remain to be developed. Intermediate (2-3 days) as well as long-lived (approximately 30 days) radionuclidic labels are highly desirable for a number of clinical procedures where /sup 99/mTc is not useful due to its short half-life. New approaches such as the use of radiolabeled antibodies to red cell antigens, or labeling specific receptor sites in the cell may lead to substantial improvements in the labeling methodology and could yield labeled cells with the least damage and maximum in vivo stability.

  5. 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.

  6. 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

  7. 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.

  8. Immunospecific red cell binding of iodine /sup 125/-labeled immunoglobulin G erythrocyte autoantibodies

    SciTech Connect

    Masouredis, S.P.; Branks, M.J.; Garratty, G.; Victoria, E.J.

    1987-09-01

    The primary interaction of autoantibodies with red cells has been studied by using labeled autoantibodies. Immunoglobulin G red cell autoantibodies obtained from IgG antiglobulin-positive normal blood donors were labeled with radioactive iodine and compared with alloanti-D with respect to their properties and binding behavior. Iodine /sup 125/-labeled IgG autoantibody migrated as a single homogeneous peak with the same relative mobility as human IgG on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric focusing pattern of labeled autoantibodies varied from donor to donor but was similar to that of alloanti-D, consisting of multiple IgG populations with isoelectric points in the neutral to alkaline range. /sup 125/I-autoantibody bound to all human red cells of common Rh phenotypes. Evidence for immunospecific antibody binding of the labeled autoantibody was based on variation in equilibrium binding to nonhuman and human red cells of common and rare phenotypes, enhanced binding after red cell protease modification, antiglobulin reactivity of cell-bound IgG comparable to that of cell-bound anti-D, and saturation binding in autoantibody excess. Scatchard analysis of two /sup 125/I-autoantibody preparations yielded site numbers of 41,500 and 53,300 with equilibrium constants of 3.7 and 2.1 X 10(8) L X mol-1. Dog, rabbit, rhesus monkey, and baboon red cells were antigen(s) negative by quantitative adsorption studies adsorbing less than 3% of the labeled autoantibody. Reduced ability of rare human D--red blood cells to adsorb the autoantibody and identification of donor autoantibodies that bind to Rh null red blood cells indicated that eluates contained multiple antibody populations of complex specificities in contrast to anti-D, which consists of a monospecific antibody population. Another difference is that less than 70% of the autoantibody IgG was adsorbed by maximum binding red blood cells as compared with greater than 85% for alloanti-D.

  9. Use of /sup 75/Se-labeled methionine to study the sequestration of senescent red blood cells

    SciTech Connect

    Smedsrod, B.; Aminoff, D.

    1985-01-01

    Labeling red blood cells with Na/sub 2//sup 51/CrO/sub 4/ enabled us to study certain aspects of red cell survival and sequestration from the circulation. As a random labeling procedure, however, the /sup 51/Cr method has certain limitations. Therefore, we developed a cohort labeling method using /sup 75/Se-methionine as a two-rat procedure. This gives a clear pulse-labeled population of rat red cells to study the dynamics of sequestration. With this labeling procedure, it was possible to demonstrate that 1) there is an increase in the density of red cells with age, 2) a significant sequestration of red cells from the circulation is apparent at the end of 48 days and essentially is complete at the end of 60 days, 3) there is a corresponding uptake of senescent red cells in the spleen, which peaks at 55 days, and 4) the 60-day end point is sharper and is more definitive when the specific activity (cpm per red blood cell) of the labeled red cells in the spleen is compared to that of the red cells still in the circulation. Asialo red cells, obtained by removal of sialic acid with sialidase, frequently have been used as a model for the study of sequestration of senescent red cells. With the technique herein described, it was possible to show that while asialo red cells will inhibit the uptake of labeled asialo red cells, they have no effect on the sequestration of senescent red cells. Presumably, different sites and mechanisms of sequestration are involved.

  10. 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.

  11. Radionuclide-labeled red blood cell imaging of vascular malformations in children

    SciTech Connect

    Sloan, G.M.; Bolton, L.L.; Miller, J.H.; Reinisch, J.F.; Nichter, L.S.

    1988-09-01

    Vascular malformations, particularly in the absence of cutaneous changes, can be difficult to distinguish from other soft tissue masses in children. We have used technetium-99m-labeled red blood cell scintigraphy to study 47 lesions in 43 children. Thirty-nine lesions showed increased flow and were, therefore, diagnosed as vascular malformations. Subsequent biopsy of 10 of these lesions confirmed that diagnosis. The other 29 lesions with increased flow were followed for 10 months to 5 years and the clinical course was consistent with vascular malformation in every case. Eight lesions showed no increased flow on technetium scan. One of these subsequently proved to be a hemangioma. The others have turned out not to be vascular malformations. Therefore, in our experience, the technetium-99m-labeled red blood cell scan has had 98% sensitivity and 100% specificity in diagnosing vascular malformations in children.

  12. Detection of gastritis by /sup 99m/Tc-labeled red-blood-cell scintigraphy

    SciTech Connect

    Wilton, G.P.; Wahl, R.L.; Juni, J.E.; Froelich, J.W.

    1984-10-01

    Gastritis is a common condition, with a variety of causes, that is diagnosed most often by barium upper gastrointestinal tract series or endoscopy. The authors report a case in which gastritis without active bleeding was apparent in scintiscans obtained during the evaluation of GI bleeding using /sup 99m/Tc-labeled red blood cells (TcRBC). The scintigraphic findings that suggest gastritis are described.

  13. Detection of gastrointestinal bleeding with /sup 99m/Tc-labeled red blood cells

    SciTech Connect

    Winzelberg, G.G.; McKusick, K.A.; Froelich, J.W.; Callahan, R.J.; Strauss, H.W.

    1982-04-01

    Using a modified in vivo /sup 99m/Tc red cell labeling technique, gastrointestinal bleeding scintigraphy was performed in 100 patients with GI bleeding. Sixty-two patients with melena or bright red blood per rectum had positive scintiscans. In comparison to results of angiography, endoscopy, surgery and contrast radiography, radionuclide scintigraphy correctly located the site of bleeding in 83% of patients. The procedures could be performed over a 24 hr period which increased the sensitivity of the test since 85% of the scintiscans were positive at one hr or greater after the onset of imaging. The procedure was more sensitive than angiography in detecting sources of GI bleeding. We conclude that GI bleeding scintigraphy /sup 99m/Tc-red cells in an accurate and effective method to detect upper and lower GI bleeding in patients with acute intermittent gastrointestinal bleeding.

  14. 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.

  15. 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.

  16. 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

  17. 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.

  18. Hepatic cavernous hemangioma: diagnosis with /sup 99m/Tc-labeled red cells and single-photon emission CT

    SciTech Connect

    Brodsky, R.I.; Friedman, A.C.; Maurer, A.H.; Radecki, P.D.; Caroline, D.F.

    1987-01-01

    During the performance of high-resolution real-time abdominal sonography, small echogenic hepatic masses are frequently discovered. A second imaging test to confirm the suspected diagnosis of hemangioma is often required. Planar labeled red-cell imaging will often not detect hemangiomas smaller than 3 cm. We studied 14 patients with labeled red-cell scintigraphy and single-photon emission CT (SPECT). Six hemangiomas were diagnosed by SPECT that would have been missed by planar imaging alone. All six were smaller than 2.5 cm. With the addition of SPECT, labeled red-cell scintigraphy has specificity and sensitivity that make it at least as reliable as dynamic CT for the noninvasive diagnosis of hepatic cavernous hemangioma.

  19. Superior sagittal sinus thrombosis: assessment with Tc-99m labeled red blood cells

    SciTech Connect

    Front, D.; Israel, O.; Even-Sapir, E.; Feinsud, M.

    1986-02-01

    The diagnostic value of scintigraphy with technetium-99m labeled red blood cells (Tc-RBC) was assessed in 19 patients with clinical suggestion of superior sagittal sinus thrombosis (SSST). Comparison of Tc-RBC static images with dynamic flow studies in the brain showed a sensitivity of 100%, specificity of 86%, and accuracy of 94% for static studies and values of 87%, 20%, and 61%, respectively, for the flow studies. Tc-RBC scintigraphy enables direct visualization of the integrity of the superior sagittal sinus, whereas CT scanning shows various but nonspecific changes in the brain associated with SSST. Single-photon emission CT study using Tc-RBC, performed in six patients, appears to have potential in the diagnosis of SSST, allowing separation of vascular structures that are superimposed on the superior sagittal sinus in planar scintigraphy study.

  20. High fluorescent and stable semiconductor quantum dots for red blood cells labeling

    NASA Astrophysics Data System (ADS)

    de Farias, Patricia M. A.; Santos, Beate S.; de Menezes, Frederico D.; Ferreira, Ricardo; Fontes, Adriana; Cesar, Carlos L.; Barjas Castro, Maria L.; Castro, Vagner; Lima, Paulo R. M.

    2005-04-01

    We present a simple and efficient method for marking living human red blood cells using CdS (Cadmium Sulfide) quantum dots (QDs). The nanocrystals were obtained via colloidal synthesis in aqueous medium with final pH=7 using sodium polyphosphate as the stabilizing agent. The methodology implementation is simple, do not requires additional capping layers nor narrow size QDs distribution. The synthesized nanoparticles were conjugated to monoclonal A anti-body. The resulting conjugates QDs/anti-A were incubated with human erythrocytes of blood groups A and O for 30 min at 37°C. The living cells in contact with the quantum dots maintained their properties for several days showing the low level of citotoxicity of the quantum dots. The conjugation of CdS QDs/anti-A show simultaneous red and green fluorescence when excited with 543 and 488 nm respectively. The efficiency of the conjugation QDs/anti-body to the erythrocytes, for each system, was monitored by confocal microscopy. The comparative analysis of the micrographs was done with the luminescence intensity maps of the samples obtained under constant capture conditions, such as, pinhole, filters, beam splitters and photomultiplier gain. The conjugates QDs/anti-A intensely marked group A erythrocytes and did not show any luminescence for group O erythrocytes, showing the sensitivity of the labeling procedure. In conclusion, we show the viability of the use of high luminescent and stable quantum dots as fluorescent labels for human erythrocytes with a methodology of simple implementation and the possibility to use them to distinguish different blood groups.

  1. 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.

  2. A label-free proteome analysis strategy for identifying quantitative changes in erythrocyte membranes induced by red cell disorders.

    PubMed

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

    2012-12-05

    Red blood cells have been extensively studied but many questions regarding membrane properties and pathophysiology remain unanswered. Proteome analysis of red cell membranes is complicated by a very wide dynamic range of protein concentrations as well as the presence of proteins that are very large, very hydrophobic, or heterogeneously glycosylated. This study investigated the removal of other blood cell types, red cell membrane extraction, differing degrees of fractionation using 1-D SDS gels, and label-free quantitative methods to determine optimized conditions for proteomic comparisons of clinical blood samples. The results showed that fractionation of red cell membranes on 1-D SDS gels was more efficient than low-ionic-strength extractions followed by 1-D gel fractionation. When gel lanes were sliced into 30 uniform slices, a good depth of analysis that included the identification of most well-characterized, low-abundance red cell membrane proteins including those present at 500 to 10,000 copies per cell was obtained. Furthermore, the size separation enabled detection of changes due to proteolysis or in vivo protein crosslinking. A combination of Rosetta Elucidator quantitation and subsequent statistical analysis enabled the robust detection of protein differences that could be used to address unresolved questions in red cell disorders. This article is part of a Special Issue entitled: Integrated omics.

  3. 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.

  4. 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

  5. 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.

  6. Glucagon in the scintigraphic diagnosis of small-bowel hemorrhage by Tc-99m-labeled red blood cells

    SciTech Connect

    Froelich, J.W.; Juni, J.

    1984-04-01

    Twelve patients undergoing scintigraphy with Tc-99m-labeled red blood cells (RBC) exhibited abnormal small-bowel activity and were given glucagon to assess its role in detecting bleeding from the small bowel. Six demonstrated focal accumulation of activity which was not identified prior to glucagon. Endoscopy, barium studies, angiography, and colonoscopy located the small-bowel bleeding site in 4 patients; in the other 2, studies of the colon failed to show the bleeding site and the origin was presumed to be the small bowel. The authors suggest that intravenous glucagon can be beneficial as an adjuvant to Tc-99m-RBC when diagnosing bleeding from the small bowel.

  7. Label-free optical sensor based on red blood cells laser tweezers Raman spectroscopy analysis for ABO blood typing.

    PubMed

    Lin, Duo; Zheng, Zuci; Wang, Qiwen; Huang, Hao; Huang, Zufang; Yu, Yun; Qiu, Sufang; Wen, Cuncheng; Cheng, Min; Feng, Shangyuan

    2016-10-17

    The clinical significance of ABO blood typing extends beyond transfusion medicine and is demonstrated to be associated with susceptibility to various diseases, even including cancer. In this study, a home-made laser tweezers Raman spectroscopy (LTRS) system was applied to detect red blood cells (RBCs) with the aim to develop a label-free, simple and objective blood typing method for the first time. High-quality Raman spectra of RBCs in the fingerprint region of 420-1700 cm-1 can be obtained, meanwhile exciting blood typing results can be achieved, especially with an accuracy of 100% for identifying Type AB from other blood types with the use of multivariate statistical analysis based on principal component analysis (PCA) combined with linear discriminant analysis (LDA). This primary work demonstrates that the label-free RBCs LTRS analysis in conjunction with PCA-LDA diagnostic algorithms has great potential as a biosensor for ABO blood typing.

  8. Clinical evaluation of a /sup 51/Cr-labeled red blood cell survival test for in vivo blood compatibility testing

    SciTech Connect

    Pineda, A.A.; Dharkar, D.D.; Wahner, H.W.

    1984-01-01

    Modified red blood cell survival studies with use of 51Cr were performed in three groups of subjects. Group 1 consisted of normal subjects who were given labeled autologous blood, group 2 were subjects in need of blood transfusions and given labeled ABO and Rh crossmatch-compatible blood, and group 3 were patients in need of blood transfusion but in whom problems arose in finding compatible blood. The results of the studies suggest that for patients with blood compatibility problems, normal red blood cell survival values at 1 hour do not exclude the possibility of severe hemolysis 24 hours later. Thus, if a 1-hour test result is normal, the procedure should be extended routinely to 24 hours. Moreover, the test can be used to evaluate the clinical importance of antibodies. We showed that anti-Yka and anti-Lan were clinically significant, but high-titer, low-avidity antibodies, anti-Kna, anti-I, and anti-HI were clinically insignificant in the cases studied. This finding emphasizes the importance of an in vivo test for the final compatibility evaluation in complicated blood replacement problems.

  9. 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.

  10. Incorporation of fluorescein conjugated function-spacer-lipid constructs into the red blood cell membrane facilitates detection of labeled cells for the duration of ex-vivo storage.

    PubMed

    Ki, Katrina K; Flower, Robert L; Faddy, Helen M; Dean, Melinda M

    2016-02-01

    The contribution of ex-vivo storage duration of packed red blood cells (PRBC) to patient outcomes and transfusion-related immunomodulation (TRIM) remains a broadly debated area in transfusion medicine. Kode™ Technology with fluorescein conjugated function-spacer-lipid (FSL-FLRO4) constructs is a tool that can aid in-vitro visualization and tracking of red blood cells (RBC) during routine storage. FSL-FLRO4 is incorporated into the RBC membrane without altering cell function. In this study, we explore the suitability of this technology to label clinical grade PRBC and to determine if the label would be retained during ex-vivo storage. Firstly, to confirm feasibility and assess the limit of detection of FSL-FLRO4 on PRBC at date of expiry (42 days post-collection), we tracked the binding of FSL-FLRO4 on PRBC at weekly intervals during routine storage. Over the time course, all cells remained labelled with FSL-FLRO4, although a decrease in the intensity of labelling was observed (P<0.0001). We then further investigated differences in FSL-FLRO4 labelling during RBC storage by labelling separated light-young and dense-old RBC from the same PRBC unit. There were no differences in the capacity of FSL-FLRO4 to label these different RBC subsets. Together, these data demonstrate that FSL-FLRO4 is a suitable reagent for labelling PRBC at any point during routine storage. This technology will facilitate the development of immunoassays and transfusion models focused on addressing the mechanisms involved in TRIM.

  11. 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.

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

    DOE PAGES

    Liu, Daniel S.; Nivon, Lucas G.; Richter, Florian; ...

    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

  13. 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.

  14. 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.

  15. Demonstration of hematobilia using technetium-99m labeled red blood cells

    SciTech Connect

    Lee, S.M.; Lee, R.G.; Clouse, M.E.; Hill, T.C.

    1986-01-01

    A 75-year-old woman, who presented with obstructive jaundice, was shown by percutaneous transhepatic cholangiography to have a markedly dilated biliary system and stones within the common bile duct. The stones were removed percutaneously using the transduodenal approach, and an internal drainage catheter was placed. Following the procedure, the patient experienced gastrointestinal bleeding manifested by melanotic stools. Blood-tinged bile was withdrawn from the biliary drainage catheter, leading to the suspicion that the bleeding might be originating from the biliary tract. A Tc-99m red blood cell (Tc-99m RBC) scan was performed to try to designate the biliary tract as the site of bleeding, and to determine if there were any other bleeding sites present. The study demonstrated bleeding from the biliary tract, which was confirmed by angiography and endoscopy. The technique for the detection of gastrointestinal bleeding using Tc-99m RBCs is well described. This case suggests that when doing studies to localize occult bleeding, the liver should be included in the field-of-view to exclude bleeding from the liver.

  16. 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

  17. 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.

  18. Definitive diagnosis of hepatic hemangiomas: MR imaging versus Tc-99m-labeled red blood cell SPECT

    SciTech Connect

    Birnbaum, B.A.; Weinreb, J.C.; Megibow, A.J.; Sanger, J.J.; Lubat, E.; Kanamuller, H.; Noz, M.E.; Bosniak, M.A. )

    1990-07-01

    Thirty-seven patients with 69 suspected hemangiomas found by means of computed tomography (CT) and/or ultrasound were studied with both 0.5-T magnetic resonance (MR) imaging and single photon emission CT (SPECT) with technetium-99m-labeled red blood cells. Using a criterion of perfusion-blood pool mismatch, SPECT readers diagnosed 50 of 64 hemangiomas and all five nonhemangiomas (sensitivity, 78% (95% confidence interval, 0.664 - 0.864); accuracy, 80% (0.69 - 0.877)). Qualitative analysis of lesion signal intensity on T2-weighted spin-echo MR images allowed readers to diagnose 58 of 64 hemangiomas and four of five nonhemangiomas (sensitivity, 91% (0.814 - 0.96); accuracy, 90% (0.807 - 0.951)). Because of the significantly higher cost of MR imaging and its inability to categorically differentiate hemangiomas from hypervascular metastases, the authors consider SPECT to be the method of choice for diagnosing hepatic hemangiomas. MR imaging should be reserved for the diagnosis of lesions smaller than 2.0 cm and for those 2.5 cm and smaller adjacent to the heart or major hepatic vessels; in such cases MR imaging was found superior to SPECT.

  19. Hybrid SPECT-CT with 99mTc-labeled red blood cell in a case of blue rubber bleb nevus syndrome: added value over planar scintigraphy.

    PubMed

    Das, Kalpa Jyoti; Sharma, Punit; Naswa, Niraj; Soundararajan, Ramya; Kumar, Rakesh; Bal, Chandrasekhar; Malhotra, Arun

    2013-01-01

    Blue rubber bleb nevus syndrome (BRBNS) is a rare clinical entity characterized by multiple venous malformations (hemangiomas) of the skin and gastrointestinal tract. These hemangiomas usually cause episodes of occult gastrointestinal bleeding leading to iron deficiency anemia, and also carry a significant potential for serious hemorrhage. The 99mechnetium (99mTc)-labeled red blood cell scintigraphy has traditionally been utilized in the localization of occult bleeding sites in patients with suspected vascular malformations, angiodysplasia, and Meckel’s diverticulum. We report the incremental value of 99mTc-labeled red blood cell hybrid single-photon emission computed tomography-computed tomography (SPECT-CT) over planar scintigraphy alone in a 12-year-old female patient with BRBNS.

  20. A case of cavernous hemangioma of the small intestine diagnosed by scintigraphy with Tc-99m-labeled red blood cells.

    PubMed

    Iwata, Y; Shiomi, S; Otso, R; Sasaki, N; Hara, J; Nakamura, S; Nishiguchi, S; Ochi, H

    2000-10-01

    Hemangioma of the small intestine is rare, and the preoperative diagnosis of it is difficult. We report a patient with gastrointestinal bleeding for whom Tc-99m-labeled red blood cell scintigraphy was useful in diagnosing cavernous hemangioma of the small intestine. A 25-year-old man was referred to our hospital for recurrent iron deficiency anemia. Because of the patient's severe anemia, imaging was performed to locate the bleeding lesion in the gastrointestinal tract. Scintigraphy with Tc-99m-labeled red blood cells revealed pooling indicating a tumor and extravasation of blood from the tumor. Scintigraphy with Tc-99m pertechnetate revealed no abnormal accumulation. Partial resection of the small intestine was done, and cavernous hemangioma of the small intestine was diagnosed by using the specimen of resected tissue.

  1. Cold hematoma visualized by technetium-99m labeled red blood cells

    SciTech Connect

    Beanblossom, M.

    1986-09-01

    A 64-yr-old male was admitted to the hospital with severe abdominal pain associated with vomiting. Upon examination, the patients Hgb was 7.8 with a WBC count of 13.3 band cells of 7 and a recticulocyte count of 3.4, no evidence of gastrointestinal bleeding. The patient's prior history revealed involvement in an automobile accident approx. 10 days prior to this admission. At that time, he suffered multiple contusions and abrasions with a fracture to his left clavicle. Apparently there were no episodes of abdominal pain or vomiting prior to the onset of illness perceived on the day of admission. A liver/spleen scan was done. Four millicuries of /sup 99m/Tc-sulfur colloid were intravenously injected using a bolus injection technique while obtaining multiple dynamic images. The flow study was unremarkable, demonstrating no abnormalities to the great vessels and good perfusion to both organs. Static images of the liver and spleen revealed a straightening or flatness to the lateral border of the spleen with a small diminished area of tracer sulfur colloid localization at the posterolateral aspect of that organ. This finding raised the suspicion that a small subcapsular hematoma had developed at the mid-posterolateral aspect of the spleen. Twenty-four hours after hospital admission, 4 units of packed RBCs were transfused into the patient. Although there was at this time still no evidence of abnormal bleeding, it was felt that because of the strong symptomatic correlation for internal bleeding, a radionuclide bleeding site study should be ordered and immediately performed.

  2. Posttransfusion Survival and Distribution of 51-CR Labeled Fresh and Liquid Preserved Syngeneic Mouse Red Blood Cells

    DTIC Science & Technology

    2007-11-02

    LABORATORY BOSTON UNIVERSITY SCHOOL OF MEDICINE 615 ALBANY STREET BOSTON, MA 02118 CO 3 JUNE 1991 ZQ CO o Reproduction in whole or in part is...Distribution unlimited. 17. DISTRIBUTION STATEMENT (ol Cha abotract ontotod In Block 20, II dlltoranl bom Report) 18. SUPPLEMENTARY NOTES 19. KEY WORDS...transfusion of nonviable red blood cells (RBC) inhibits the reticuloendothelial system. The study reported here examines the in vivo distribution of

  3. Pharmacokinetics and red cell utilization of 52Fe/59Fe-labelled iron polymaltose in anaemic patients using positron emission tomography.

    PubMed

    Beshara, Soheir; Sörensen, Jens; Lubberink, Mark; Tolmachev, Vladimir; Långström, Bengt; Antoni, Gunnar; Danielson, Bo G; Lundqvist, Hans

    2003-03-01

    Parenteral iron-polysaccharide complexes are increasingly applied. The pharmacokinetics of iron sucrose have been assessed by our group using positron emission tomography (PET). A single intravenous injection of 100 mg iron as iron (III) hydroxide-polymaltose complex, labelled with a tracer in the form of 52Fe/59Fe, was similarly assessed in six patients using PET for about 8 h. Red cell utilization was followed for 4 weeks. Iron polymaltose was similarly distributed to the liver, spleen and bone marrow. However, a larger proportion of this complex was rapidly distributed to the bone marrow. The shorter equilibration phase for the liver, about 25 min, indicates the minimal role of the liver for direct distribution. Splenic uptake also reflected the reticuloendothelial handling of this complex. Red cell utilization ranged from 61% to 99%. Despite the relatively higher uptake by the bone marrow, there was no saturation of marrow transport systems at this dose level. In conclusion, high red cell utilization of iron polymaltose occurred in anaemic patients. The major portion of the injected dose was rapidly distributed to the bone marrow. In addition, the reticuloendothelial uptake of this complex may reflect the safety of polysaccharide complexes. Non-saturation of transport systems to the bone marrow indicated the presence of a large interstitial transport pool, which might possibly be transferrin.

  4. Red blood cell production

    MedlinePlus

    ... hemocytoblasts give rise to all of the formed elements in blood. If a hemocytoblast commits to becoming a cell called a proerythroblast, it will develop into a new red blood cell. The formation of a red ...

  5. Study of the influence of laser of low potency to label red blood cells and plasmatic protein with 99mTc in vitro

    NASA Astrophysics Data System (ADS)

    Rolim, Ricardo Q.; Carvalho, Elaine F.; Nascimento, Edgar V.; Souza, Grace M.; Magnata, Simey S.; Bernardo-Filho, Mario; Catanho, Maria T.

    2004-09-01

    This work aims to verify the effect of laser of low potency to label red blood cells and plasmatic protein with 99mTc in vitro. The experiments were carried out by incubating of anticoagulant whole blood. Differents doses of gallium arsenide laser with energy density of 3j/cm2, 6j/cm2, 9 j/cm2, 18 j/cm2 and 804nm wavelength was applied. A stannous chloride solution of 1,2μg/mL was added the incubation for 60 minutes. After this the 99mTc was added and the incubation was continued for another 10 minutes. Those were centrifuged, precipitated with thrichoroacetic acid 5% and mensured in a counter. The results shows that there is a significant decrease in the fixation of 99mTc in red blood cells when the concentration of 3j/ cm2 and (from 93 to 32.2%) and in occurs an increase %ATI from 12 to 54,6% of plasma protein. The laser promote modification on some properties of the red blood cellular membrane, probably, due to the metabolization of cellular that could be capable to generation the active metabolites.

  6. 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.

  7. High Red Blood Cell Count

    MedlinePlus

    Symptoms High red blood cell count By Mayo Clinic Staff A high red blood cell count is an increase in oxygen-carrying cells in your bloodstream. Red blood cells transport oxygen from your lungs to tissues throughout ...

  8. 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.

  9. Technetium-99m-labelled red blood cell imaging in the diagnosis of hepatic haemangiomas: the role of SPECT/CT with a hybrid camera.

    PubMed

    Schillaci, Orazio; Danieli, Roberta; Manni, Carlo; Capoccetti, Francesca; Simonetti, Giovanni

    2004-07-01

    Delayed liver single-photon emission computed tomography (SPECT) after (99m)Tc red blood cell (RBC) labelling is helpful in detecting hepatic haemangiomas; however, diagnosis can be difficult when lesions are situated adjacent to structures like the inferior vena cava, the heart or hepatic vessels, where blood activity persists. The aims of this study were to evaluate the usefulness of RBC SPECT and transmission computed tomography (RBC SPECT/CT) performed simultaneously with a hybrid imaging system for correct characterisation of hepatic lesions in patients with suspected haemangioma, and to assess the additional value of fused images compared with SPECT alone. Twelve patients with 24 liver lesions were studied. The acquisitions of both anatomical (CT) and functional (SPECT) data were performed during a single session. SPECT images were first interpreted alone and then re-evaluated after adding the transmission anatomical maps. Image fusion was successful in all patients, with perfect correspondence between SPECT and CT data, allowing the precise anatomical localisation of sites of increased blood pool activity. SPECT/CT had a significant impact on results in four patients (33.3%) with four lesions defined as indeterminate on SPECT images, accurately characterising the hot spot foci located near vascular structures. In conclusion, RBC SPECT/CT imaging using this hybrid SPECT/CT system is feasible and useful in the identification or exclusion of suspected hepatic haemangiomas located near regions with high vascular activity.

  10. 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.

  11. 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

  12. 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.

  13. Freeze-Dried Human Red Blood Cells

    DTIC Science & Technology

    1992-04-15

    freeze-dried and rehydrated blood cells will be made radioactive with " chromium and infused into my other arm through a hypodermic needle . No more than...directed at: (1) development of buffer formulations based on the glass transition and water replacement theory : (2) establishing standard...survival of transfused red blood cells. The labelled RBC were infused through a 20 gauge needle into the volunteer via a scalp vein in the right arm

  14. Oxygen delivery from red cells.

    PubMed Central

    Clark, A; Federspiel, W J; Clark, P A; Cokelet, G R

    1985-01-01

    This paper deals with the theoretical analysis of the unloading of oxygen from a red cell. A scale analysis of the governing transport equations shows that the solutions have a boundary layer structure near the red-cell membrane. The boundary layer is a region of chemical nonequilibrium, and it owes its existence to the fact that the kinetic time scales are shorter than the diffusion time scales in the red cell. The presence of the boundary layer allows an analytical solution to be obtained by the method of matched asymptotic expansions. A very useful result from the analysis is a simple, lumped-parameter description of the oxygen delivery from a red cell. The accuracy of the lumped-parameter description has been verified by comparing its predictions with results obtained by numerical integration of the full equations for a one-dimensional slab. As an application, we calculate minimum oxygen unloading times for red cells. PMID:3978198

  15. A novel synthesis of polyacrolein microspheres and their application for cell labeling and cell separation.

    PubMed

    Margel, S; Beitler, U; Ofarim, M

    1981-01-01

    A novel method for the synthesis of polyacrolein microspheres with fluorescent or magnetic properties is described. These microspheres carry reactive aldehyde groups on their surface, which are used for covalent binding of various proteins at physiological pH. Polyacrolein microspheres may be used as a simple tool for cell labeling and cell separation. The feasibility of specific labeling of fresh human red blood cells and of the separation of human red blood cells from turkey red blood cells by means of a magnetic field is discussed.

  16. Pediatric red cell disorders and pure red cell aplasia.

    PubMed

    Perkins, Sherrie L

    2004-12-01

    Anemia in children may arise from a wide variety of pathogenetic mechanisms that include congenital and acquired disorders. Often the diagnostic considerations include disorders that are not seen commonly in adults and lifelong disorders that arise in children and persist throughout life. Consideration of diverse causes of anemia such as red cell membrane disorders, red cell enzymopathies, congenital dyserythropoietic anemias, congenital sideroblastic anemias, and hereditary pure red cell aplasia (Diamond-Blackfan anemia), as well as infectious causes such as parvovirus B19 infection, often is required when diagnosing anemia in an infant or young child. Knowledge of these entities that are important causes of anemia in the pediatric population, including clinical manifestations and laboratory workup, will aid in recognition of the specific disease entities and effective workup of pediatric red cell disorders.

  17. Red blood cells, spherocytosis (image)

    MedlinePlus

    Spherocytosis is a hereditary disorder of the red blood cells (RBCs), which may be associated with a mild anemia. Typically, the affected RBCs are small, spherically shaped, and lack the light centers seen ...

  18. Red Blood Cell Antibody Identification

    MedlinePlus

    ... name: Red Blood Cell Antibody Identification Related tests: Direct Antiglobulin Test ; RBC Antibody Screen ; Blood Typing ; Type ... a positive RBC antibody screen or a positive direct antiglobulin test (DAT) . It is used to identify ...

  19. Isotope Labeling in Insect Cells

    PubMed Central

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

    2011-01-01

    Recent years have seen remarkable progress in applying nuclear magnetic resonance (NMR) spectroscopy to proteins that have traditionally been difficult to study due to issues with folding, posttranslational modification, and expression levels or combinations thereof. In particular, insect cells have proved useful in allowing large quantities of isotope-labeled, functional proteins to be obtained and purified to homogeneity, allowing study of their structures and dynamics by using NMR. Here, we provide protocols that have proven successful in such endeavors. PMID:22167667

  20. 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.

  1. Red Blood Cell Magnetophoresis

    PubMed Central

    Zborowski, Maciej; Ostera, Graciela R.; Moore, Lee R.; Milliron, Sarah; Chalmers, Jeffrey J.; Schechter, Alan N.

    2003-01-01

    The existence of unpaired electrons in the four heme groups of deoxy and methemoglobin (metHb) gives these species paramagnetic properties as contrasted to the diamagnetic character of oxyhemoglobin. Based on the measured magnetic moments of hemoglobin and its compounds, and on the relatively high hemoglobin concentration of human erythrocytes, we hypothesized that differential migration of these cells was possible if exposed to a high magnetic field. With the development of a new technology, cell tracking velocimetry, we were able to measure the migration velocity of deoxygenated and metHb-containing erythrocytes, exposed to a mean magnetic field of 1.40 T and a mean gradient of 0.131 T/mm, in a process we call cell magnetophoresis. Our results show a similar magnetophoretic mobility of 3.86 × 10−6 mm3 s/kg for erythrocytes with 100% deoxygenated hemoglobin and 3.66 × 10−6 mm3 s/kg for erythrocytes containing 100% metHb. Oxygenated erythrocytes had a magnetophoretic mobility of from −0.2 × 10−6 mm3 s/kg to +0.30 × 10−6 mm3 s/kg, indicating a significant diamagnetic component relative to the suspension medium, in agreement with previous studies on the hemoglobin magnetic susceptibility. Magnetophoresis may open up an approach to characterize and separate cells for biochemical analysis based on intrinsic and extrinsic magnetic properties of biological macromolecules. PMID:12668472

  2. Red blood cell magnetophoresis.

    PubMed

    Zborowski, Maciej; Ostera, Graciela R; Moore, Lee R; Milliron, Sarah; Chalmers, Jeffrey J; Schechter, Alan N

    2003-04-01

    The existence of unpaired electrons in the four heme groups of deoxy and methemoglobin (metHb) gives these species paramagnetic properties as contrasted to the diamagnetic character of oxyhemoglobin. Based on the measured magnetic moments of hemoglobin and its compounds, and on the relatively high hemoglobin concentration of human erythrocytes, we hypothesized that differential migration of these cells was possible if exposed to a high magnetic field. With the development of a new technology, cell tracking velocimetry, we were able to measure the migration velocity of deoxygenated and metHb-containing erythrocytes, exposed to a mean magnetic field of 1.40 T and a mean gradient of 0.131 T/mm, in a process we call cell magnetophoresis. Our results show a similar magnetophoretic mobility of 3.86 x 10(-6) mm(3) s/kg for erythrocytes with 100% deoxygenated hemoglobin and 3.66 x 10(-6) mm(3) s/kg for erythrocytes containing 100% metHb. Oxygenated erythrocytes had a magnetophoretic mobility of from -0.2 x 10(-6) mm(3) s/kg to +0.30 x 10(-6) mm(3) s/kg, indicating a significant diamagnetic component relative to the suspension medium, in agreement with previous studies on the hemoglobin magnetic susceptibility. Magnetophoresis may open up an approach to characterize and separate cells for biochemical analysis based on intrinsic and extrinsic magnetic properties of biological macromolecules.

  3. Leukemic cell labeling with indium-111-oxine

    SciTech Connect

    Uchida, T.; Takagi, Y.; Matsuda, S.; Yui, T.; Ishibashi, T.; Kimura, H.; Kariyone, S.

    1984-01-01

    Leukemic cells were labeled with In-111-oxine in patients with acute leukemia. In vitro labeling studies revealed that labeling efficiency reached maximum 80.8 +- 3.6% (mean +- 1SD) by 2 times washes after 20 minutes incubation time. Cell viability was assessed by trypan blue exclusion test and in vitro culture of leukemic cells, which showed no cellular damage during labeling procedure. Elution of In-111 from the labeled cells was 10.0 +- 1.2% at 12 hours after labeling. For in vivo leukemic cell kinetic studies, more than 10/sup 8/ leukemic cells separated from Ficoll-Hypacque sedimentation were labeled by 30 minutes of In-111-oxine incubation and two times washes at 37/sup 0/C. In vivo studies were performed in 7 patients with acute myeloblastic, lymphoblastic leukemia and blastic crisis of chronic myelocytic leukemia. Labeled leukemic cells disappeared in single exponential fashion with half life of 9.6 to 31.8 hours. Total leukemic cell pool in peripheral circulation was calculated, which correlated well with peripheral leukemic cell counts (r=0.99). No relationship was observed between total leukemic cell pool and leukemic cell turnover rate. Migration patterns of labeled leukemic cells showed that pulmonary uptake was evident within 15 minutes after the infusion and returned to base-line. Splenic and hepatic uptake showed gradual increase up to 24 hours. Bone marrow accumulation was shown only in 2 cases. Presently, there are no suitable radionuclides for leukemic cell labeling. In-111-oxine labeled leukemic cells would overcome this difficulty.

  4. 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.

  5. Synthesis and optical properties of pyrrolidinyl peptide nucleic acid carrying a clicked Nile red label

    PubMed Central

    Yotapan, Nattawut; Charoenpakdee, Chayan; Wathanathavorn, Pawinee; Ditmangklo, Boonsong

    2014-01-01

    Summary DNA or its analogues with an environment-sensitive fluorescent label are potentially useful as a probe for studying the structure and dynamics of nucleic acids. In this work, pyrrolidinyl peptide nucleic acid (acpcPNA) was labeled at its backbone with Nile red, a solvatochromic benzophenoxazine dye, by means of click chemistry. The optical properties of the Nile red-labeled acpcPNA were investigated by UV–vis and fluorescence spectroscopy in the absence and in the presence of DNA. In contrast to the usual quenching observed in Nile red-labeled DNA, the hybridization with DNA resulted in blue shifting and an enhanced fluorescence regardless of the neighboring bases. More pronounced blue shifts and fluorescence enhancements were observed when the DNA target carried a base insertion in close proximity to the Nile red label. The results indicate that the Nile red label is located in a more hydrophobic environment in acpcPNA–DNA duplexes than in the single-stranded acpcPNA. The different fluorescence properties of the acpcPNA hybrids of complementary DNA and DNA carrying a base insertion are suggestive of different interactions between the Nile red label and the duplexes. PMID:25246975

  6. Synthesis and optical properties of pyrrolidinyl peptide nucleic acid carrying a clicked Nile red label.

    PubMed

    Yotapan, Nattawut; Charoenpakdee, Chayan; Wathanathavorn, Pawinee; Ditmangklo, Boonsong; Wagenknecht, Hans-Achim; Vilaivan, Tirayut

    2014-01-01

    DNA or its analogues with an environment-sensitive fluorescent label are potentially useful as a probe for studying the structure and dynamics of nucleic acids. In this work, pyrrolidinyl peptide nucleic acid (acpcPNA) was labeled at its backbone with Nile red, a solvatochromic benzophenoxazine dye, by means of click chemistry. The optical properties of the Nile red-labeled acpcPNA were investigated by UV-vis and fluorescence spectroscopy in the absence and in the presence of DNA. In contrast to the usual quenching observed in Nile red-labeled DNA, the hybridization with DNA resulted in blue shifting and an enhanced fluorescence regardless of the neighboring bases. More pronounced blue shifts and fluorescence enhancements were observed when the DNA target carried a base insertion in close proximity to the Nile red label. The results indicate that the Nile red label is located in a more hydrophobic environment in acpcPNA-DNA duplexes than in the single-stranded acpcPNA. The different fluorescence properties of the acpcPNA hybrids of complementary DNA and DNA carrying a base insertion are suggestive of different interactions between the Nile red label and the duplexes.

  7. 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.

  8. Nitric oxide scavenging by red cell microparticles.

    PubMed

    Liu, Chen; Zhao, Weixin; Christ, George J; Gladwin, Mark T; Kim-Shapiro, Daniel B

    2013-12-01

    Red cell microparticles form during the storage of red blood cells and in diseases associated with red cell breakdown and asplenia, including hemolytic anemias such as sickle cell disease. These small phospholipid vesicles that are derived from red blood cells have been implicated in the pathogenesis of transfusion of aged stored blood and hemolytic diseases, via activation of the hemostatic system and effects on nitric oxide (NO) bioavailability. Red cell microparticles react with the important signaling molecule NO almost as fast as cell-free hemoglobin, about 1000 times faster than red-cell-encapsulated hemoglobin. The degree to which this fast reaction with NO by red cell microparticles influences NO bioavailability depends on several factors that are explored here. In the context of stored blood preserved in ADSOL, we find that both cell-free hemoglobin and red cell microparticles increase as a function of duration of storage, and the proportion of extra erythrocytic hemoglobin in the red cell microparticle fraction is about 20% throughout storage. Normalized by hemoglobin concentration, the NO-scavenging ability of cell-free hemoglobin is slightly higher than that of red cell microparticles as determined by a chemiluminescence NO-scavenging assay. Computational simulations show that the degree to which red cell microparticles scavenge NO will depend substantially on whether they enter the cell-free zone next to the endothelial cells. Single-microvessel myography experiments performed under laminar flow conditions demonstrate that microparticles significantly enter the cell-free zone and inhibit acetylcholine, endothelial-dependent, and NO-dependent vasodilation. Taken together, these data suggest that as little as 5 μM hemoglobin in red cell microparticles, an amount formed after the infusion of one unit of aged stored packed red blood cells, has the potential to reduce NO bioavailability and impair endothelial-dependent vasodilation.

  9. Born approximation model for light scattering by red blood cells.

    PubMed

    Lim, Joonoh; Ding, Huafeng; Mir, Mustafa; Zhu, Ruoyu; Tangella, Krishnarao; Popescu, Gabriel

    2011-10-01

    The primary role of a red blood cell (RBC) is delivering oxygen throughout our body. Abnormalities of this basic function lead to anemia and are caused by numerous diseases such as malaria and sickle cell anemia. As prompt and inexpensive tests for blood screening are in demand, we have developed a faster and reliable way to measure morphological parameters associated with the structure of red blood cells and the size distribution of the cells in a whole blood smear. Modeling the RBC shape under Born approximation, we are able to determine parameters of clinical relevance, such as the diameter, thickness and dimple size. From a measured quantitative phase image of a blood smear, we can determine the average and standard deviation of the red blood cell volume simultaneously, i.e., without analyzing each cell individually. This approach may open the door for a new generation of label-free, high-throughput blood testing.

  10. Mechanisms of immune red cell destruction, and red cell compatibility testing

    SciTech Connect

    Garratty, G.

    1983-03-01

    The immune destruction of red cells can occur as a complement-mediated intravascular process, or extravascularly, where the red cells are destroyed by macrophages following interaction with cell-bound IgG1, IgG3, and/or C3b. Many of the factors that affect this in vivo destruction are not taken into account during in vitro pretransfusion compatibility testing. At present, even by use of more elaborate tests, it is difficult to accurately predict the fate of a transfused unit of blood. By using some simple information, such as antibody specificity and thermal range, it is sometimes possible to predict the outcome of transfusing a unit of blood that is incompatible in vitro. At other times it may be necessary to utilize /sup 51/Cr-labeled red cells to determine the risk of transfusing such units. Because of the paucity of reported clinical correlations, macrophage/monocyte monolayer assays are of little practical value at present.

  11. 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.

  12. Reversibility of cell surface label rearrangement

    PubMed Central

    1976-01-01

    Cell surface labeling can cause rearrangements of randomly distributed membrane components. Removal of the label bound to the cell surface allows the membrane components to return to their original random distribution, demonstrating that label is necessary to maintain as well as to induce rearrangements. With scanning electron microscopy, the rearrangement of concanavalin A (con A) and ricin binding sites on LA-9 cells has been followed by means of hemocyanin, a visual label. The removal of con A from its binding sites at the cell surface with alpha- methyl mannoside, and the return of these sites to their original distribution are also followed in this manner. There are labeling differences with con A and ricin. Under some conditions, however, the same rearrangements are seen with both lectins. The disappearance of labeled sites from areas of ruffling activity is a major feature of the rearrangements seen. Both this ruffling activity and the rearrangement of label are sensitive to cytochalasin B, and ruffling activity, perhaps along with other cytochalasin-sensitive structure, may play a role in the rearrangements of labeled sites. PMID:1025154

  13. 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.

  14. 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.

  15. 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.

  16. Visualization of cutaneous hemangioma with Tc-99m tagged red blood cells

    SciTech Connect

    Gordon, L.; Vujic, I.; Spicer, K.M.

    1981-10-01

    Scintigraphy with Tc-99m labeled red blood cells (RBCs) was used to evaluate a patient with a large cutaneous hemangioma. The usefulness of this procedure when combined with arteriography is discussed.

  17. 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

  18. Red cell transfusion "trigger": a review.

    PubMed

    Petrides, Marian

    2003-07-01

    Despite the publication of several consensus guidelines that set forth recommendations for the transfusion of red cells, actual clinical practice continues to vary widely. Animal data and studies in human volunteers and patients support a red cell transfusion threshold of 7 to 8 g/dl in most patients. However, conflicting data, particularly in cardiac patients and in the elderly, suggest that it may be impossible to define a single red cell "trigger" for all patients. A well-designed, randomized, controlled trial is still needed to establish a safe threshold for red cell transfusion in adults with coronary artery disease.

  19. 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.

  20. 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.

  1. Labeling Cells with Silver/Dendrimer Nanocomposites

    DTIC Science & Technology

    2005-01-01

    labeling. A PAMAME5.NH 2 dendrimer was used as a template to prepare first a silver -dendrimer complex in an aqueous solution at biologic pH=7.4...electron microscope operating at 200 kV. Samples were prepared by mounting a drop of aqueous solutions of nanoparticles on carbon-coated copper grids...UNCLASSIFIED Defense Technical Information Center Compilation Part Notice ADP019741 TITLE: Labeling Cells with Silver /Dendrimer Nanocomposites

  2. 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.

  3. 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...

  4. 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...

  5. 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...

  6. 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...

  7. 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...

  8. Uptake of carnitine by red blood cells

    SciTech Connect

    Campa, M.; Borum, P.

    1986-05-01

    A significant amount of blood carnitine (70% of cord blood and 40% of blood from healthy adults) is partitioned into the red blood cell compartment of whole blood. Data indicate that the plasma compartment and the red blood cell compartment of whole blood represent different metabolic pools of carnitine. There are no data to indicate that red blood cells synthesize carnitine, but our understanding of the uptake of carnitine by red blood cells is negligible. Red blood cells were obtained from healthy adults, washed twice with normal saline, and used for uptake experiments. When the cells were incubated at 37/sup 0/C in the presence of /sup 14/C-carnitine, radioactivity was found both in the soluble cytosolic and membrane fractions of the cells following lysis. The uptake was dependent upon the time of incubation, temperature of incubation, and carnitine concentration in the incubation medium. Washed red blood cell membranes incubated with /sup 14/C-carnitine showed specific binding of radioactivity. These data are consistent with the hypothesis that red blood cells have an uptake mechanism for L-carnitine.

  9. 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

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

    PubMed

    Mohandas, Narla; Gallagher, Patrick G

    2008-11-15

    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.

  11. 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.

  12. Human spleen and red blood cells

    NASA Astrophysics Data System (ADS)

    Pivkin, Igor; Peng, Zhangli; Karniadakis, George; Buffet, Pierre; Dao, Ming

    2016-11-01

    Spleen plays multiple roles in the human body. Among them is removal of old and altered red blood cells (RBCs), which is done by filtering cells through the endothelial slits, small micron-sized openings. There is currently no experimental technique available that allows us to observe RBC passage through the slits. It was previously noticed that people without a spleen have less deformable red blood cells, indicating that the spleen may play a role in defining the size and shape of red blood cells. We used detailed RBC model implemented within the Dissipative Particle Dynamics (DPD) simulation framework to study the filter function of the spleen. Our results demonstrate that spleen indeed plays major role in defining the size and shape of the healthy human red blood cells.

  13. Malaria and human red blood cells.

    PubMed

    Mohandas, Narla; An, Xiuli

    2012-11-01

    Invasion by the malaria parasite, Plasmodium falciparum, brings about extensive changes in the host red cells. These include loss of the normal discoid shape, increased rigidity of the membrane, elevated permeability to a wide variety of ionic and other species and increased adhesiveness, most notably to endothelial surfaces. These effects facilitate survival of the parasite within the host cell and tend to increase the virulence of disease that includes cerebral malaria and anemia. Numerous proteins secreted by the internalized parasite and interacting with red cell membrane proteins are responsible for the changes occurring to the host cell. Anemia, a serious clinical manifestation of malaria, is due to increased destruction of both infected and uninfected red cells due to membrane alterations, as well as ineffective erythropoiesis. There is very good evidence that various red cell disorders including hemoglobinopathies and hereditary ovalocytosis decrease the virulence of disease following parasite infection. A number of mechanism(s) are likely responsible for the protective effect of various red cell abnormalities including decreased invasion, impaired intraerythrocytic development of the parasites and altered interaction between exported parasite proteins and the red cell membrane skeleton.

  14. 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.

  15. Autoimmune Lymphoproliferative Syndrome with Red Cell Aplasia.

    PubMed

    Meena, K R; Bisht, Supriya; Tamaria, K C

    2015-12-01

    Autoimmune Lymphoproliferative Syndrome (ALPS) is a rare inherited disorder of abnormal lymphocyte apoptosis, leading to chronic lymphoproliferation. It presents as lymphadenopathy, hepatosplenomegaly and autoimmune phenomena. Pure red cell aplasia is characterized by normochromic normocytic anemia, reticulocytopenia, and absence of erythroblasts from a normal bone marrow. Only few lymphoproliferative disorders have been associated with erythroid aplasia. The authors are reporting a case of ALPS associated with red cell aplasia in a 7-y-old girl.

  16. Diphenylhydantoin-induced pure red cell aplasia.

    PubMed

    Rusia, Usha; Malhotra, Purnima; Joshi, Panul

    2006-01-01

    Pure red cell aplasia is an uncommon complication of diphenylhydantoin therapy. It has not been reported in Indian literature. Awareness of the entity helps in establishing the cause of anaemia in these patients and alerts the physicians to the need of comprehensive haematological monitoring in these patients. A case of 58-year-old male who developed pure red cell aplasia following three months of diphenylhydantoin therapy is reported here.

  17. Labeled cells in patients with malignancy

    SciTech Connect

    Dutcher, J.P.

    1984-07-01

    Recently, new approaches for radiosotopic cell labeling have gained prominence in the investigation of various aspects of malignant diseases and in the clinical care of such patients. Isotopes such as indium-111 can be visualized with standard scanning techniques providing further information about the migration of normal and malignant cells has been discovered. In vivo studies have been performed with indium-111 in animals and humans, including comparisons of the migration of abnormal cells (malignant) and of lymphocytes to abnormal nodes. Evaluation and comparison of the migration of carcinoma cells, normal lymphoid cells, and malignant lymphoid cells in animals show markedly different patterns of distribution, which could have bearing on investigations of mechanisms of metastasis. In vivo human studies also have evaluated the migration patterns of lymphoid cells from patients with chronic lymphocytic leukemia and well-differentiated lymphoma, showing very different migrating behavior between these two polarities of a similar diseases. There are concerns about the use of an isotope such as indium-111 for the labeling of long-lived cells such as lymphocytes. Laboratory studies have demonstrated impaired cell function at high concentrations of radioactivity. Some workers have expressed concern about long-term changes in cells that recirculate. Others cite precedents of other long-term uses of isotopes, therapeutically, without detrimental effects. These concerns continue to be investigated. Finally, an area of much interest in the use of indium-111 is the labeling of granulocytes. This technique has been useful diagnostically, to localize infections. The major value in patients with malignancy, primarily with hematologic malignancies, is to evaluate the potential benefit of granulocyte transfusions. Many of these patients develop prolonged granulocytopenia and become infected, and granulocyte transfusions may become a therapeutic consideration.

  18. Adhesion of platelets to artificial surfaces: effect of red cells.

    PubMed

    Brash, J L; Brophy, J M; Feuerstein, I A

    1976-05-01

    Adhesion of platelets to several polymer- and protein-coated glass surfaces has been studied in vitro. The apparatus consists of a cylindrical probe rotating in a test tube containing the platelet medium and allows close control of fluid shear and mass transport. Suspensions of washed pig platelets constitute the basic platelet medium, and can be modified by adding back red cells and plasma proteins. Adhesion is measured via 51Cr-labeling of platelets. In the absence of red cells, identical low levels of adhesion were seen on all surfaces and saturation was reached within 2 min. In the presence of red cells, adhesion was greater. Saturation on all surfaces except fibrinogen and collagen again occurred within 2 min. The adhesion levels on polymer surfaces and glass were indistinguishable, while those on albumin were lower and those on fibrinogen were higher. Collagen was the most reactive surface. It did not equilibrate within 15 min., and kinetic data indicated a platelet diffusivity strongly dependent on hematocrit. These effects were attributed to rotational and translational motion of the red cells causing increased diffusion and surface-platelet collision energy.

  19. Quenching of red cell tryptophan fluorescence by mercurial compounds.

    PubMed

    Verkman, A S; Lukacovic, M F; Tinklepaugh, M S; Dix, J A

    1986-01-01

    Intrinsic tryptophan fluorescence in red cell ghost membranes labeled with N-ethylmaleimide (N-EM) is quenched in a dose-dependent manner by the organic mercurial p-chloromercuribenzene sulfonate (p-CMBS). Fluorescence lifetime analysis shows that quenching occurs by a static mechanism. Binding of p-CMBS occurs by a rapid (less than 5 s) biomolecular association (dissociation constant K1 = 1.8 mM) followed by a slower unimolecular transition with forward rate constant k2 = 0.015 s-1 and reverse rate constant k-2 = 0.0054 s-1. Analysis of the temperature dependence of k2 gives delta H = 6.5 kcal/mol and delta S = -21 eu. The mercurial compounds p-chloromercuribenzoic acid, p-aminophenylmercuric acetate, and mercuric chloride quench red cell tryptophan fluorescence by the same mechanism as p-CMBS does; the measured k2 value was the same for each compound, whereas K1 varied. p-CMBS also quenches the tryptophan fluorescence in vesicles reconstituted with purified band 3, the red cell anion exchange protein, in a manner similar to that in ghost membranes. These experiments define a mercurial binding site on band 3 in ghosts treated with N-EM and establish the binding mechanism to this site. The characteristics of this p-CMBS binding site on band 3 differ significantly from those of the p-CMBS binding site involved in red cell water and urea transport inhibition.

  20. Synthesis of 5-nitro-2-(N-3-(4-azidophenyl)-propylamino)-benzoic acid: Photoaffinity labeling of human red blood cell ghosts with a 5-nitro-2-(3-phenylpropylamino)-benzoic acid analog

    SciTech Connect

    Branchini, B.R.; Murtiashaw, M.H.; Egan, L.A. )

    1991-04-15

    A photoaffinity analog of the potent epithelial chloride channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoic acid has been synthesized and characterized. In the dark, this reagent, 5-nitro-2-(N-3-(4-azidophenyl)-propylamino)-benzoic acid, and the parent compound reversibly inhibited chloride efflux in human red blood cell ghosts. Irradiation of ghost membranes with 350 microM arylazide analog reduced the rate of chloride efflux to 33% of the control value. The photoinactivation process was not reversed by exhaustive washing of ghost membranes. Covalent incorporation of the photoaffinity reagent was supported by difference ultraviolet spectroscopy, which indicated the attachment of the substituted 2-amino-5-nitrobenzoic acid chromophore to ghost membranes. The novel photolabeling agent described here should be a useful structural probe for chloride channels in erythrocyte membranes and epithelial cells.

  1. Genomic Typing of Red Cell Antigens

    DTIC Science & Technology

    2011-09-01

    Antigen‐Matched  Red  Cells   for  Sickle   Cell   Anemia  Patients  Using  Molecular Typing to Augment Testing: Meghan Delaney, Prashant Gaur, Askale...H, Constans J, Quilici JC, Lefevre‐Witier P, Sevin J, Stevens M: Study of red blood  cell  and serum enzymes in  five  Pyrenean communities and in a...Antigen‐Matched Red  Cells  for  Sickle   Cell  Anemia Patients  Using Molecular Typing to Augment Testing: AABB (poster) 2009.  Background: Patients with  sickle

  2. 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...

  3. 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...

  4. 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...

  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 864.5300 - 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...

  7. 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...

  8. 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...

  9. 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...

  10. 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...

  11. 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...

  12. Cell-selective metabolic labeling of biomolecules with bioorthogonal functionalities.

    PubMed

    Xie, Ran; Hong, Senlian; Chen, Xing

    2013-10-01

    Metabolic labeling of biomolecules with bioorthogonal functionalities enables visualization, enrichment, and analysis of the biomolecules of interest in their physiological environments. This versatile strategy has found utility in probing various classes of biomolecules in a broad range of biological processes. On the other hand, metabolic labeling is nonselective with respect to cell type, which imposes limitations for studies performed in complex biological systems. Herein, we review the recent methodological developments aiming to endow metabolic labeling strategies with cell-type selectivity. The cell-selective metabolic labeling strategies have emerged from protein and glycan labeling. We envision that these strategies can be readily extended to labeling of other classes of biomolecules.

  13. Theory of non-Newtonian viscosity of red blood cell suspension: effect of red cell deformation.

    PubMed

    Murata, T

    1983-01-01

    The effects of the deformation of red blood cells on non-Newtonian viscosity of a concentrated red cell suspension are investigated theoretically. To simplify the problem an elastic spherical shell filled with an incompressible Newtonian fluid is considered as a model of a normal red cell. The equation of the surface of the shell suspended in a steady simple shear flow is calculated on the assumption that the deformation from a spherical shape is very small. The relative viscosity of a concentrated suspension of such particles is obtained based on the "free surface cell" method proposed by Happel. It is shown that the relative viscosity decreases as the shear rate increases.

  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. Freeze-Dried Human Red Blood Cells

    DTIC Science & Technology

    1991-07-12

    starting cells are lost at rehydration). Hypotonic or hypertonic lysis of red cells can disrupt the normal asymmetric distribution of phospholipids between...remove the buffy coat and plasma. The packed RBC were washed in isotonic dextrose saline according to standard washing procedures (11] using an automated...cell washer ( Model 2991, COBE, Lakewood, CO). The washed and packed RBC (about 85% hematocrit) were resuspended to about 40% in hypertonic phosphate

  16. Isotope labeling of proteins in insect cells.

    PubMed

    Skora, Lukasz; Shrestha, Binesh; Gossert, Alvar D

    2015-01-01

    Protein targets of contemporary research are often membrane proteins, multiprotein complexes, secreted proteins, or other proteins of human origin. These are difficult to express in the standard expression host used for most nuclear magnetic resonance (NMR) studies, Escherichia coli. Insect cells represent an attractive alternative, since they have become a well-established expression system and simple solutions have been developed for generation of viruses to efficiently introduce the target protein DNA into cells. Insect cells enable production of a larger fraction of the human proteome in a properly folded way than bacteria, as insect cells have a very similar set of cytosolic chaperones and a closely related secretory pathway. Here, the limited and defined glycosylation pattern that insect cells produce is an advantage for structural biology studies. For these reasons, insect cells have been established as the most widely used eukaryotic expression host for crystallographic studies. In the past decade, significant advancements have enabled amino acid type-specific as well as uniform isotope labeling of proteins in insect cells, turning them into an attractive expression host for NMR studies.

  17. Metabolic dependence of red cell deformability

    PubMed Central

    Weed, Robert I.; LaCelle, Paul L.; Merrill, Edward W.

    1969-01-01

    The contribution of the metabolic state of human erythrocytes to maintenance of cellular deformability was studied during and after in vitro incubation in serum for periods up to 28 hr. An initial loss of membrane deformability became apparent between 4 and 6 hr when cellular adenosine triphosphate (ATP) levels were approximately 70% of initial values. Membrane deformability then remained stable between 6 and 10 hr. After 10 hr, when cellular ATP had decreased to < 15% of initial values, progressive parallel changes occurred in red cell calcium which increased 400% by 24 hr and in the viscosity of red cell suspensions which had risen 500-750% at 24 hr. A further progressive decrease in membrane deformability also occurred and was reflected by a 1000% increase in negative pressure required to deform the membrane. Red cell filterability decreased to zero as the disc-sphere shape transformation ensued. These changes were accompanied by an increase in ghost residual hemoglobin and nonhemoglobin protein. Regeneration of ATP in depleted cells by incubation with adenosine produced significant reversal of these changes, even in the presence of ouabain. Introduction of calcium into reconstituted ghosts prepared from fresh red cells mimicked the depleted state, and introduction of ATP, ethylenediamine tetraacetate (EDTA), and magnesium into depleted cells mimicked the adenosine effects in intact depleted cells. ATP added externally to 24-hr depleted cells was without effect. Simultaneous introduction of EDTA, ATP, or magnesium along with calcium into reconstituted ghosts prevented the marked decrease in deformability produced by calcium alone. Incorporation of adenosine diphosphate (ADP), nicotinamide adenine dinucleotide (NAD), NAD phosphate (NADP), NADP, reduced form (NADPH), glutatione, reduced form (GSH), inosine triphosphate (ITP), guanosine triphosphate (GTP), and uridine triphosphate (UTP) was without effect. These data suggest that a major role of ATP in maintenance

  18. Investigation of red blood cell antigens with highly fluorescent and stable semiconductor quantum dots.

    PubMed

    de Farias, Patrícia Maria Albuquerque; Santos, Beate Saegesser; de Menezes, Frederico Duarte; de Carvalho Ferreira, Ricardo; Barjas-Castro, Maria Lourdes; Castro, Vagner; Lima, Paulo Roberto Moura; Fontes, Adriana; Cesar, Carlos Lenz

    2005-01-01

    We report a new methodology for red blood cell antigen expression determination by a simple labeling procedure employing luminescent semiconductor quantum dots. Highly luminescent and stable core shell cadmium sulfide/cadmium hydroxide colloidal particles are obtained, with a predominant size of 9 nm. The core-shell quantum dots are functionalized with glutaraldehyde and conjugated to a monoclonal anti-A antibody to target antigen-A in red blood cell membranes. Erythrocyte samples of blood groups A+, A2+, and O+ are used for this purpose. Confocal microscopy images show that after 30 min of conjugation time, type A+ and A2+ erythrocytes present bright emission, whereas the O+ group cells show no emission. Fluorescence intensity maps show different antigen expressions for the distinct erythrocyte types. The results obtained strongly suggest that this simple labeling procedure may be employed as an efficient tool to investigate quantitatively the distribution and expression of antigens in red blood cell membranes.

  19. Storage Lesion. Role of Red Cell Breakdown

    PubMed Central

    Kim-Shapiro, Daniel B.; Lee, Janet; Gladwin, Mark T.

    2011-01-01

    As stored blood ages intraerythrocytic energy sources are depleted resulting in reduced structural integrity of the membrane. Thus, stored red cells become less deformable and more fragile as they age. This fragility leads to release of cell-free hemoglobin and formation of microparticles, sub-micron hemoglobin-containing vesicles. Upon transfusion, it is likely that additional hemolysis and microparticle formation occurs due to breakdown of fragile red blood cells. Release of cell-free hemoglobin and microparticles leads to increased consumption of nitric oxide (NO), an important signaling molecule that modulates blood flow, and may promote inflammation. Stored blood may also be deficient in recently discovered blood nitric oxide synthase activity. We hypothesize that these factors play a potential role in the blood storage lesion. PMID:21496045

  20. Anti-galactose antibodies do not bind to normal human red cells

    SciTech Connect

    Kay, M.M.B.; Bosman, G.J.C.G.M.

    1986-03-01

    The authors investigated the possibility that senescent cell IgG might have an anti-galactose (anti-gal) specificity as suggested by others. Anti-gal was isolated from normal human serum with ..cap alpha.. melibiose-agarose. The assays used were hemagglutination, rosetting, phagocytosis, and /sup 125/I protein A binding assay, immunoblotting, and glycine/HCL, pH 2.3, versus sugar elutions. Results revealed binding of anti-gal to rabbit but not human RBC. Immunoblotting of anti-gal revealed labeling of approx.29 bands in rabbit red cell membranes and no labeling of autologous human red cell membranes. The authors attempted to inhibit binding of anti-gal with various sugars. Melibiose caused enhancement rather than inhibition of agglutination when used at concentrations reported by previous investigators to cause inhibition. Neither ..cap alpha.. melibiose or galactose caused inhibition of phagocytosis of senescent cells. Senescent cell IgG was not displaced from freshly isolated old red cells by incubation with melibiose or galactose as determined by an /sup 125/I protein A binding assay. The authors were also unable to elute IgG from stored red cells with galactose. The authors conclude that senescent cell IgG does not have an anti-galactose specificity. The authors were unable to demonstrate an anti-gal antibody to normal human red cells.

  1. Interferometric phase microscopy of red blood cells

    NASA Astrophysics Data System (ADS)

    Xue, Liang; Sun, Nan; Tang, Xian; Wang, Yin; Wang, Shouyu

    2013-12-01

    Quantitative phase imaging of cells with high accuracy in a completely noninvasive manner is a challenging task. To provide a proper solution to this important need, interferometric phase microscopy is described which relies on the off-axis interferometry, confocal microscopy and high-speed image capture technology. Phase retrieval from the single interferogram is done by algorithms based on the fast Fourier transform, traditional Hilbert transform and two-step Hilbert transform, respectively. Furthermore, a phase aberrations compensation approach is applied to correct the phase distribution of the red blood cells obtained via the three methods mentioned before without the pre-known knowledge for removing the wave front curvature introduced by the microscope objectives, off-axis imaging, etc., which otherwise hinders the phase reconstruction. The improved results reveal the better inner structures of the red blood cells. The development of quantitative phase imaging technique is shedding light on their future directions and applications for basic and clinical research.

  2. 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.

  3. Incidental detection of a bleeding gastrointestinal stromal tumor on Tc-99m red blood cell scintigraphy.

    PubMed

    Santhosh, Sampath; Bhattacharya, Anish; Gupta, Vikas; Singh, Rajinder; Radotra, Bishan Dass; Mittal, Bhagwant Rai

    2012-10-01

    The role of 99m-technetium labeled red blood cell (RBC) scintigraphy in acute gastro-intestinal bleed is well-established. The authors report a case of a bleeding gastrointestinal stromal tumor (GIST) incidentally discovered on Tc-99m RBC scintigraphy.

  4. Detection of an ileal cavernous hemangioma by technetium-99m red blood cell imaging

    SciTech Connect

    Holloway, H.; Johnson, J.; Sandler, M.

    1988-01-01

    Patients with arteriovenous malformations of the bowel may have multiple symptoms secondary to chronic blood loss. A case of ileal cavernous hemangioma detected by Tc-99m labeled red blood cell imaging in the absence of active gastrointestinal bleeding is presented.

  5. Synthetic Glycosphingolipids for Live-Cell Labeling.

    PubMed

    Dauner, Martin; Batroff, Ellen; Bachmann, Verena; Hauck, Christof R; Wittmann, Valentin

    2016-07-20

    Glycosphingolipids are an important component of cell membranes that are involved in many biological processes. Fluorescently labeled glycosphingolipids are frequently used to gain insight into their localization. However, the attachment of a fluorophore to the glycan part or-more commonly-to the lipid part of glycosphingolipids is known to alter the biophysical properties and can perturb the biological function of the probe. Presented here is the synthesis of novel glycosphingolipid probes with mono- and disaccharide head groups and ceramide moieties containing fatty acids of varying chain length (C4 to C20). These glycosphingolipids bear an azide or an alkyne group as chemical reporter to which a fluorophore can be attached through a bioorthogonal ligation reaction. The fluorescent tag and any linker connected to it can be chosen in a flexible manner. We demonstrate the suitability of the probes by selective visualization of the plasma membrane of living cells by confocal microscopy techniques. Whereas the derivatives with the shorter fatty acids can be directly applied to HEK 293T cells, the hydrophobic glycosphingolipids with longer fatty acids can be delivered to cells using fusogenic liposomes.

  6. 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...

  7. 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...

  8. 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...

  9. 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...

  10. 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...

  11. Label-fracture: a method for high resolution labeling of cell surfaces.

    PubMed

    Pinto da Silva, P; Kan, F W

    1984-09-01

    We introduce here a technique, "label-fracture," that allows the observation of the distribution of a cytochemical label on a cell surface. Cell surfaces labeled with an electron-dense marker (colloidal gold) are freeze-fractured and the fracture faces are replicated by plantinum/carbon evaporation. The exoplasmic halves of the membrane, apparently stabilized by the deposition of the Pt/C replica, are washed in distilled water. The new method reveals the surface distribution of the label coincident with the Pt/C replica of the exoplasmic fracture face. Initial applications indicate high resolution (less than or equal to 15 nm) and exceedingly low background. "Label-fracture" provides extensive views of the distribution of the label on membrane surfaces while preserving cell shape and relating to the freeze-fracture morphology of exoplasmic fracture faces. The regionalization of wheat germ agglutinin receptors on the plasma membranes of boar sperm cells is illustrated. The method and the interpretation of its results are straightforward. Label-fracture is appropriate for routine use as a surface labeling technique.

  12. 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...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7100 Red blood cell enzyme assay. (a) Identification. Red blood cell enzyme assay is a device used to measure the activity...

  13. 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...

  14. 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...

  15. 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...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7100 Red blood cell enzyme assay. (a) Identification. Red blood cell enzyme assay is a device used to measure the activity...

  16. 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...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7100 Red blood cell enzyme assay. (a) Identification. Red blood cell enzyme assay is a device used to measure the activity...

  17. 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...

  18. 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...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7100 Red blood cell enzyme assay. (a) Identification. Red blood cell enzyme assay is a device used to measure the activity...

  19. 21 CFR 660.30 - Reagent Red Blood Cells.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 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 § 660.30 Reagent Red Blood Cells. (a) Proper name and definition. The proper name of the product shall...

  20. 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...

  1. 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...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7100 Red blood cell enzyme assay. (a) Identification. Red blood cell enzyme assay is a device used to measure the activity...

  2. Red cell distribution width and cancer

    PubMed Central

    Danese, Elisa

    2016-01-01

    Red cell distribution width (RDW) is an index which primarily reflects impaired erythropoiesis and abnormal red blood cell survival. In last years the interest in this marker has considerably grown and now a lot of data are available indicating that this simple and inexpensive parameter is a strong and independent risk factor for death in the general population. Moreover, several investigations have been performed to investigate the role of RDW in cardiovascular and thrombotic disorders. Contrarily, there are relatively few reports focusing on RDW in the area of oncology and to date none review have been performed in this specific field. As such, the aim of this narrative review is to summarize some interesting results obtained in studies performed in patients affected by solid and hematological tumors. Even if larger studies are needed before these preliminary findings can be generalized, it seems plausible to affirm that RDW can be useful by adding prognostic information in patients with oncologic disease. PMID:27867951

  3. Fluorescent Photo-conversion: A second chance to label unique cells.

    PubMed

    Mellott, Adam J; Shinogle, Heather E; Moore, David S; Detamore, Michael S

    2015-03-01

    Not all cells behave uniformly after treatment in tissue engineering studies. In fact, some treated cells display no signs of treatment or show unique characteristics not consistent with other treated cells. What if the "unique" cells could be isolated from a treated population, and further studied? Photo-convertible reporter proteins, such as Dendra2, allow for the ability to selectively identify unique cells with a secondary label within a primary labeled treated population. In the current study, select cells were identified and labeled through photo-conversion of Dendra2-transfected human Wharton's Jelly cells (hWJCs) for the first time. Robust photo-conversion of green-to-red fluorescence was achieved consistently in arbitrarily selected cells, allowing for precise cell identification of select hWJCs. The current study demonstrates a method that offers investigators the opportunity to selectively label and identify unique cells within a treated population for further study or isolation from the treatment population. Photo-convertible reporter proteins, such as Dendra2, offer the ability over non-photo-convertible reporter proteins, such as green fluorescent protein, to analyze unique individual cells within a treated population, which allows investigators to gain more meaningful information on how a treatment affects all cells within a target population.

  4. 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.”

  5. 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.

  6. Reversibility of red blood cell deformation.

    PubMed

    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."

  7. Glycolate kinase activity in human red cells.

    PubMed

    Fujii, S; Beutler, E

    1985-02-01

    Human red cells manifest glycolate kinase activity. This activity copurifies with pyruvate kinase and is decreased in the red cells of subjects with hereditary pyruvate kinase deficiency. Glycolate kinase activity was detected in the presence of FDP or glucose-1,6-P2. In the presence of 1 mmol/L FDP, the Km for adenosine triphosphate (ATP) was 0.28 mmol/L and a half maximum velocity for glycolate was obtained at 40 mmol/L. The pH optimum of the reaction was over 10.5 With 10 mumol/L FDP, 500 mumol/L glucose-1,6-P2, 2 mmol/L ATP, 5 mmol/L MgCl2, and 50 mmol/L glycolate at pH 7.5, glycolate kinase activity was calculated to be approximately 0.0013 U/mL RBC. In view of this low activity even in the presence of massive amounts of glycolate, the glycolate kinase reaction cannot account for the maintenance of the reported phosphoglycolate level in human red cells.

  8. Red blood cell transfusion in newborn infants

    PubMed Central

    Whyte, Robin K; Jefferies, Ann L

    2014-01-01

    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

  9. 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.

  10. Osmotic properties of human red cells.

    PubMed

    Solomon, A K; Toon, M R; Dix, J A

    1986-01-01

    When an osmotic pressure gradient is applied to human red cells, the volume changes anomalously, as if there were a significant fraction of "nonosmotic water" which could not serve as solvent for the cell solutes, a finding which has been discussed widely in the literature. In 1968, Gary-Bobo and Solomon (J. Gen. Physiol. 52:825) concluded that the anomalies could not be entirely explained by the colligative properties of hemoglobin (Hb) and proposed that there was an additional concentration dependence of the Hb charge (ZHb). A number of investigators, particularly Freedman and Hoffman (1979, J. Gen. Physiol. 74:157) have been unable to confirm Gary-Bobo and Solomon's experimental evidence for this concentration dependence of ZHb and we now report that we are also unable to repeat the earlier experiments. Nonetheless, there still remains a significant anomaly which amounts to 12.5 +/- 0.8% of the total isosmotic cell water (P much less than 0.0005, t test), even after taking account of the concentration dependence of the Hb osmotic coefficient and all the other known physical chemical constraints, ideal and nonideal. It is suggested that the anomalies at high Hb concentration in shrunken cells may arise from the ionic strength dependence of the Hb osmotic coefficient. In swollen red cells at low ionic strength, solute binding to membrane and intracellular proteins is increased and it is suggested that this factor may account, in part, for the anomalous behavior of these cells.

  11. Non-tuberculous Mycobacteriosis with T-cell Lymphoma in a Red Panda (Ailurus fulgens).

    PubMed

    Fuke, N; Hirai, T; Makimura, N; Goto, Y; Habibi, W A; Ito, S; Trang, N T; Koshino, K; Takeda, M; Yamaguchi, R

    2016-01-01

    A 9-year-old male red panda (Ailurus fulgens) became emaciated and died. Necropsy examination revealed systemic lymphadenomegaly. The liver, lungs and left kidney contained multifocal yellow nodules. Microscopical examination revealed granulomatous inflammation in the liver, lungs, kidney, spleen and lymph nodes, with numerous acid-fast bacilli. Sequencing of genetic material isolated from the tissues classified the pathogen as Mycobacterium gastri. Lymphoma was found in the liver, lungs, kidney and lymph nodes. The neoplastic cells were strongly labelled for expression of CD3, Ki67 and proliferating cell nuclear antigen by immunohistochemistry. This is the first report of M. gastri infection with T-cell lymphoma in a red panda.

  12. Osmotic water permeability of human red cells

    PubMed Central

    1981-01-01

    The osmotic water permeability of human red cells has been reexamined with a stopped-flow device and a new perturbation technique. Small osmotic gradients are used to minimize the systematic error caused by nonlinearities in the relationship between cell volume and light scattering. Corrections are then made for residual systematic error. Our results show that the hydraulic conductivity, Lp, is essentially independent of the direction of water flow and of osmolality in the range 184-365 mosM. the mean value of Lp obtained obtained was 1.8 +/- 0.1 (SEM) X 10-11 cm3 dyne -1 s-1. PMID:7229611

  13. 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.

  14. 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

  15. 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.

  16. Impact of glycocalyx structure on red cell-red cell affinity in polymer suspensions.

    PubMed

    Rad, Samar; Meiselman, Herbert J; Neu, Björn

    2014-11-01

    A theoretical framework based on macromolecular depletion has been utilized in order to examine the energetics of red blood cell interactions. Three different glycocalyx structures are considered and cell-cell affinities are calculated by superposition of depletion, steric and electrostatic interactions. The theoretical model predicts a non-monotonic dependence of the interaction energies on polymer size. Further, our results indicate that the glycocalyx segment distribution has a large impact on adhesion energies between cells: a linear segment distribution induces the strongest adhesion between cells followed by pseudo-tail and uniform distributions. Our approach confirms the concept of a depletion mechanism for RBC aggregation, and also provides new insights that may eventually help to understand and quantify cellular factors that control red blood cell interactions in health and disease.

  17. 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.

  18. Argon laser radiation of human clots: differential photoabsorption in red cell rich and red cell poor clots

    SciTech Connect

    Lee, G.; Chan, M.C.; Seckinger, D.L.; Vazquez, A.; Rosenthal, P.K.; Lee, K.K.; Ikeda, R.M.; Reis, R.L.; Hanna, E.S.; Mason, D.T.

    1985-06-01

    Since argon laser radiation (454-514 nm) can vaporize human clots, the authors determined whether the absorption of laser energies can differ among different types of blood clots. Thus, they performed spectrophotometric studies and examined the ability of this laser to penetrate red cell rich and red cell poor clots. Fifty-four red cell rich and red cell poor clot samples, varying in depth from 1.8 to 5.0 mm, were subjected to 3, 5 and 7 watts from an argon laser beam. At a given power intensity, the deeper the red cell rich clot, the longer was the time needed to penetrate the clot. The higher the power used, the shorter was the red clot penetration time. In contrast, all power levels used up to 5 minutes did not penetrate any of the varying depths of red cell poor clots. Spectrophotometrically, the red cell rich clot had an absorption curve typical of hemoglobin pigment while the red cell poor clot, in the absence of hemoglobin, had poor absorption between 350 and 600 nm and was unable to absorb argon laser energies. Thus, the argon laser provides a therapeutic modality for human red cell rich clot dissolution but the present approach does not appear to be effective against red cell poor clots.

  19. 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.

  20. Open Gradient Magnetic Red Blood Cell Sorter Evaluation on Model Cell Mixtures

    PubMed Central

    Moore, Lee R.; Nehl, Franzisca; Dorn, Jenny; Chalmers, Jeffrey J.; Zborowski, Maciej

    2014-01-01

    The emerging applications of biological cell separation to rare circulating tumor cell (CTC) detection and separation from blood rely on efficient methods of red blood cell (RBC) debulking. The two most widely used methods of centrifugation and RBC lysis have been associated with the concomitant significant losses of the cells of interest (such as progenitor cells or circulating tumor cells). Moreover, RBC centrifugation and lysis are not well adapted to the emerging diagnostic applications, relying on microfluidics and micro-scale total analytical systems. Therefore, magnetic RBC separation appears a logical alternative considering the high iron content of the RBC (normal mean 105 fg) as compared to the white blood cell iron content (normal mean 1.6 fg). The typical magnetic forces acting on a RBC are small, however, as compared to typical forces associated with centrifugation or the forces acting on synthetic magnetic nanoparticles used in current magnetic cell separations. This requires a significant effort in designing and fabricating a practical magnetic RBC separator. Applying advanced designs to the low cost, high power permanent magnets currently available, and building on the accumulated knowledge of the immunomagnetic cell separation methods and devices, an open gradient magnetic red blood cell (RBC) sorter was designed, fabricated and tested on label-free cell mixtures, with potential applications to RBC debulking from whole blood samples intended for diagnostic tests. PMID:24910468

  1. Open Gradient Magnetic Red Blood Cell Sorter Evaluation on Model Cell Mixtures.

    PubMed

    Moore, Lee R; Nehl, Franzisca; Dorn, Jenny; Chalmers, Jeffrey J; Zborowski, Maciej

    2013-02-01

    The emerging applications of biological cell separation to rare circulating tumor cell (CTC) detection and separation from blood rely on efficient methods of red blood cell (RBC) debulking. The two most widely used methods of centrifugation and RBC lysis have been associated with the concomitant significant losses of the cells of interest (such as progenitor cells or circulating tumor cells). Moreover, RBC centrifugation and lysis are not well adapted to the emerging diagnostic applications, relying on microfluidics and micro-scale total analytical systems. Therefore, magnetic RBC separation appears a logical alternative considering the high iron content of the RBC (normal mean 105 fg) as compared to the white blood cell iron content (normal mean 1.6 fg). The typical magnetic forces acting on a RBC are small, however, as compared to typical forces associated with centrifugation or the forces acting on synthetic magnetic nanoparticles used in current magnetic cell separations. This requires a significant effort in designing and fabricating a practical magnetic RBC separator. Applying advanced designs to the low cost, high power permanent magnets currently available, and building on the accumulated knowledge of the immunomagnetic cell separation methods and devices, an open gradient magnetic red blood cell (RBC) sorter was designed, fabricated and tested on label-free cell mixtures, with potential applications to RBC debulking from whole blood samples intended for diagnostic tests.

  2. Image analysis of nucleated red blood cells.

    PubMed

    Zajicek, G; Shohat, M; Melnik, Y; Yeger, A

    1983-08-01

    Bone marrow smears stained with Giemsa were scanned with a video camera under computer control. Forty-two cells representing the six differentiation classes of the red bone marrow were sampled. Each cell was digitized into 70 X 70 pixels, each pixel representing a square area of 0.4 micron2 in the original image. The pixel gray values ranged between 0 and 255. Zero stood for white, 255 represented black, while the numbers in between stood for the various shades of gray. After separation and smoothing the images were processed with a Sobel operator outlining the points of steepest gray level change in the cell. These points constitute a closed curve denominated as inner cell boundary, separating the cell into an inner and an outer region. Two types of features were extracted from each cell: form features, e.g., area and length, and gray level features. Twenty-two features were tested for their discriminative merit. After selecting 16, the discriminant analysis program classified correctly all 42 cells into the 6 classes.

  3. Hereditary spherocytosis, elliptocytosis, and other red cell membrane disorders.

    PubMed

    Da Costa, Lydie; Galimand, Julie; Fenneteau, Odile; Mohandas, Narla

    2013-07-01

    Hereditary spherocytosis and elliptocytosis are the two most common inherited red cell membrane disorders resulting from mutations in genes encoding various red cell membrane and skeletal proteins. Red cell membrane, a composite structure composed of lipid bilayer linked to spectrin-based membrane skeleton is responsible for the unique features of flexibility and mechanical stability of the cell. Defects in various proteins involved in linking the lipid bilayer to membrane skeleton result in loss in membrane cohesion leading to surface area loss and hereditary spherocytosis while defects in proteins involved in lateral interactions of the spectrin-based skeleton lead to decreased mechanical stability, membrane fragmentation and hereditary elliptocytosis. The disease severity is primarily dependent on the extent of membrane surface area loss. Both these diseases can be readily diagnosed by various laboratory approaches that include red blood cell cytology, flow cytometry, ektacytometry, electrophoresis of the red cell membrane proteins, and mutational analysis of gene encoding red cell membrane proteins.

  4. Sequential Immunoprecipitation of Secretory Vesicle Proteins from Biosynthetically Labelled Cells.

    PubMed

    Guest, Paul C

    2017-01-01

    Pulse radiolabelling of cells with radioactive amino acids is a common method for studying the biosynthesis of proteins. The labelled proteins can then be immunoprecipitated and analysed by electrophoresis and imaging techniques. This chapter presents a protocol for the biosynthetic labelling and immunoprecipitation of pancreatic islet proteins which are known to be affected in psychiatric disorders such as schizophrenia.

  5. 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.

  6. Red cell antigens: Structure and function

    PubMed Central

    Pourazar, Abbasali

    2007-01-01

    Landsteiner and his colleagues demonstrated that human beings could be classified into four groups depending on the presence of one (A) or another (B) or both (AB) or none (O) of the antigens on their red cells. The number of the blood group antigens up to 1984 was 410. In the next 20 years, there were 16 systems with 144 antigens and quite a collection of antigens waiting to be assigned to systems, pending the discovery of new information about their relationship to the established systems. The importance of most blood group antigens had been recognized by immunological complications of blood transfusion or pregnancies; their molecular structure and function however remained undefined for many decades. Recent advances in molecular genetics and cellular biochemistry resulted in an abundance of new information in this field of research. In this review, we try to give some examples of advances made in the field of ‘structure and function of the red cell surface molecules.’ PMID:21938229

  7. Functions of red cell surface proteins.

    PubMed

    Daniels, G

    2007-11-01

    The external membrane of the red cell contains numerous proteins that either cross the lipid bilayer one or more times or are anchored to it through a lipid tail. Many of these proteins express blood group activity. The functions of some of these proteins are known; in others their function can only be surmised from the protein structure or from limited experimental evidence. They are loosely divided into four categories based on their functions: membrane transporters; adhesion molecules and receptors; enzymes; and structural proteins that link the membrane with the membrane skeleton. Some of the proteins carry out more than one of these functions. Some proteins may complete their major functions during erythropoiesis or may only be important under adverse physiological conditions. Furthermore, some might be evolutionary relics and may no longer have significant functions. Polymorphisms or rare changes in red cell surface proteins are often responsible for blood groups. The biological significance of these polymorphisms or the selective pressures responsible for their stability within populations are mostly not known, although exploitation of the proteins by pathogenic micro-organisms has probably played a major role.

  8. Cell labeling with magnetic nanoparticles: opportunity for magnetic cell imaging and cell manipulation.

    PubMed

    Kolosnjaj-Tabi, Jelena; Wilhelm, Claire; Clément, Olivier; Gazeau, Florence

    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.

  9. Double-labelled HIV-1 particles for study of virus-cell interaction

    SciTech Connect

    Lampe, Marko; Briggs, John A.G.; Endress, Thomas; Glass, Baerbel; Riegelsberger, Stefan; Kraeusslich, Hans-Georg; Lamb, Don C.; Braeuchle, Christoph; Mueller, Barbara . E-mail: Barbara_Mueller@med.uni-heidelberg.de

    2007-03-30

    Human immunodeficiency virus (HIV) delivers its genome to a host cell through fusion of the viral envelope with a cellular membrane. While the viral and cellular proteins involved in entry have been analyzed in detail, the dynamics of virus-cell fusion are largely unknown. Single virus tracing (SVT) provides the unique opportunity to visualize viral particles in real time allowing direct observation of the dynamics of this stochastic process. For this purpose, we developed a double-coloured HIV derivative carrying a green fluorescent label attached to the viral matrix protein combined with a red label fused to the viral Vpr protein designed to distinguish between complete virions and subviral particles lacking MA after membrane fusion. We present here a detailed characterization of this novel tool together with exemplary live cell imaging studies, demonstrating its suitability for real-time analyses of HIV-cell interaction.

  10. The Effects of Oxidative Damage on In Vivo Aging, Density, and Survival of Baboon Red Blood Cells

    DTIC Science & Technology

    2007-11-02

    the plate. The biotin-treated and avidin-isolated red blood cells were washed three times in glucose phosphate buffered saline and labeled with 51Cr...OF BABOON RED BLOOD CELLS BY J.B. McKENNEY, C.R. VALERI, A. GIORGIO, G. RAGNO, J. TRAINOR, N. FORTIER, B. WODA, AND L.M. SNYDER NAVAL BLOOD ...TITLE (and Subtitle) THE EFFECTS OF OX I DATIVE DAMAGE ON IN VIVO AGING, DENSITY, AND SURVIVAL OF BABOON RED BLOOD CELLS 7. AUTHORf«; J.B

  11. 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.

  12. Ektacytometry: Instrumentation and Applications in Red Blood Cell Preservation Studies.

    DTIC Science & Technology

    1982-05-10

    that it was necessary to maintain an isotonic environment, since the red blood cells respond differently to shear stress under hypertonic or...the red blood cells respond differently to shear stress under hypertonic or hypotonic conditions. The most unexpected observation was an absence of...required to compare samples analyzed the same day. 2. Treatment of Human Red Blood Cells With Hypotonic and Hypertonic Solutions and Glutaraldehyde

  13. Nucleoside analog labeling of neural stem cells and their progeny.

    PubMed

    DeBoer, Erik Michael; Rasin, Mladen-Roko

    2013-01-01

    Nucleoside analog pulse labeling is an important technique which can assess the birthdate, cell cycle maintenance, or cycling rates of cells during development. This method has evolved over several decades of use and is now applied to a multitude of tissue subtypes and systems. The methodology in this chapter covers the classic uses for analog pulse labeling as well as their use in conjunction with the newly characterized technique of in utero electroporation (IUE).

  14. 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.

  15. 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.

  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. 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.

  18. 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...

  19. 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...

  20. 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...

  1. 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.

  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. Red blood cell nitric oxide synthase modulates red blood cell deformability in sickle cell anemia.

    PubMed

    Mozar, Anaïs; Connes, Philippe; Collins, Bianca; Hardy-Dessources, Marie-Dominique; Romana, Marc; Lemonne, Nathalie; Bloch, Wilhelm; Grau, Marijke

    2016-11-04

    Sickle cell anemia (SCA) is an inherited red blood cells (RBC) disorder characterized by significantly decreased RBC deformability. The present study aimed to assess whether modulation of RBC Nitric Oxide Synthase (RBC-NOS) activation could affect RBC deformability in SCA.Blood of twenty-five SCA patients was treated for 1 hour at 37°C with Phosphate Buffered Saline (PBS) or PBS containing 1% of Dimethylsulfoxyde as control, L-arginine or N(5)-(1-Iminoethyl)-L-ornithine (L-NIO) to directly stimulate or inhibit RBC-NOS, insulin or wortmannin to indirectly stimulate or inhibit RBC-NOS through their effects on the PI3 Kinase/Akt pathway, and sodium nitroprusside (SNP) and 2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) as NO donor and NO scavenger, respectively. RBC deformability was measured by ektacytometry at 3 Pa.RBC deformability significantly increased after insulin treatment and significantly decreased after L-NIO and wortmannin incubation. The other conditions did not affect deformability. Significantly increased nitrotyrosine levels, a marker of enhanced free radical generation, were detected by immunohistochemistry in SNP and insulin treated samples.These data suggest that RBC deformability of SCA can be modulated by RBC-NOS activity but also that oxidative stress may impair effectiveness of RBC-NOS produced NO.

  4. Hypoalbuminemia causes high blood viscosity by increasing red cell lysophosphatidylcholine.

    PubMed

    Joles, J A; Willekes-Koolschijn, N; Koomans, H A

    1997-09-01

    Albumin deficiency is accompanied by a reduction in red cell deformability and blood hyperviscosity. Albumin deficiency increases plasma fibrinogen and triglyceride levels and may alter red cell membrane lipid composition. These options, which could all contribute to reduced red cell deformability (RCD) and hyperviscosity, were studied in the Nagase analbuminemic rat (NAR), a mutant Sprague Dawley rat (CON), characterized by normal total protein levels, with an absolute deficiency of albumin, but elevated levels of non-albumin proteins and hyperlipidemia. Plasma protein-binding of the polar phopholipid lysophosphatidylcholine (LPC) was markedly decreased. LPC comprised only 26 +/- 1% of total plasma phospholipids as compared to 42 +/- 2% in CON. NAR red cells in CON plasma had a viscosity that was similar to CON red cells in CON plasma. Conversely, CON red cells in NAR plasma show an increased viscosity as compared to CON red cells in CON plasma. The maximum deformation index of both NAR and CON red cells was markedly decreased in NAR plasma as compared to either NAR or CON cells in CON plasma (0.04 +/- 0.03 and 0.02 +/- 0.02 vs. 0.22 +/- 0.06 and 0.15 +/- 0.04, respectively; P < 0.05). Thus, plasma composition causes hyperviscosity and reduced RCD in NAR. Fibrinogen is not responsible since red cells in serum and red cells in plasma had a similar viscosity and differences in viscosity and RCD between NAR and CON were maintained. Plasma triglycerides are also not responsible since the viscosity of red cells in serum with a 50% reduction in triglycerides was not reduced. LPC levels in red cells were increased in NAR (8.7 +/- 0.2 vs. 5.5 +/- 0.3% of total phospholipids; P < 0.01). Adding albumin to NAR blood dose-dependently decreased whole blood viscosity, despite marked increases in plasma viscosity, and increased RCD of NAR cells (from 0.04 +/- 0.03 to 0.21 +/- 0.01; P < 0.05). There was also some effect on CON RCD of similar albumin addition to CON blood (from 0

  5. 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.

  6. Core-shell CdS/Cd(OH)2 quantum dots: synthesis and bioconjugation to target red cells antigens.

    PubMed

    de Farias, P M Albuquerque; Santos, B Saegesser; de Menezes, F Duarte; Ferreira, R de Carvalho; Barjas-Castro, M de Lourdes; Castro, V; Lima, P R Moura; Fontes, A; Cesar, C L

    2005-09-01

    We report a new and efficient methodology of labelling red blood cells, in order to investigate the expression of anti-A antigen, employing luminescent semiconductor nanocrystals. Highly luminescent and stable core-shell cadmium sulphide/cadmium hydroxide [CdS/CdS(OH)2] colloidal particles were obtained in the nanometre size range. The surface of these particles was characterized by using a monoclonal anti-A antibody via a one-step glutaraldehyde cross-linking procedure, followed by conjugation of the particles to red cells of blood groups A+, and O+. Laser scanning confocal microscopy images indicated that after conjugation for 30 min, A+ and erythrocytes presented different patterns of dual bright emission whereas the O+ group cells showed no emission. We suggest that this labelling procedure may be applied as a quantitative tool to investigate the distribution and expression of alloantigen in red blood cells.

  7. 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.

  8. Efflux of red cell water into buffered hypertonic solutions.

    PubMed

    OLMSTEAD, E G

    1960-03-01

    Buffered NaCl solutions hypertonic to rabbit serum were prepared and freezing point depressions of each determined after dilution with measured amounts of water. Freezing point depression of these dilutions was a linear function of the amount of water added. One ml. of rabbit red cells was added to each 4 ml. of the hypertonic solutions and after incubation at 38 degrees C. for 30 minutes the mixture was centrifuged and a freezing point depression determined on the supernatant fluid. The amount of water added to the hypertonic solutions by the red cells was calcuated from this freezing point depression. For each decrease in the freezing point of -0.093 degrees C. of the surrounding solution red cells gave up approximately 5 ml. of water per 100 ml. of red cells in the range of -0.560 to -0.930 degrees C. Beyond -0.930 degrees C. the amount of water given up by 100 ml. of red cells fits best a parabolic equation. The maximum of this equation occurred at a freezing point of the hypertonic solution of -2.001 degrees C. at which time the maximum amount of water leaving the red cells would be 39.9 ml. per 100 ml. of red cells. The data suggest that only about 43 per cent of the red cell water is available for exchange into solutions of increasing tonicity.

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

    PubMed Central

    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

  10. Red blood cell vesiculation in hereditary hemolytic anemia.

    PubMed

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

    2013-12-13

    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.

  11. 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

  12. Red blood cell transfusion in clinical practice.

    PubMed

    Klein, Harvey G; Spahn, Donat R; Carson, Jeffrey L

    2007-08-04

    Every year, about 75 million units of blood are collected worldwide. Red blood cell (RBC) transfusion is one of the few treatments that adequately restore tissue oxygenation when oxygen demand exceeds supply. Although the respiratory function of blood has been studied intensively, the trigger for RBC transfusion remains controversial, and doctors rely primarily on clinical experience. Laboratory assays that indicate failing tissue oxygenation would be ideal to guide the need for transfusion, but none has proved easy, reproducible, and sensitive to regional tissue hypoxia. The clinical importance of the RBCs storage lesion (ie, the time-dependent metabolic, biochemical, and molecular changes that stored blood cells undergo) is poorly understood. RBCs can be filtered, washed, frozen, or irradiated for specific indications. Donor screening and testing have dramatically reduced infectious risks in the developed world, but infection remains a major hazard in developing countries, where 13 million units of blood are not tested for HIV or hepatitis viruses. Pathogen inactivation techniques are in clinical trials for RBCs, but none is available for use. Despite serious immunological and non-immunological complications, RBC transfusion holds a therapeutic index that exceeds that of many common medications.

  13. Hemodynamic effects of red blood cell aggregation.

    PubMed

    Baskurt, Oguz K; Meiselman, Herbert J

    2007-01-01

    The influence of red blood cell (RBC) aggregation on blood flow in vivo has been under debate since early 1900's, yet a full understanding has still has not been reached. Enhanced RBC aggregation is well known to increase blood viscosity measured in rotational viscometers. However, it has been demonstrated that RBC aggregation may decrease flow resistance in cylindrical tubes, due to the formation of a cell-poor zone near the tube wall which results from the enhanced central accumulation of RBC. There is also extensive discussion regarding the effects of RBC aggregation on in vivo blood flow resistance. Several groups have reported increased microcirculatory flow resistance with enhanced RBC aggregation in experiments that utilized intravital microscopy. Alternatively, whole organ studies revealed that flow resistance may be significantly decreased if RBC aggregation is enhanced. Recently, new techniques have been developed to achieve well-controlled, graded alterations in RBC aggregation without influencing suspending phase properties. Studies using this technique revealed that the effects of RBC aggregation are determined by the degree of aggregation changes, and that this relationship can be explained by different hemodynamic mechanisms.

  14. Understanding red blood cell alloimmunization triggers.

    PubMed

    Hendrickson, Jeanne E; Tormey, Christopher A

    2016-12-02

    Blood group alloimmunization is "triggered" when a person lacking a particular antigen is exposed to this antigen during transfusion or pregnancy. Although exposure to an antigen is necessary for alloimmunization to occur, it is not alone sufficient. Blood group antigens are diverse in structure, function, and immunogenicity. In addition to red blood cells (RBCs), a recipient of an RBC transfusion is exposed to donor plasma, white blood cells, and platelets; the potential contribution of these elements to RBC alloimmunization remains unclear. Much attention in recent years has been placed on recipient factors that influence RBC alloantibody responses. Danger signals, identified in murine and human studies alike as being risk factors for alloimmunization, may be quite diverse in nature. In addition to exogenous or condition-associated inflammation, autoimmunity is also a risk factor for alloantibody formation. Triggers for alloimmunization in pregnancy are not well-understood beyond the presence of a fetal/maternal bleed. Studies using animal models of pregnancy-induced RBC alloimmunization may provide insight in this regard. A better understanding of alloimmunization triggers and signatures of "responders" and "nonresponders" is needed for prevention strategies to be optimized. A common goal of such strategies is increased transfusion safety and improved pregnancy outcomes.

  15. Red blood cells in retinal vascular disorders.

    PubMed

    Agrawal, Rupesh; Sherwood, Joseph; Chhablani, Jay; Ricchariya, Ashutosh; Kim, Sangho; Jones, Philip H; Balabani, Stavroula; Shima, David

    2016-01-01

    Microvascular circulation plays a vital role in regulating physiological functions, such as vascular resistance, and maintaining organ health. Pathologies such as hypertension, diabetes, or hematologic diseases affect the microcirculation posing a significant risk to human health. The retinal vasculature provides a unique window for non-invasive visualisation of the human circulation in vivo and retinal vascular image analysis has been established to predict the development of both clinical and subclinical cardiovascular, metabolic, renal and retinal disease in epidemiologic studies. Blood viscosity which was otherwise thought to play a negligible role in determining blood flow based on Poiseuille's law up to the 1970s has now been shown to play an equally if not a more important role in controlling microcirculation and quantifying blood flow. Understanding the hemodynamics/rheology of the microcirculation and its changes in diseased states remains a challenging task; this is due to the particulate nature of blood, the mechanical properties of the cells (such as deformability and aggregability) and the complex architecture of the microvasculature. In our review, we have tried to postulate a possible role of red blood cell (RBC) biomechanical properties and laid down future framework for research related to hemorrheological aspects of blood in patients with retinal vascular disorders.

  16. Production of Alexa Fluor 488-labeled reovirus and characterization of target cell binding, competence, and immunogenicity of labeled virions.

    PubMed

    Fecek, Ronald J; Busch, Ryan; Lin, Hong; Pal, Kasturi; Cunningham, Cynthia A; Cuff, Christopher F

    2006-07-31

    Respiratory enteric orphan virus (reovirus) has been used to study many aspects of the biology and genetics of viruses, viral infection, pathogenesis, and the immune response to virus infection. This report describes the functional activity of virus labeled with Alexa Fluor 488, a stable fluorescent dye. Matrix assisted laser desorption-time of flight analysis indicated that Alexa Fluor 488 labeled the outer capsid proteins of reovirus. Labeled virus bound to murine L929 fibroblasts as determined by flow cytometry and fluorescence microscopy, and the specificity of binding were demonstrated by competitive inhibition with non-labeled virus. Labeled reovirus induced apoptosis and cytopathic effect in infected L929 cells. Mice infected with labeled virus mounted robust serum antibody and CD8(+) T-cell responses, indicating that labeled virus retained immunogenicity in vivo. These results indicate that Alexa Fluor 488-labeled virus provides a powerful new tool to analyze reovirus infection in vitro and in vivo.

  17. 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.

  18. Photochemical decontamination of red cell concentrates with the silicon phthalocyanine Pc 4 and red light

    NASA Astrophysics Data System (ADS)

    Ben-Hur, Ehud; Zuk, Maria M.; Oetjen, Joyce; Chan, Wai-Shun; Lenny, Leslie; Horowitz, Bernard

    1999-07-01

    Virus inactivation in red blood cells concentrates (RBCC) is being studied in order to increase the safety of the blood supply. For this purpose we have been studying the silicon phthalocyanine (Pc 4), a photosensitizer activated with red light. Two approaches were used to achieve enhanced selectivity of Pc 4 for virus inactivation. One was formulation of Pc 4 in liposomes that reduce its binding to red cells. The other was the use of a light emitting diode (LED) array emitting at 700 nm. Vesicular stomatitis virus (VSV) infectivity served as an endpoint for virus kill in treated RBCC. Red cell hemolysis and circulatory survival in rabbits served as measures for red cell damage. Treatment of small aliquots of human RBCC with 2 (mu) M Pc 4 in liposomes and 10 J/cm2 of 700 nm LED light in the presence of the quenches of reactive oxygen species glutathione and trolox resulted in 6 log10 inactivation of VSV. Under these conditions hemolysis of treated red cells stored at 4 degree(s)C for 21 days was only slightly above that of control cells. Rabbit RBCC similarly treated circulated with a half life of 7.5 days compared with 10.5 days of control. It is concluded that Pc 4 used as described here may be useful for viral decontamination of RBCC, pending toxicological and clinical studies.

  19. 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.

  20. Identification of autoreactive B cells with labeled nucleosomes.

    PubMed

    Gies, Vincent; Wagner, Alain; Seifert, Cécile; Guffroy, Aurélien; Fauny, Jean-D; Knapp, Anne-M; Pasquali, Jean-L; Martin, Thierry; Dumortier, Hélène; Korganow, Anne-S; Soulas-Sprauel, Pauline

    2017-04-04

    The pathogenesis of autoimmune diseases has not been completely elucidated yet, and only a few specific treatments have been developed so far. In autoimmune diseases mediated by pathogenic autoantibodies, such as systemic lupus erythematosus, the specific detection and analysis of autoreactive B cells is crucial for a better understanding of the physiopathology. Biological characterization of these cells may help to define new therapeutic targets. Very few techniques allowing the precise detection of autoreactive B cells have been described so far. Herein we propose a new flow cytometry technique for specific detection of anti-nucleosome B cells, which secrete autoantibodies in systemic lupus erythematosus, using labeled nucleosomes. We produced different fluorochrome-labeled nucleosomes, characterized them, and finally tested them in flow cytometry. Nucleosomes labeled via the cysteines present in H3 histone specifically bind to autoreactive B cells in the anti-DNA transgenic B6.56R mice model. The present work validates the use of fluorochrome-labeled nucleosomes via cysteines to identify anti-nucleosome B cells and offers new opportunities for the description of autoreactive B cell phenotype.

  1. Cell Labeling via Membrane-Anchored Lipophilic MR Contrast Agents

    PubMed Central

    2015-01-01

    Cell tracking in vivo with MR imaging requires the development of contrast agents with increased sensitivity that effectively label and are retained by cells. Most clinically approved Gd(III)-based contrast agents require high incubation concentrations and prolonged incubation times for cellular internalization. Strategies to increase contrast agent permeability have included conjugating Gd(III) complexes to cell penetrating peptides, nanoparticles, and small molecules which have greatly improved cell labeling but have not resulted in improved cellular retention. To overcome these challenges, we have synthesized a series of lipophilic Gd(III)-based MR contrast agents that label cell membranes in vitro. Two of the agents were synthesized with a multiplexing strategy to contain three Gd(III) chelates (1 and 2) while the third contains a single Gd(III) chelate (3). These new agents exhibit significantly enhanced labeling and retention in HeLa and MDA-MB-231-mcherry cells compared to agents that are internalized by cells (4 and Prohance). PMID:24787689

  2. 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.

  3. 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.

  4. Optofluidic device for label-free cell classification from whole blood.

    PubMed

    Wu, Tsung-Feng; Lo, Yu-Hwa

    2015-01-01

    A unique optofluidic lab-on-a-chip device that can detect optically encoded forward scattering signals is demonstrated. With a unique design of a spatial mask that patterns the intensity distribution of the illuminating light, the position and velocity of each travelling cell in the flow can be measured with submicrometer resolution, which enables the generation of a cell distribution plot over the cross section of the channel. The distribution of cells is highly sensitive to its size and stiffness, both being important biomarkers for cell classification without cell labelling. The optical-coding technique offers an easy route to classify cells based on their size and stiffness. Because the stiffness and size of neutrophils are distinct from other types of white blood cells, the number of neutrophils can be detected from other white blood cells and red blood cells. Above all, the enumeration of neutrophil concentration can be obtained from only 5 μL of human blood with a simple blood preparation process saving the usual steps of anticoagulation, centrifugation, antibody labelling, or filtering. The optofluidic system is compact, inexpensive, and simple to fabricate and operate. The system uses a commodity laser diode and a Si PIN photoreceiver and digital signal processing to extract vital information about cells and suppress the noise from the encoded optical scattering signals. The optofluidic device holds promise to be a point-of-care and home care device to measure neutrophil concentration, which is the key indicator of the immune functions for cancer patients undergoing chemotherapy.

  5. Lack of Erythropoietic Inhibitory Effect of Serum From Patients with Congenital Pure Red Cell Aplasia

    ERIC Educational Resources Information Center

    Geller, Gary; And Others

    1975-01-01

    Serum of five children ages 1 to 19 months with congenital pure red cell aplasia (incomplete or defective development of red blood cells) was injected in normal mice to determine possible inhibition of red blood cell formulating stimulants. (CL)

  6. 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

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

    PubMed

    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.

  8. 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.

  9. Selective in vivo metabolic cell-labeling-mediated cancer targeting.

    PubMed

    Wang, Hua; Wang, Ruibo; Cai, Kaimin; He, Hua; Liu, Yang; Yen, Jonathan; Wang, Zhiyu; Xu, Ming; Sun, Yiwen; Zhou, Xin; Yin, Qian; Tang, Li; Dobrucki, Iwona T; Dobrucki, Lawrence W; Chaney, Eric J; Boppart, Stephen A; Fan, Timothy M; Lezmi, Stéphane; Chen, Xuesi; Yin, Lichen; Cheng, Jianjun

    2017-02-13

    Distinguishing cancer cells from normal cells through surface receptors is vital for cancer diagnosis and targeted therapy. Metabolic glycoengineering of unnatural sugars provides a powerful tool to manually introduce chemical receptors onto the cell surface; however, cancer-selective labeling still remains a great challenge. Herein we report the design of sugars that can selectively label cancer cells both in vitro and in vivo. Specifically, we inhibit the cell-labeling activity of tetraacetyl-N-azidoacetylmannosamine (Ac4ManAz) by converting its anomeric acetyl group to a caged ether bond that can be selectively cleaved by cancer-overexpressed enzymes and thus enables the overexpression of azido groups on the surface of cancer cells. Histone deacetylase and cathepsin L-responsive acetylated azidomannosamine, one such enzymatically activatable Ac4ManAz analog developed, mediated cancer-selective labeling in vivo, which enhanced tumor accumulation of a dibenzocyclooctyne-doxorubicin conjugate via click chemistry and enabled targeted therapy against LS174T colon cancer, MDA-MB-231 triple-negative breast cancer and 4T1 metastatic breast cancer in mice.

  10. 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

  11. 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

  12. Endothelial cell labeling with indium-111-oxine as a marker of cell attachment to bioprosthetic surfaces

    SciTech Connect

    Sharefkin, J.B.; Lather, C.; Smith, M.; Rich, N.M.

    1983-03-01

    Canine vascular endothelium labeled with indium-111-oxine was used as a marker of cell attachment to vascular prosthetic surfaces with complex textures. Primarily cultured and freshly harvested endothelial cells both took up the label rapidly. An average of 72% of a 32 micro Ci labeling dose was taken up by 1.5 X 10(6) cells in 10 min in serum-free medium. Over 95% of freshly labeled cells were viable by trypan blue tests and only 5% of the label was released after 1 h incubations at 37 degrees C. Labeled and unlabeled cells had similar rates of attachment to plastic dishes. Scanning electron microscopic studies showed that labeled cells retained their ability to spread on tissue culture dishes even at low (1%) serum levels. Labeled endothelial cells seeded onto Dacron or expanded polytetrafluoroethylene vascular prostheses by methods used in current surgical models could be identified by autoradiography of microscopic sections of the prostheses, and the efficiency of cell attachment to the prosthesis could be measured by gamma counting. Indium-111 labeling affords a simple and rapid way to measure initial cell attachment to, and distribution on, vascular prosthetic materials. The method could also allow measurement of early cell loss from a flow surface in vivo by using external gamma imaging.

  13. Red cell exchange: special focus on sickle cell disease.

    PubMed

    Kim, Haewon C

    2014-12-05

    The primary function of red blood cells (RBCs) is to deliver oxygen from the lungs to tissues. Tissue hypoxia occurs when the oxygen-carrying capacity of RBCs is compromised due primarily to 3 causes: (1) a reduction in circulating RBC mass, (2) an increase in circulating RBC mass, or (3) abnormal hemoglobin (Hb) that either does not sufficiently release oxygen to tissues (high-oxygen-affinity hemoglobin) or occludes the microvasculature due to deformed RBCs (sickled RBCs). To improve oxygenation in patients with reduced or increased RBC mass, RBC administration (simple transfusion) or RBC removal (RBC depletion) is performed, respectively. However, for patients with abnormal Hb, RBCs containing abnormal Hb are removed and replaced by healthy volunteer donor RBCs by red cell exchange (RCE). RCE can be performed by manual exchange or by automated exchange using a blood cell separator (erythrocytapheresis). In this review, indications for RCE in sickle cell disease using the evidence-based American Society for Apheresis categories(1) are presented and the rationale for RCE in each disorder are discussed. Simple transfusion versus RCE and manual RCE versus automated RCE are compared. Finally, this review briefly presents some of the challenges of performing erythrocytapheresis in small children and discusses various choices for central venous access during RCE.(2.)

  14. Deep Learning in Label-free Cell Classification

    DOE PAGES

    Chen, Claire Lifan; Mahjoubfar, Ata; Tai, Li-Chia; ...

    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

  15. 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

  16. 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.

  17. Deep Learning in Label-free Cell Classification

    SciTech Connect

    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 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. In conclusion, this system opens up a new path to data-driven phenotypic diagnosis and better understanding of the heterogeneous gene expressions in cells.

  18. 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

    2016-03-15

    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 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.

  19. 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.

  20. Dysferlin and other non-red cell proteins accumulate in the red cell membrane of Diamond-Blackfan Anemia patients.

    PubMed

    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.

  1. 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...

  2. 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.

  3. Label-free density difference amplification-based cell sorting.

    PubMed

    Song, Jihwan; Song, Minsun; Kang, Taewook; Kim, Dongchoul; Lee, Luke P

    2014-11-01

    The selective cell separation is a critical step in fundamental life sciences, translational medicine, biotechnology, and energy harvesting. Conventional cell separation methods are fluorescent activated cell sorting and magnetic-activated cell sorting based on fluorescent probes and magnetic particles on cell surfaces. Label-free cell separation methods such as Raman-activated cell sorting, electro-physiologically activated cell sorting, dielectric-activated cell sorting, or inertial microfluidic cell sorting are, however, limited when separating cells of the same kind or cells with similar sizes and dielectric properties, as well as similar electrophysiological phenotypes. Here we report a label-free density difference amplification-based cell sorting (dDACS) without using any external optical, magnetic, electrical forces, or fluidic activations. The conceptual microfluidic design consists of an inlet, hydraulic jump cavity, and multiple outlets. Incoming particles experience gravity, buoyancy, and drag forces in the separation chamber. The height and distance that each particle can reach in the chamber are different and depend on its density, thus allowing for the separation of particles into multiple outlets. The separation behavior of the particles, based on the ratio of the channel heights of the inlet and chamber and Reynolds number has been systematically studied. Numerical simulation reveals that the difference between the heights of only lighter particles with densities close to that of water increases with increasing the ratio of the channel heights, while decreasing Reynolds number can amplify the difference in the heights between the particles considered irrespective of their densities.

  4. Trypsinization-dependent cell labeling with fluorescent nanoparticles

    PubMed Central

    2014-01-01

    Trypsin is often used to detach adhered cell subculture from a substrate. However, the proteolytic activity of trypsin may harm cells by cleaving the cell membrane proteins. The present study shows that cellular uptake of fluorescent nanoparticles is remarkably increased within 24 h after trypsinization. These results highlight the trypsin-induced protein digestion, provoking leaky cell plasma membrane which leads to the strongly enhanced cellular uptake of the nanoparticles. To prevent this effect, one should expose cells to the nanoparticle (NP)-based fluorescent labels at least 48 h after trypsinization. PMID:25328505

  5. 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.

  6. Labeling proteins inside living cells using external fluorophores for microscopy

    PubMed Central

    Teng, Kai Wen; Ishitsuka, Yuji; Ren, Pin; Youn, Yeoan; Deng, Xiang; Ge, Pinghua; Lee, Sang Hak; Belmont, Andrew S; Selvin, Paul R

    2016-01-01

    Site-specific fluorescent labeling of proteins inside live mammalian cells has been achieved by employing Streptolysin O, a bacterial toxin which forms temporary pores in the membrane and allows delivery of virtually any fluorescent probes, ranging from labeled IgG’s to small ligands, with high efficiency (>85% of cells). The whole process, including recovery, takes 30 min, and the cell is ready to be imaged immediately. A variety of cell viability tests were performed after treatment with SLO to ensure that the cells have intact membranes, are able to divide, respond normally to signaling molecules, and maintains healthy organelle morphology. When combined with Oxyrase, a cell-friendly photostabilizer, a ~20x improvement in fluorescence photostability is achieved. By adding in glutathione, fluorophores are made to blink, enabling super-resolution fluorescence with 20–30 nm resolution over a long time (~30 min) under continuous illumination. Example applications in conventional and super-resolution imaging of native and transfected cells include p65 signal transduction activation, single molecule tracking of kinesin, and specific labeling of a series of nuclear and cytoplasmic protein complexes. DOI: http://dx.doi.org/10.7554/eLife.20378.001 PMID:27935478

  7. 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

  8. 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.

  9. [Effects of infusion media on human red blood cell morphology].

    PubMed

    Burova, O O; Gusev, A A; Petrikov, S S; Gusev, S A; Basyreva, L Iu

    2006-01-01

    The effect of various infusion media on the structure of human red blood cells was evaluated in vitro and in vivo. The in vitro experiments used 10% sodium chloride (NaCl) solution, 10% glucose solution, 20% albumin solution, Rheopolyglucin, HyperHAES solution (18 g of NaCl in combination with 60 g of hydroxyethylstarch (HES), 200/0.5), Voluven (HES 130/0.4/9:1), and a combination of hypertensive NaCl solution and Rheopolyglucin. The morphofunctional response of red blood cells was studied in the clinical setting when 6% Voluven solution (HES 130/0.4/ 9:1) and hypertensive NaCl and glucose solutions were used. It was established that 10% NaCl solution caused considerable changes in the morphology of red blood cells both in the experiment and in patients with severe brain injury. The magnitude of structural changes increased as blood NaCl concentrations became higher. 10% glucose solution, Voluven, Rheopolyglucin, and albumin did not virtually affect the structure of red blood cells. Infusion of Voluven (500 ml of 6% solution for 40 minutes) induced no changes in the morphology of red blood cells in the clinical setting. Among the test solutions used to correct intracranial hypertension (HyperHAES, 10% NaCl, a combination of rheopolyglucin and 10% NaCl), HyperHAES exerted the least effect on the morphology of red blood cells.

  10. 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.

  11. Cinnamomum zeylanicum extract on the radiolabelling of blood constituents and the morphometry of red blood cells: in vitro assay.

    PubMed

    Benarroz, M O; Fonseca, A S; Rocha, G S; Frydman, J N G; Rocha, V C; Pereira, M O; Bernardo-Filho, M

    2008-02-01

    Effects of Cinnamomum zeylanicum (cinnamon) on the labelling of blood constituents with technetium-99m(99mTc) and on the morphology of red blood cells were studied. Blood samples from Wistar rats were incubated with cinnamon extract for 1 hour or with 0.9% NaCl, as control. Labelling of blood constituents with 99mTc was performed. Plasma (P) and blood cells (BC), soluble (SF-P and SF-BC) and insoluble (IF-P and IF-BC) fractions were separated. The radioactivity in each fraction was counted and the percentage of radioactivity incorporated (%ATI) was calculated. Blood smears were prepared, fixed, stained and the qualitative and quantitative morphological analysis of the red blood cells was evaluated. The data showed that the cinnamon extract decreased significantly (p<0.05) the %ATI on BC, IF-P and IF-BC. No modifications were verified on shape of red blood cells. Cinnamon extracts could alter the labelling of blood constituents with 99mTc, and although our results were obtained with animals, precaution is suggested in interpretations of nuclear medicine examinations involving the labelling of blood constituents in patients who are using cinnamon.

  12. In vivo quantification of magnetically labelled cells by MRI relaxometry.

    PubMed

    Gimenez, Ulysse; Lajous, Hélène; El Atifi, Michèle; Bidart, Marie; Auboiroux, Vincent; Fries, Pascal Henry; Berger, François; Lahrech, Hana

    2016-11-01

    Cellular MRI, which visualizes magnetically labelled cells (cells*), is an active research field for in vivo cell therapy and tracking. The simultaneous relaxation rate measurements (R2 *, R2 , R1 ) are the basis of a quantitative cellular MRI method proposed here. U937 cells were labelled with Molday ION Rhodamine B, a bi-functional superparamagnetic and fluorescent nanoparticle (U937*). U937* viability and proliferation were not affected in vitro. In vitro relaxometry was performed in a cell concentration range of [2.5 × 10(4) -10(8) ] cells/mL. These measurements show the existence of complementary cell concentration intervals where these rates vary linearly. The juxtaposition of these intervals delineates a wide cell concentration range over which one of the relaxation rates in a voxel of an in vivo image can be converted into an absolute cell concentration. The linear regime was found at high concentrations for R1 in the range of [10(6) - 2 × 10(8) ] cells/mL, at intermediate concentrations for R2 in [2.5 × 10(5) - 5 × 10(7) ] cells/mL and at low concentrations for R2 * in [8 × 10(4) - 5 × 10(6) ] cells/mL. In vivo relaxometry was performed in a longitudinal study, with labelled U937 cells injected into a U87 glioma mouse model. Using in vitro data, maps of in vivo U937* concentrations were obtained by converting one of the in vivo relaxation rates to cell concentration maps. MRI results were compared with the corresponding optical images of the same brains, showing the usefulness of our method to accurately follow therapeutic cell biodistribution in a longitudinal study. Results also demonstrate that the method quantifies a large range of magnetically labelled cells*. Copyright © 2016 John Wiley & Sons, Ltd.

  13. Stem Cells and Labeling for Spinal Cord Injury

    PubMed Central

    Gazdic, Marina; Volarevic, Vladislav; Arsenijevic, Aleksandar; Erceg, Slaven; Moreno-Manzano, Victoria; Arsenijevic, Nebojsa; Stojkovic, Miodrag

    2016-01-01

    Spinal cord injury (SCI) is a devastating condition that usually results in sudden and long-lasting locomotor and sensory neuron degeneration below the lesion site. During the last two decades, the search for new therapies has been revolutionized with the improved knowledge of stem cell (SC) biology. SCs therapy offers several attractive strategies for spinal cord repair. The transplantation of SCs promotes remyelination, neurite outgrowth and axonal elongation, and activates resident or transplanted progenitor cells across the lesion cavity. However, optimized growth and differentiation protocols along with reliable safety assays should be established prior to the clinical application of SCs. Additionally, the ideal method of SCs labeling for efficient cell tracking after SCI remains a challenging issue that requires further investigation. This review summarizes the current findings on the SCs-based therapeutic strategies, and compares different SCs labeling approaches for SCI. PMID:28035961

  14. Detection threshold of single SPIO-labeled cells with FIESTA.

    PubMed

    Heyn, Chris; Bowen, Chris V; Rutt, Brian K; Foster, Paula J

    2005-02-01

    MRI of superparamagnetic iron oxide (SPIO)-labeled cells has become a valuable tool for studying the in vivo trafficking of transplanted cells. Cellular detection with MRI is generally considered to be orders of magnitude less sensitive than other techniques, such as positron emission tomography (PET), single photon emission-computed tomography (SPECT), or optical fluorescence microscopy. However, an analytic description of the detection threshold for single SPIO-labeled cells and the parameters that govern detection has not been adequately provided. In the present work, the detection threshold for single SPIO-labeled cells and the effect of resolution and SNR were studied for a balanced steady-state free precession (SSFP) sequence (3D-FIESTA). Based on the results from both theoretical and experimental analyses, an expression that predicts the minimum detectable mass of SPIO (m(c)) required to detect a single cell against a uniform signal background was derived: m(c) = 5v/(K(fsl) x SNR), where v is the voxel volume, SNR is the image signal-to-noise ratio, and K(fsl) is an empirical constant measured to be 6.2 +/- 0.5 x 10(-5) microl/pgFe. Using this expression, it was shown that the sensitivity of MRI is not very different from that of PET, requiring femtomole quantities of SPIO iron for detection under typical micro-imaging conditions (100 microm isotropic resolution, SNR = 60). The results of this work will aid in the design of cellular imaging experiments by defining the lower limit of SPIO labeling required for single cell detection at any given resolution and SNR.

  15. Non-label immune cell state prediction using Raman spectroscopy

    PubMed Central

    Ichimura, Taro; Chiu, Liang-da; Fujita, Katsumasa; Machiyama, Hiroaki; Yamaguchi, Tomoyuki; Watanabe, Tomonobu M.; Fujita, Hideaki

    2016-01-01

    The acquired immune system, mainly composed of T and B lymphocytes, plays a key role in protecting the host from infection. It is important and technically challenging to identify cell types and their activation status in living and intact immune cells, without staining or killing the cells. Using Raman spectroscopy, we succeeded in discriminating between living T cells and B cells, and visualized the activation status of living T cells without labeling. Although the Raman spectra of T cells and B cells were similar, they could be distinguished by discriminant analysis of the principal components. Raman spectra of activated T cells with anti-CD3 and anti-CD28 antibodies largely differed compared to that of naïve T cells, enabling the prediction of T cell activation status at a single cell level. Our analysis revealed that the spectra of individual T cells gradually change from the pattern of naïve T cells to that of activated T cells during the first 24 h of activation, indicating that changes in Raman spectra reflect slow changes rather than rapid changes in cell state during activation. Our results indicate that the Raman spectrum enables the detection of dynamic changes in individual cell state scattered in a heterogeneous population. PMID:27876845

  16. 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.

  17. Determination of lipid asymmetry in human red cells by resonance energy transfer

    SciTech Connect

    Connor, J.; Schroit, A.J.

    1987-08-11

    This report describes the application of a resonance energy transfer assay to determine the transbilayer distribution of /sup 125/I-labelled 7-nitro-2,1,3-benzoxadiazol-4-yl (NBD)-labelled lipid analogues. The validity of this technique was established by determining the relationship between the distance of separation of lissamine rhodamine B labeled phosphatidylethanolamine (N-Rho-PE) acceptor lipid and NBD-labeled donor lipid and energy transfer efficiency. By determination of the distance between probes at 50% transfer efficiency (R/sub 0/), the distance between fluorophores distributed symmetrically (outer leaflet label) and asymmetrically in artificially generated vesicles was determined. Calculation of the average distance between probes revealed a 14-A difference between NBD-lipid and N-Rho-PE localized in the same leaflet and in opposing leaflets, respectively. Application of this technique to the study of the transbilayer distribution of NBD-lipid in human red blood cells (RBC) showed that exogenously supplied NBD-phosphatidylserine (NBD-PS) was selectively transported to the inner leaflet, whereas NBD-phosphatidylcholine remained in outer leaflet. In contrast, pretreatment of the RBC with diamide (a SH cross-linking reagent) blocked the transport of NBD-PS. The absence or presence of NBD-PS in the outer leaflet was independently verified by employing back-exchange, trinitrobenzenesulfonic acid derivatization, and decarboxylation with PS decarboxylase experiments. These control experiments yielded results which confirmed the lipid distributions determined by the resonance energy transfer assay.

  18. Prolonged red cell storage before transfusion increases extravascular hemolysis

    PubMed Central

    Rapido, Francesca; Brittenham, Gary M.; Bandyopadhyay, Sheila; La Carpia, Francesca; L’Acqua, Camilla; McMahon, Donald J.; Rebbaa, Abdelhadi; Wojczyk, Boguslaw S.; Netterwald, Jane; Wang, Hangli; Schwartz, Joseph; Eisenberger, Andrew; Soffing, Mark; Yeh, Randy; Divgi, Chaitanya; Ginzburg, Yelena Z.; Shaz, Beth H.; Sheth, Sujit; Francis, Richard O.; Spitalnik, Steven L.; Hod, Eldad A.

    2016-01-01

    BACKGROUND. Some countries have limited the maximum allowable storage duration for red cells to 5 weeks before transfusion. In the US, red blood cells can be stored for up to 6 weeks, but randomized trials have not assessed the effects of this final week of storage on clinical outcomes. METHODS. Sixty healthy adult volunteers were randomized to a single standard, autologous, leukoreduced, packed red cell transfusion after 1, 2, 3, 4, 5, or 6 weeks of storage (n = 10 per group). 51-Chromium posttransfusion red cell recovery studies were performed and laboratory parameters measured before and at defined times after transfusion. RESULTS. Extravascular hemolysis after transfusion progressively increased with increasing storage time (P < 0.001 for linear trend in the AUC of serum indirect bilirubin and iron levels). Longer storage duration was associated with decreasing posttransfusion red cell recovery (P = 0.002), decreasing elevations in hematocrit (P = 0.02), and increasing serum ferritin (P < 0.0001). After 6 weeks of refrigerated storage, transfusion was followed by increases in AUC for serum iron (P < 0.01), transferrin saturation (P < 0.001), and nontransferrin-bound iron (P < 0.001) as compared with transfusion after 1 to 5 weeks of storage. CONCLUSIONS. After 6 weeks of refrigerated storage, transfusion of autologous red cells to healthy human volunteers increased extravascular hemolysis, saturated serum transferrin, and produced circulating nontransferrin-bound iron. These outcomes, associated with increased risks of harm, provide evidence that the maximal allowable red cell storage duration should be reduced to the minimum sustainable by the blood supply, with 35 days as an attainable goal. REGISTRATION. ClinicalTrials.gov NCT02087514. FUNDING. NIH grant HL115557 and UL1 TR000040. PMID:27941245

  19. Modelling the structure of the red cell membrane.

    PubMed

    Burton, Nicholas M; Bruce, Lesley J

    2011-04-01

    The red cell membrane has long been the focus of extensive study. The macromolecules embedded within the membrane carry the blood group antigens and perform many functions including the vital task of gas exchange. Links between the intramembrane macromolecules and the underlying cytoskeleton stabilize the biconcave morphology of the red cell and allow deformation during microvascular transit. Much is now known about the proteins of the red cell membrane and how they are organised. In many cases we have an understanding of which proteins are expressed, the number of each protein per cell, their oligomeric state(s), and how they are collected in large multi-protein complexes. However, our typical view of these structures is as cartoon shapes in schematic figures. In this study we have combined knowledge of the red cell membrane with a wealth of protein structure data from crystallography, NMR, and homology modelling to generate the first, tentative models of the complexes which link the membrane to the cytoskeleton. Measurement of the size of these complexes and comparison with known cytoskeletal distance parameters suggests the idea of interaction between the membrane complexes, which may have profound implications for understanding red cell function and deformation.

  20. Labeled cells in the investigation of hematologic disorders

    SciTech Connect

    Alavi, J.B.; Hansell, J.

    1984-07-01

    Radiolabeling techniques for white cells, platelets, and erythrocytes are reviewed. The early studies using diisopropylfluoro-32P contributed to an understanding of the production and circulation of the blood elements, and 51Cr proved useful in localizing sites of cell migration or destruction. 111In-oxine has further improved the understanding of blood cell organ sequestration, and permitted combined kinetic and organ imaging studies. Radionuclide labels have been essential for the elucidation of various hematologic disorders, such as the neutropenias, thrombocytopenias, anemias, and polycythemia. Many new treatments, including monoclonal antibodies, have been evaluated with radionuclides.

  1. Label-free classification of cultured cells through diffraction imaging.

    PubMed

    Dong, Ke; Feng, Yuanming; Jacobs, Kenneth M; Lu, Jun Q; Brock, R Scott; Yang, Li V; Bertrand, Fred E; Farwell, Mary A; Hu, Xin-Hua

    2011-06-01

    Automated classification of biological cells according to their 3D morphology is highly desired in a flow cytometer setting. We have investigated this possibility experimentally and numerically using a diffraction imaging approach. A fast image analysis software based on the gray level co-occurrence matrix (GLCM) algorithm has been developed to extract feature parameters from measured diffraction images. The results of GLCM analysis and subsequent classification demonstrate the potential for rapid classification among six types of cultured cells. Combined with numerical results we show that the method of diffraction imaging flow cytometry has the capacity as a platform for high-throughput and label-free classification of biological cells.

  2. Red blood cell dynamics: from cell deformation to ATP release.

    PubMed

    Wan, Jiandi; Forsyth, Alison M; Stone, Howard A

    2011-10-01

    The mechanisms of red blood cell (RBC) deformation under both static and dynamic, i.e., flow, conditions have been studied extensively since the mid 1960s. Deformation-induced biochemical reactions and possible signaling in RBCs, however, were proposed only fifteen years ago. Therefore, the fundamental relationship between RBC deformation and cellular signaling dynamics i.e., mechanotransduction, remains incompletely understood. Quantitative understanding of the mechanotransductive pathways in RBCs requires integrative studies of physical models of RBC deformation and cellular biochemical reactions. In this article we review the physical models of RBC deformation, spanning from continuum membrane mechanics to cellular skeleton dynamics under both static and flow conditions, and elaborate the mechanistic links involved in deformation-induced ATP release.

  3. Blood cell labelling. Theory and methods: radiation hazards.

    PubMed

    Trott, N G; Akbari, R B

    1984-02-03

    The chief physical properties of the radionuclide In111 are outlined, and compared with those of three other radionuclides, Tc99m, I131 and Cr51 which have similar applications. It is pointed out that the gamma-rays of In111 are appreciably more penetrating in lead than those of Tc99m and the significance of this, both in the use of shielding on syringes and in the effectiveness of lead glass screens is discussed. Examples are given of the dosimetry for In111 labelled cells in humans and it is noted that the absorbed dose in the spleen per mCi (37 MBq) injected may be some 10 rad (0.1 Gy). The problems that have been noted of damage to cells arising from oxine labelling and now considered to be due to radiation damage are briefly reviewed.

  4. Mechanisms Linking Red Blood Cell Disorders and Cardiovascular Diseases

    PubMed Central

    2015-01-01

    The present paper aims to review the main pathophysiological links between red blood cell disorders and cardiovascular diseases, provides a brief description of the latest studies in this area, and considers implications for clinical practice and therapy. Anemia is associated with a special risk in proatherosclerotic conditions and heart disease and became a new therapeutic target. Guidelines must be updated for the management of patients with red blood cell disorders and cardiovascular diseases, and targets for hemoglobin level should be established. Risk scores in several cardiovascular diseases should include red blood cell count and RDW. Complete blood count and hemorheological parameters represent useful, inexpensive, widely available tools for the management and prognosis of patients with coronary heart disease, heart failure, hypertension, arrhythmias, and stroke. Hypoxia and iron accumulation cause the most important cardiovascular effects of sickle cell disease and thalassemia. Patients with congenital chronic hemolytic anemia undergoing splenectomy should be monitored, considering thromboembolic and cardiovascular risk. PMID:25710019

  5. Mechanisms linking red blood cell disorders and cardiovascular diseases.

    PubMed

    Mozos, Ioana

    2015-01-01

    The present paper aims to review the main pathophysiological links between red blood cell disorders and cardiovascular diseases, provides a brief description of the latest studies in this area, and considers implications for clinical practice and therapy. Anemia is associated with a special risk in proatherosclerotic conditions and heart disease and became a new therapeutic target. Guidelines must be updated for the management of patients with red blood cell disorders and cardiovascular diseases, and targets for hemoglobin level should be established. Risk scores in several cardiovascular diseases should include red blood cell count and RDW. Complete blood count and hemorheological parameters represent useful, inexpensive, widely available tools for the management and prognosis of patients with coronary heart disease, heart failure, hypertension, arrhythmias, and stroke. Hypoxia and iron accumulation cause the most important cardiovascular effects of sickle cell disease and thalassemia. Patients with congenital chronic hemolytic anemia undergoing splenectomy should be monitored, considering thromboembolic and cardiovascular risk.

  6. Cell Labeling and Injection in Developing Embryonic Mouse Hearts

    PubMed Central

    Dirschinger, Ralf J.; Evans, Sylvia M.; Puceat, Michel

    2014-01-01

    Testing the fate of embryonic or pluripotent stem cell-derivatives in in vitro protocols has led to controversial outcomes that do not necessarily reflect their in vivo potential. Preferably, these cells should be placed in a proper embryonic environment in order to acquire their definite phenotype. Furthermore, cell lineage tracing studies in the mouse after labeling cells with dyes or retroviral vectors has remained mostly limited to early stage mouse embryos with still poorly developed organs. To overcome these limitations, we designed standard and ultrasound-mediated microinjection protocols to inject various agents in targeted regions of the heart in mouse embryos at E9.5 and later stages of development.  Embryonic explant or embryos are then cultured or left to further develop in utero. These agents include fluorescent dyes, virus, shRNAs, or stem cell-derived progenitor cells. Our approaches allow for preservation of the function of the organ while monitoring migration and fate of labeled and/or injected cells. These technologies can be extended to other organs and will be very helpful to address key biological questions in biology of development. PMID:24797676

  7. 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.

  8. Computational Biomechanics of Human Red Blood Cells in Hematological Disorders.

    PubMed

    Li, Xuejin; Li, He; Chang, Hung-Yu; Lykotrafitis, George; Em Karniadakis, George

    2017-02-01

    We review recent advances in multiscale modeling of the biomechanical characteristics of red blood cells (RBCs) in hematological diseases, and their relevance to the structure and dynamics of defective RBCs. We highlight examples of successful simulations of blood disorders including malaria and other hereditary disorders, such as sickle-cell anemia, spherocytosis, and elliptocytosis.

  9. 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

  10. 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

  11. 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

  12. 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.

  13. 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

  14. A Fast Multi-Object Extraction Algorithm Based on Cell-Based Connected Components Labeling

    NASA Astrophysics Data System (ADS)

    Gu, Qingyi; Takaki, Takeshi; Ishii, Idaku

    We describe a cell-based connected component labeling algorithm to calculate the 0th and 1st moment features as the attributes for labeled regions. These can be used to indicate their sizes and positions for multi-object extraction. Based on the additivity in moment features, the cell-based labeling algorithm can label divided cells of a certain size in an image by scanning the image only once to obtain the moment features of the labeled regions with remarkably reduced computational complexity and memory consumption for labeling. Our algorithm is a simple-one-time-scan cell-based labeling algorithm, which is suitable for hardware and parallel implementation. We also compared it with conventional labeling algorithms. The experimental results showed that our algorithm is faster than conventional raster-scan labeling algorithms.

  15. 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

  16. 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.

  17. 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.

  18. 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.

  19. 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

  20. Novel single-cell functional analysis of red blood cells using laser tweezers Raman spectroscopy: application for sickle cell disease.

    PubMed

    Liu, Rui; Mao, Ziliang; Matthews, Dennis L; Li, Chin-Shang; Chan, James W; Satake, Noriko

    2013-07-01

    Laser tweezers Raman spectroscopy was used to characterize the oxygenation response of single normal adult, sickle, and cord blood red blood cells (RBCs) to an applied mechanical force. Individual cells were subjected to different forces by varying the laser power of a single-beam optical trap, and the intensities of several oxygenation-specific Raman spectral peaks were monitored to determine the oxygenation state of the cells. For all three cell types, an increase in laser power (or mechanical force) induced a greater deoxygenation of the cell. However, sickle RBCs deoxygenated more readily than normal RBCs when subjected to the same optical forces. Conversely, cord blood RBCs were able to maintain their oxygenation better than normal RBCs. These results suggest that differences in the chemical or mechanical properties of fetal, normal, and sickle cells affect the degree to which applied mechanical forces can deoxygenate the cell. Populations of normal, sickle, and cord RBCs were identified and discriminated based on this mechanochemical phenomenon. This study demonstrates the potential application of laser tweezers Raman spectroscopy as a single-cell, label-free analytical tool to characterize the functional (e.g., mechanical deformability, oxygen binding) properties of normal and diseased RBCs.

  1. Fibrinogen, red blood cells, and factor XIII in venous thrombosis.

    PubMed

    Walton, B L; Byrnes, J R; Wolberg, A S

    2015-06-01

    Cardiovascular disease is the leading cause of death and disability worldwide. Among cardiovascular causes of death, venous thrombosis (VT) is ranked third most common in the world. Venous thrombi have high red blood cell and fibrin content; however, the pathophysiologic mechanisms that contribute to venous thrombus composition and stability are still poorly understood. This article reviews biological, biochemical, and biophysical contributions of fibrinogen, factor XIII, and red blood cells to VT, and new evidence suggesting interactions between these components mediate venous thrombus composition and size.

  2. 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.

  3. 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.

  4. Squeezing red blood cells on an optical waveguide to monitor cell deformability during blood storage.

    PubMed

    Ahluwalia, Balpreet Singh; McCourt, Peter; Oteiza, Ana; Wilkinson, James S; Huser, Thomas R; Hellesø, Olav Gaute

    2015-01-07

    Red blood cells squeeze through micro-capillaries as part of blood circulation in the body. The deformability of red blood cells is thus critical for blood circulation. In this work, we report a method to optically squeeze red blood cells using the evanescent field present on top of a planar waveguide chip. The optical forces from a narrow waveguide are used to squeeze red blood cells to a size comparable to the waveguide width. Optical forces and pressure distributions on the cells are numerically computed to explain the squeezing process. The proposed technique is used to quantify the loss of blood deformability that occurs during blood storage lesion. Squeezing red blood cells using waveguides is a sensitive technique and works simultaneously on several cells, making the method suitable for monitoring stored blood.

  5. 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.

  6. 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.

  7. 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.

  8. In Vitro Osteogenic Potential of Green Fluorescent Protein Labelled Human Embryonic Stem Cell-Derived Osteoprogenitors

    PubMed Central

    Sriram, Gopu; Li, Mingming; Zou, Yu; Li, Lulu; Handral, Harish K.; Rosa, Vinicus; Cao, Tong

    2016-01-01

    Cellular therapy using stem cells in bone regeneration has gained increasing interest. Various studies suggest the clinical utility of osteoprogenitors-like mesenchymal stem cells in bone regeneration. However, limited availability of mesenchymal stem cells and conflicting evidence on their therapeutic efficacy limit their clinical application. Human embryonic stem cells (hESCs) are potentially an unlimited source of healthy and functional osteoprogenitors (OPs) that could be utilized for bone regenerative applications. However, limited ability to track hESC-derived progenies in vivo greatly hinders translational studies. Hence, in this study, we aimed to establish hESC-derived OPs (hESC-OPs) expressing green fluorescent protein (GFP) and to investigate their osteogenic differentiation potential in vitro. We fluorescently labelled H9-hESCs using a plasmid vector encoding GFP. The GFP-expressing hESCs were differentiated into hESC-OPs. The hESC-OPsGFP+ stably expressed high levels of GFP, CD73, CD90, and CD105. They possessed osteogenic differentiation potential in vitro as demonstrated by increased expression of COL1A1, RUNX2, OSTERIX, and OPG transcripts and mineralized nodules positive for Alizarin Red and immunocytochemical expression of osteocalcin, alkaline phosphatase, and collagen-I. In conclusion, we have demonstrated that fluorescently labelled hESC-OPs can maintain their GFP expression for the long term and their potential for osteogenic differentiation in vitro. In future, these fluorescently labelled hESC-OPs could be used for noninvasive assessment of bone regeneration, safety, and therapeutic efficacy. PMID:28003831

  9. Endothelial Lu/BCAM glycoproteins are novel ligands for red blood cell alpha4beta1 integrin: role in adhesion of sickle red blood cells to endothelial cells.

    PubMed

    El Nemer, Wassim; Wautier, Marie-Paule; Rahuel, Cécile; Gane, Pierre; Hermand, Patricia; Galactéros, Frédéric; Wautier, Jean-Luc; Cartron, Jean-Pierre; Colin, Yves; Le Van Kim, Caroline

    2007-04-15

    The Lutheran (Lu) blood group and basal cell adhesion molecule (BCAM) antigens are both carried by 2 glycoprotein isoforms of the immunoglobulin superfamily representing receptors for the laminin alpha(5) chain. In addition to red blood cells, Lu/BCAM proteins are highly expressed in endothelial cells. Abnormal adhesion of red blood cells to the endothelium could potentially contribute to the vaso-occlusive episodes in sickle cell disease. Considering the presence of integrin consensus-binding sites in Lu/BCAM proteins, we investigated their potential interaction with integrin alpha(4)beta(1), the unique integrin expressed on immature circulating sickle red cells. Using cell adhesion assays under static and flow conditions, we demonstrated that integrin alpha(4)beta(1) expressed on transfected cells bound to chimeric Lu-Fc protein. We showed that epinephrine-stimulated sickle cells, but not control red cells, adhered to Lu-Fc via integrin alpha(4)beta(1) under flow conditions. Antibody-mediated activation of integrin alpha(4)beta(1) induced adhesion of sickle red cells to primary human umbilical vein endothelial cells; this adhesion was inhibited by soluble Lu-Fc and vascular cell adhesion molecule-1 (VCAM-1)-Fc proteins. This novel interaction between integrin alpha(4)beta(1) in sickle red cells and endothelial Lu/BCAM proteins could participate in sickle cell adhesion to endothelium and potentially play a role in vaso-occlusive episodes.

  10. 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.

  11. Artificial Red Cells with Polyhemoglobin Membranes.

    DTIC Science & Technology

    1981-09-01

    preparing emulsions and ejecting cells from the oil phase. IX. REFERENCES 1. Wallace, H. W., Asher, W. J., and Li, N. N. Liquid - liquid oxygenation: a...1S. KEY WORDS (Continue, an reverse side if naceoay mnd identify by block number) Artificial Blood, Hemoglobin, Polyhemoglobin, Biotonometry Liquid ...cell-size microdroplets containing 30% of hemoglobin were held in liquid membrane capsules and treated with glutaralddhyde that cross linked the

  12. Chronic red blood cell exchange to prevent clinical complications in sickle cell disease.

    PubMed

    Cabibbo, Sergio; Fidone, Carmelo; Garozzo, Giovanni; Antolino, Agostino; Manenti, Giovanna Oriella; Bennardello, Francesco; Licitra, Vincenzo; Calabrese, Salvatore; Costantino, Francesco; Travali, Simone; Distefano, Roberto; Bonomo, Pietro

    2005-06-01

    We tracked the results of 394 manual or automatic red blood cell exchanges done with a cell separator in 20 sickle cell patients at high risk for recurrent complications. Over an average of 6 years, none of the patients developed complications related to the procedure or to the increased blood use. It was safe and effective in preventing complications of sickle cell disease, and if done automatically, reduced iron overload. Ferritin levels also decreased in patients treated with automatic red blood cell exchange. Furthermore, using Single Donor Red Blood Cell units (SDRC) we reduced the potential exposure to transfusion transmitted infectious diseases (TTI).

  13. Deoxygenation affects tyrosine phosphoproteome of red cell membrane from patients with sickle cell disease.

    PubMed

    Siciliano, Angela; Turrini, Franco; Bertoldi, Mariarita; Matte, Alessandro; Pantaleo, Antonella; Olivieri, Oliviero; De Franceschi, Lucia

    2010-04-15

    Sickle cell disease (SCD) is a worldwide distributed hereditary red cell disorder related to the production of a defective form of hemoglobin, hemoglobin S (HbS). One of the hallmarks of SCD is the presence of dense, dehydrate highly adhesive sickle red blood cells (RBCs) that result from persistent membrane damage associated with HbS polymerization, abnormal activation of membrane cation transports and generation of distorted and rigid red cells with membrane perturbation and cytoskeleton dysfunction. Although modulation of phosphorylation state of the proteins from membrane and cytoskeleton networks has been proposed to participate in red cell homeostasis, much still remains to be investigated in normal and diseased red cells. Here, we report that tyrosine (Tyr-) phosphoproteome of sickle red cells was different from normal controls and was affected by deoxygenation. We found proteins, p55 and band 4.1, from the junctional complex, differently Tyr-phosphorylated in SCD RBCs compared to normal RBCs under normoxia and modulated by deoxygenation, while band 4.2 was similarly Tyr-phosphorylated in both conditions. In SCD RBCs we identified the phosphopeptides for protein 4.1R located in the protein FERM domain (Tyr-13) and for alpha-spectrin located near or in a linker region (Tyr-422 and Tyr-1498) involving protein areas crucial for their functions in the context of red cell membrane properties, suggesting that Tyr-phosphorylation may be part of the events involved in maintaining membrane mechanical stability in SCD red cells.

  14. Shape of red blood cells in contact with artificial surfaces.

    PubMed

    Grzhibovskis, Richards; Krämer, Elisabeth; Bernhardt, Ingolf; Kemper, Björn; Zanden, Carl; Repin, Nikolay V; Tkachuk, Bogdan V; Voinova, Marina V

    2017-03-01

    The phenomenon of physical contact between red blood cells and artificial surfaces is considered. A fully three-dimensional mathematical model of a bilayer membrane in contact with an artificial surface is presented. Numerical results for the different geometries and adhesion intensities are found to be in agreement with experimentally observed geometries obtained by means of digital holographic microscopy.

  15. Hereditary red cell membrane disorders and laboratory diagnostic testing.

    PubMed

    King, M-J; Zanella, A

    2013-06-01

    This overview describes two groups of nonimmune hereditary hemolytic anemias caused by defects in membrane proteins located in distinct layers of the red cell membrane. Hereditary spherocytosis (HS), hereditary elliptocytosis (HE), and hereditary pyropoikilocytosis (HPP) represent disorders of the red cell cytoskeleton. Hereditary stomatocytoses represents disorders of cation permeability in the red cell membrane. The current laboratory screening tests for HS are the osmotic fragility test, acid glycerol lysis time test (AGLT), cryohemolysis test, and eosin-5'-maleimide (EMA)-binding test. For atypical HS, SDS-polyacrylamide gel electrophoresis of erythrocyte membrane proteins is carried out to confirm the diagnosis. The diagnosis of HE/HPP is based on abnormal red cell morphology and the detection of protein 4.1R deficiency or spectrin variants using gel electrophoresis. None of screening tests can detect all HS cases. Some testing centers (a survey of 25 laboratories) use a combination of tests (e.g., AGLT and EMA). No specific screening test for hereditary stomatocytoses is available. The preliminary diagnosis is based on presenting a compensated hemolytic anemia, macrocytosis, and a temperature or time dependent pseudohyperkalemia in some patients. Both the EMA-binding test and the osmotic fragility test may help in differential diagnosis of HS and hereditary stomatocytosis.

  16. 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

  17. 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.

  18. Lipid traffic between high density lipoproteins and Plasmodium falciparum-infected red blood cells

    PubMed Central

    1991-01-01

    Several intraerythrocytic growth cycles of Plasmodium falciparum could be achieved in vitro using a serum free medium supplemented only with a human high density lipoprotein (HDL) fraction (d = 1.063-1.210). The parasitemia obtained was similar to that in standard culture medium containing human serum. The parasite development was incomplete with the low density lipoprotein (LDL) fraction and did not occur with the VLDL fraction. The lipid traffic from HDL to the infected erythrocytes was demonstrated by pulse labeling experiments using HDL loaded with either fluorescent NBD-phosphatidylcholine (NBD-PC) or radioactive [3H]palmitoyl-PC. At 37 degrees C, the lipid probes rapidly accumulated in the infected cells. After incubation in HDL medium containing labeled PC, a subsequent incubation in medium with either an excess of native HDL or 20% human serum induced the disappearance of the label from the erythrocyte plasma membrane but not from the intraerythrocytic parasite. Internalization of lipids did not occur at 4 degrees C. The mechanism involved a unidirectional flux of lipids but no endocytosis. The absence of labeling of P. falciparum, with HDL previously [125I]iodinated on their apolipoproteins or with antibodies against the apolipoproteins AI and AII by immunofluorescence and immunoblotting, confirmed that no endocytosis of the HDL was involved. A possible pathway of lipid transport could be a membrane flux since fluorescence videomicroscopy showed numerous organelles labeled with NBD-PC moving between the erythrocyte and the parasitophorous membranes. TLC analysis showed that a partial conversion of the PC to phosphatidylethanolamine was observed in P. falciparum-infected red cells after pulse with [3H]palmitoyl-PC-HDL. The intensity of the lipid traffic was stage dependent with a maximum at the trophozoite and young schizont stages (38th h of the erythrocyte life cycle). We conclude that the HDL fraction appears to be a major lipid source for Plasmodium

  19. 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.

  20. 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

  1. 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.

  2. 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

  3. 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.

  4. The red cell storage lesion(s): of dogs and men

    PubMed Central

    Klein, Harvey G.

    2017-01-01

    The advent of preservative solutions permitted refrigerated storage of red blood cells. However, the convenience of having red blood cell inventories was accompanied by a disadvantage. Red cells undergo numerous physical and metabolic changes during cold storage, the “storage lesion(s)”. Whereas controlled clinical trials have not confirmed the clinical importance of such changes, ethical and operational issues have prevented careful study of the oldest stored red blood cells. Suggestions of toxicity from meta-analyses motivated us to develop pre-clinical canine models to compare the freshest vs the oldest red blood cells. Our model of canine pneumonia with red blood cell transfusion indicated that the oldest red blood cells increased mortality, that the severity of pneumonia is important, but that the dose of transfused red blood cells is not. Washing the oldest red blood cells reduces mortality by removing senescent cells and remnants, whereas washing fresher cells increases mortality by damaging the red blood cell membrane. An opposite effect was found in a model of haemorrhagic shock with reperfusion injury. Physiological studies indicate that release of iron from old cells is a primary mechanism of toxicity during infection, whereas scavenging of cell-free haemoglobin may be beneficial during reperfusion injury. Intravenous iron appears to have toxicity equivalent to old red blood cells in the pneumonia model, suggesting that intravenous iron and old red blood cells should be administered with caution to infected patients. PMID:28263166

  5. The red cell storage lesion(s): of dogs and men.

    PubMed

    Klein, Harvey G

    2017-03-01

    The advent of preservative solutions permitted refrigerated storage of red blood cells. However, the convenience of having red blood cell inventories was accompanied by a disadvantage. Red cells undergo numerous physical and metabolic changes during cold storage, the "storage lesion(s)". Whereas controlled clinical trials have not confirmed the clinical importance of such changes, ethical and operational issues have prevented careful study of the oldest stored red blood cells. Suggestions of toxicity from meta-analyses motivated us to develop pre-clinical canine models to compare the freshest vs the oldest red blood cells. Our model of canine pneumonia with red blood cell transfusion indicated that the oldest red blood cells increased mortality, that the severity of pneumonia is important, but that the dose of transfused red blood cells is not. Washing the oldest red blood cells reduces mortality by removing senescent cells and remnants, whereas washing fresher cells increases mortality by damaging the red blood cell membrane. An opposite effect was found in a model of haemorrhagic shock with reperfusion injury. Physiological studies indicate that release of iron from old cells is a primary mechanism of toxicity during infection, whereas scavenging of cell-free haemoglobin may be beneficial during reperfusion injury. Intravenous iron appears to have toxicity equivalent to old red blood cells in the pneumonia model, suggesting that intravenous iron and old red blood cells should be administered with caution to infected patients.

  6. 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.

  7. Characterization of dsRed2-positive cells in the doublecortin-dsRed2 transgenic adult rat retina.

    PubMed

    Trost, A; Schroedl, F; Marschallinger, J; Rivera, F J; Bogner, B; Runge, C; Couillard-Despres, S; Aigner, L; Reitsamer, H A

    2014-12-01

    Doublecortin (DCX) is predominantly expressed in neuronal precursor cells and young immature neurons of the developing and adult brain, where it is involved in neuronal differentiation, migration and plasticity. Moreover, its expression pattern reflects neurogenesis, and transgenic DCX promoter-driven reporter models have been previously used to investigate adult neurogenesis. In this study, we characterize dsRed2 reporter protein-expressing cells in the adult retina of the transgenic DCX promoter-dsRed2 rat model, with the aim to identify cells with putative neurogenic activity. Additionally, we confirmed the expression of the dsRed2 protein in DCX-expressing cells in the adult hippocampal dentate gyrus. Adult DCX-dsRed2 rat retinas were analyzed by immunohistochemistry for expression of DCX, NF200, Brn3a, Sox2, NeuN, calbindin, calretinin, PKC-a, Otx2, ChAT, PSA-NCAM and the glial markers GFAP and CRALBP, followed by confocal laser-scanning microscopy. In addition, brain sections of transgenic rats were analyzed for dsRed2 expression and co-localization with DCX, NeuN, GFAP and Sox2 in the cortex and dentate gyrus. Endogenous DCX expression in the adult retina was confined to horizontal cells, and these cells co-expressed the DCX promoter-driven dsRed2 reporter protein. In addition, we encountered dsRed2 expression in various other cell types in the retina: retinal ganglion cells (RGCs), a subpopulation of amacrine cells, a minority of bipolar cells and in perivascular cells. Since also RGCs expressed dsRed2, the DCX-dsRed2 rat model might offer a useful tool to study RGCs in vivo under various conditions. Müller glial cells, which have previously been identified as cells with stem cell features and with neurogenic potential, did express neither endogenous DCX nor the dsRed2 reporter. However, and surprisingly, we identified a perivascular glial cell type expressing the dsRed2 reporter, enmeshed with the glia/stem cell marker GFAP and colocalizing with the

  8. Cell-free measurements of brightness of fluorescently labeled antibodies

    NASA Astrophysics Data System (ADS)

    Zhou, Haiying; Tourkakis, George; Shi, Dennis; Kim, David M.; Zhang, Hairong; Du, Tommy; Eades, William C.; Berezin, Mikhail Y.

    2017-02-01

    Validation of imaging contrast agents, such as fluorescently labeled imaging antibodies, has been recognized as a critical challenge in clinical and preclinical studies. As the number of applications for imaging antibodies grows, these materials are increasingly being subjected to careful scrutiny. Antibody fluorescent brightness is one of the key parameters that is of critical importance. Direct measurements of the brightness with common spectroscopy methods are challenging, because the fluorescent properties of the imaging antibodies are highly sensitive to the methods of conjugation, degree of labeling, and contamination with free dyes. Traditional methods rely on cell-based assays that lack reproducibility and accuracy. In this manuscript, we present a novel and general approach for measuring the brightness using antibody-avid polystyrene beads and flow cytometry. As compared to a cell-based method, the described technique is rapid, quantitative, and highly reproducible. The proposed method requires less than ten microgram of sample and is applicable for optimizing synthetic conjugation procedures, testing commercial imaging antibodies, and performing high-throughput validation of conjugation procedures.

  9. Cell-free measurements of brightness of fluorescently labeled antibodies

    PubMed Central

    Zhou, Haiying; Tourkakis, George; Shi, Dennis; Kim, David M.; Zhang, Hairong; Du, Tommy; Eades, William C.; Berezin, Mikhail Y.

    2017-01-01

    Validation of imaging contrast agents, such as fluorescently labeled imaging antibodies, has been recognized as a critical challenge in clinical and preclinical studies. As the number of applications for imaging antibodies grows, these materials are increasingly being subjected to careful scrutiny. Antibody fluorescent brightness is one of the key parameters that is of critical importance. Direct measurements of the brightness with common spectroscopy methods are challenging, because the fluorescent properties of the imaging antibodies are highly sensitive to the methods of conjugation, degree of labeling, and contamination with free dyes. Traditional methods rely on cell-based assays that lack reproducibility and accuracy. In this manuscript, we present a novel and general approach for measuring the brightness using antibody-avid polystyrene beads and flow cytometry. As compared to a cell-based method, the described technique is rapid, quantitative, and highly reproducible. The proposed method requires less than ten microgram of sample and is applicable for optimizing synthetic conjugation procedures, testing commercial imaging antibodies, and performing high-throughput validation of conjugation procedures. PMID:28150730

  10. 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

  11. Experimental study of super paramagnetic iron oxide labeled synovial mesenchymal stem cells.

    PubMed

    Yu, Fang-Yuan; Li, Hong-Hang; Chen, Chang-Hui; Bi, Sheng-Rong

    2015-01-01

    To investigate the feasibility and changes of biological characteristics before and after synovial mesenchymal stem cells (SMSCs) labelled by super paramagnetic iron oxide (SPIO). The rabbit SMSCs were isolated, cultured, purified and identified in vitro. After adding the different concentrations of SPIO-labelled liquid, the cells were incubated 24 h in 37°C carbon dioxide incubator. The labeled-cell samples were observed by Prussian blue staining, transmission electron microscope (TEM) and the cell biology before and after the labeling was compared. The blue stained particles could be seen in the cytoplasm; the SPIO label was positive in 95% SMSC cells. With the concentration of the label liquid increasing, the blue-stained cytoplasm became darker. A large number of high electron density particles could be seen in the cytoplasm and in the pinocytosis vesicles by TEM, which suggested SPIO label positive. When the SPIO concentration was (12.5~50) μg/mL, the differences in cell proliferation and cell viability between the SMSCs after labelling and the SMSCs before labelling were not significant; when the concentration was over 100 μg/mL, the cell proliferation and cell viability were inhibited. A certain concentration range of SPIO can safely label the rabbit SMSC according to this study, which is important for solving the problem of tracing SMSCs in the joints.

  12. Partitioning of red blood cell aggregates in bifurcating microscale flows

    NASA Astrophysics Data System (ADS)

    Kaliviotis, E.; Sherwood, J. M.; Balabani, S.

    2017-03-01

    Microvascular flows are often considered to be free of red blood cell aggregates, however, recent studies have demonstrated that aggregates are present throughout the microvasculature, affecting cell distribution and blood perfusion. This work reports on the spatial distribution of red blood cell aggregates in a T-shaped bifurcation on the scale of a large microvessel. Non-aggregating and aggregating human red blood cell suspensions were studied for a range of flow splits in the daughter branches of the bifurcation. Aggregate sizes were determined using image processing. The mean aggregate size was marginally increased in the daughter branches for a range of flow rates, mainly due to the lower shear conditions and the close cell and aggregate proximity therein. A counterintuitive decrease in the mean aggregate size was apparent in the lower flow rate branches. This was attributed to the existence of regions depleted by aggregates of certain sizes in the parent branch, and to the change in the exact flow split location in the T-junction with flow ratio. The findings of the present investigation may have significant implications for microvascular flows and may help explain why the effects of physiological RBC aggregation are not deleterious in terms of in vivo vascular resistance.

  13. Partitioning of red blood cell aggregates in bifurcating microscale flows

    PubMed Central

    Kaliviotis, E.; Sherwood, J. M.; Balabani, S.

    2017-01-01

    Microvascular flows are often considered to be free of red blood cell aggregates, however, recent studies have demonstrated that aggregates are present throughout the microvasculature, affecting cell distribution and blood perfusion. This work reports on the spatial distribution of red blood cell aggregates in a T-shaped bifurcation on the scale of a large microvessel. Non-aggregating and aggregating human red blood cell suspensions were studied for a range of flow splits in the daughter branches of the bifurcation. Aggregate sizes were determined using image processing. The mean aggregate size was marginally increased in the daughter branches for a range of flow rates, mainly due to the lower shear conditions and the close cell and aggregate proximity therein. A counterintuitive decrease in the mean aggregate size was apparent in the lower flow rate branches. This was attributed to the existence of regions depleted by aggregates of certain sizes in the parent branch, and to the change in the exact flow split location in the T-junction with flow ratio. The findings of the present investigation may have significant implications for microvascular flows and may help explain why the effects of physiological RBC aggregation are not deleterious in terms of in vivo vascular resistance. PMID:28303921

  14. 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

  15. Correlative fluorescence and electron microscopy of quantum dot labeled proteins on whole cells in liquid.

    PubMed

    Peckys, Diana B; Dukes, Madeline J; de Jonge, Niels

    2014-01-01

    Correlative fluorescence microscopy and scanning transmission electron microscopy (STEM) of cells fully immersed in liquid is a new methodology with many application areas. Proteins, in live cells immobilized on microchips, are labeled with fluorescent quantum dot (QD) nanoparticles. In this protocol, the epidermal growth factor receptor (EGFR) is labeled. The cells are fixed after a selected labeling time, for example, 5 min as needed to form EGFR dimers. The microchip with cells is then imaged with fluorescence microscopy. Thereafter, the microchip with the labeled cells and one with a spacer are assembled in a special microfluidic device and imaged with STEM.

  16. The membrane topology of the amino-terminal domain of the red cell calcium pump.

    PubMed Central

    Castello, P. R.; González Flecha, F. L.; Caride, A. J.; Fernández, H. N.; Delfino, J. M.; Rossi, J. P.

    1997-01-01

    A systematic study of the membrane-associated regions in the plasma membrane Ca2+ pump of erythrocytes has been performed by hydrophobic photolabeling. Purified Ca2+ pump was labeled with 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)-diazirine ([125I]TID), a generic photoactivatable hydrophobic probe. These results were compared with the enzyme labeled with a strictly membrane-bound probe, [3H]bis-phosphatidylethanolamine (trifluoromethyl) phenyldiazirine. A significant light-dependent labeling of an M(r) 135,000-140,000 peptide, corresponding to the full Ca2+ pump, was observed with both probes. After proteolysis of the pump labeled with each probe and isolation of fragments by SDS-PAGE, a common pattern of labeled peptides was observed. Similarly, labeling of the Ca2+ pump with [125I]TID, either in isolated red blood cell membranes or after the enzyme was purified, yields a similar pattern of labeled peptides. Taken together, these results validate the use of either probe to study the lipid interface of the membrane-embedded region of this protein, and sustain the notion that the conformation of the pump is maintained throughout the procedures of solubilization, affinity purification, and reconstitution into proteoliposomes. In this work, we put special emphasis on a detailed analysis of the N-terminal domain of the Ca2+ pump. A labeled peptide of M(r) 40,000 belonging to this region was purified and further digested with V8 protease. The specific incorporation of [125I]TID to proteolytic fragments pertaining to the amino-terminal region indicates the existence of two transmembrane stretches in this domain. A theoretical analysis based on the amino acid sequence 1-322 predicts two segments with high probability of membrane insertion, in agreement with the experimental data. Each segment shows a periodicity pattern of hydrophobicity and variability compatible with alpha-helical structure. These results strongly suggest the existence of a transmembrane helical hairpin

  17. 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.

  18. Blood volume and red cell life span (M113), part C

    NASA Technical Reports Server (NTRS)

    Johnson, P. C., Jr.

    1973-01-01

    Prechamber, in-chamber, and postchamber blood samples taken from Skylab simulation crewmembers did not indicate significant shortening of the red cell life span during the mission. This does not suggest that the space simulation environment could not be associated with red cell enzyme changes. It does show that any changes in enzymes were not sufficiently great to significantly shorten red cell survival. There was no evidence of bone marrow erythropoetic suppression nor was there any evidence of increased red cell destruction.

  19. Quantitative measurement of red blood cell central pallor and hypochromasia.

    PubMed

    Bacus, J W

    1980-06-01

    A quantitataive definition and techniques of measurement for central pallor of red blood cells are proposed. These are based on high-resolution measurements of absorbance across the center of the cell. Thus, the measurements reflect both variations in cell thickness and hemoglobin concentration. Although contributions of thickness and concentration may differ in individual cells, to a first approximation, a specific cell may be considered as having a similar concentration of hemoglobin throughout, and thus the major contribution to the central pallor is that due to the difference in thickness between the edges of the cell and the center. The definition proposed expresses central pallor as the percentage volume of indentation, comparing the red cell to a disc of uniform absorbance equal to the maximum found at the cell edges. Population distributions of central pallor then provide a basis for quantitation of hypochromasia. The mean and standard deviation of such distributions are proposed as quantitative descriptors. Sample distributions from 27 normal persons, 8 patients with spherocytic anemia and 26 patients with iron deficiency anemia were studied.

  20. 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.

  1. Expression of blood group antigens on red cell microvesicles.

    PubMed

    Oreskovic, R T; Dumaswala, U J; Greenwalt, T J

    1992-01-01

    The purpose of this study was to determine whether epitopes of the A, B, D, Fya, M, N, S, s, and K blood group antigens are present on microvesicle membranes shed by red cells during storage. Vesicles were isolated from outdated units of blood having and lacking the specified antigens. Diluted antisera were absorbed with fixed quantities of vesicles from red cells with the test antigen and red cells lacking that antigen (controls). The adsorbed and unadsorbed antisera were titrated and scored by using panel cells from persons known to be heterozygous for all the non-AB antigens. The mean titration scores following adsorption with the vesicles from A, B, D, M+N-, M-N+, S+s-, S-s+, and Fy(a+b-) units were appreciably lower than the control scores (0, 0, 3, 2, 2, 0, 4, and 4 vs. 19, 23, 34, 13, 12, 16, 18, and 29, respectively), which indicated the presence of these epitopes on the membrane of shed vesicles. The results following adsorption with K:1,2 vesicles were equivocal.

  2. Multiscale Modeling of Red Blood Cells Squeezing through Submicron Slits

    NASA Astrophysics Data System (ADS)

    Peng, Zhangli; Lu, Huijie

    2016-11-01

    A multiscale model is applied to study the dynamics of healthy red blood cells (RBCs), RBCs in hereditary spherocytosis, and sickle cell disease squeezing through submicron slits. This study is motivated by the mechanical filtration of RBCs by inter-endothelial slits in the spleen. First, the model is validated by comparing the simulation results with experiments. Secondly, the deformation of the cytoskeleton in healthy RBCs is investigated. Thirdly, the mechanisms of damage in hereditary spherocytosis are investigated. Finally, the effects of cytoplasm and membrane viscosities, especially in sickle cell disease, are examined. The simulations results provided guidance for future experiments to explore the dynamics of RBCs under extreme deformation.

  3. Photoacoustic tomography of unlabelled red blood cell at the nanoscale

    NASA Astrophysics Data System (ADS)

    Samant, Pratik; Chen, Jian; Xiang, Liangzhong

    2016-09-01

    In this letter, we present the principle behind nanoscale photoacoustic tomography (nPAT), in addition to simulation results demonstrating the thermal safety and the diagnostic potential of such a modality. Nanoscale photoacoustic tomography is a novel biomedical imaging modality that can allow for the 3D imaging of cells at nanometer resolutions. This modality also allows for the imaging of single red blood cells (RBCs) such that the hemoglobin concentration quantities can be visualized within the cell. As a result, we believe that nPAT can allow for diagnostic information at unprecedented resolutions and enable the visualization of previously unseen phenomenon in RBCs.

  4. Is pure red cell aplasia (PRCA) a clonal disorder?

    PubMed

    Sivakumaran, M; Bhavnani, M; Stewart, A; Roberts, B E; Geary, G C

    1993-01-01

    Pure red cell aplasia (PRCA) is an uncommon disorder, many cases lacking a well defined aetiology. This report describes three cases of PRCA (two idiopathic and one associated with B-CLL) who were investigated to assess the possibility of their PRCA being associated with a clonal proliferation of T-lymphocytes. The results show that one patient had evidence of T-cell receptor (TCR) gamma chain rearrangement, and the other had a TCR delta chain rearrangement. These two cases raise the possibility of PRCA being associated with a clonal proliferation of T-cells and further studies are warranted.

  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. 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.

  7. 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.

  8. Photodynamic treatment of red blood cell concentrates for virus inactivation enhances red blood cell aggregation: protection with antioxidants.

    PubMed

    Ben-Hur, E; Barshtein, G; Chen, S; Yedgar, S

    1997-10-01

    Photodynamic treatment (PDT) using phthalocyanines and red light appears to be a promising procedure for decontamination of red blood cell (RBC) concentrates for transfusion. A possible complication of this treatment may be induced aggregation of RBC. The production of RBC aggregates was measured with a novel computerized cell flow properties analyzer (CFA). The PDT of RBC concentrates with sulfonated aluminum phthalocyanine (AIPcS4) and the silicon phthalocyanine Pc 4 under virucidal conditions markedly enhanced RBC aggregation and higher shear stress was required to disperse these aggregates. The clusters of cells were huge and abnormally shaped, unlike the rouleaux formed by untreated RBC. This aggregation was prevented when a mixture of antioxidants was included during PDT. Addition of the antioxidants after PDT reduced aggregation only partially. It is concluded that inclusion of antioxidants during PDT of RBC concentrates prior to transfusion may reduce or eliminate the hemodynamic risk that the virucidal treatment may present to the recipient.

  9. Online Biomedical Resources for Malaria-Related Red Cell Disorders

    PubMed Central

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

    2013-01-01

    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

  10. THE BIREFRINGENCE OF THE HUMAN RED CELL GHOSTS

    PubMed Central

    Ponder, Eric; Barreto, Delia

    1956-01-01

    The type of birefringence described by Mitchison, which extends some 0.5 µ in from the surface of the human red cell ghost in glycerol and which shows a maximum retardation of about 7 A, is only found in ghosts which are sufficiently well hemoglobinised to be seen with the ordinary microscope. Ghosts from which all hemoglobin has been lost are not visible with the ordinary microscope and are not birefringent, although they are clearly visible with phase contrast. About 90 per cent of the ghosts in glycerol preparations are of the latter type, the exact percentage being a function of time. Mitchison's measurements of birefringence, although reproducible, accordingly apply only to ghosts in which some hemoglobin still remains complexed with the lipoprotein layers of the red cell ultrastructure, and do not enable one to draw conclusions as to the thickness and orientation of the lipoprotein surface layers. PMID:13286451

  11. Patterns of Nonelectrolyte Permeability in Human Red Blood Cell Membrane

    PubMed Central

    Naccache, P.; Sha'afi, R. I.

    1973-01-01

    The permeability of human red cell membrane to 90 different molecules has been measured. These solutes cover a wide spectrum of nonelectrolytes with varying chemical structure, chain length, lipid solubility, chemical reactive group, ability to form hydrogen bonds, and other properties. In general, the present study suggests that the permeability of red cell membrane to a large solute is determined by lipid solubility, its molecular size, and its hydrogen-bonding ability. The permeability coefficient increases with increasing lipid solubility and decreasing ability to form hydrogen bonds, whereas it decreases with increasing molecular size. In the case of small solutes, the predominant diffusion factor is steric hindrance augmented by lipid solubility. It is also found that replacement of a hydroxyl group by a carbonyl group or an ether linkage tends to increase permeability. On the other hand, replacement of a hydroxyl group by an amide group tends to decrease the permeability coefficient. PMID:4804758

  12. 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.

  13. Comparison of human red cell lysis by hypochlorous and hypobromous acids: insights into the mechanism of lysis.

    PubMed Central

    Vissers, M C; Carr, A C; Chapman, A L

    1998-01-01

    Human red blood cells are lysed by the neutrophil-derived oxidant hypochlorous acid (HOCl), although the mechanism of lysis is unknown. Hypobromous acid (HOBr), a similarly reactive oxidant, lysed red cells approx. 10-fold faster than HOCl. Therefore we compared the effects of these oxidants on thiols, membrane lipids and proteins to determine which reactions are associated with lysis. There was no difference in the loss of reduced glutathione or membrane thiols with either oxidant, but HOBr reacted more readily with membrane lipids and proteins. Bromohydrin derivatives of phospholipids and cholesterol were seen at approx. one-tenth the level of oxidant than chlorohydrins were. However, these products were detected only with high concentrations of HOCl or HOBr, which caused instant haemolysis. Membrane protein modification occurred at much lower doses of oxidant and was more closely correlated with lysis. SDS/PAGE analysis showed that band 3, the anion transport protein, was lost at the lowest dose of HOBr and at the higher concentrations of HOCl. Labelling the red cells with eosin 5-maleimide, a fluorescent label for band 3, suggested possible clustering of this protein in oxidant-exposed cells. There was also irreversible cross-linking of all the major membrane proteins; this reaction occurred more readily with HOBr. The results indicate that membrane protein modification is the reaction responsible for HOCl-mediated lysis. These effects, and particularly cross-link formation, might result in clustering of band 3 and other membrane and cytoskeletal proteins to form haemolytic pores. PMID:9461501

  14. 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.

  15. 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.

  16. 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.

  17. Pure Red Cell Aplasia Associated with Good Syndrome

    PubMed Central

    Okui, Masayuki; Yamamichi, Takashi; Asakawa, Ayaka; Harada, Masahiko; Horio, Hirotoshi

    2017-01-01

    Pure red cell aplasia (PRCA) and hypogammaglobulinemia are paraneoplastic syndromes that are rarer than myasthenia gravis in patients with thymoma. Good syndrome coexisting with PRCA is an extremely rare pathology. We report the case of a 50-year-old man with thymoma and PRCA associated with Good syndrome who achieved complete PRCA remission after thymectomy and postoperative immunosuppressive therapy, and provide a review of the pertinent literature. PMID:28382272

  18. My passion and passages with red blood cells.

    PubMed

    Hoffman, Joseph F

    2008-01-01

    This article mainly presents, in sequential panels of time, an overview of my professional involvements and laboratory experiences. I became smitten with red blood cells early on, and this passion remains with me to this day. I highlight certain studies, together with those who performed the work, recognizing that it was necessary to limit the details and the topics chosen for discussion. I am uncertain of the interest a personal account has for others, but at least it's here for the record.

  19. Role of Complement in Red Cell Dysfunction in Trauma

    DTIC Science & Technology

    2012-12-01

    3 Introduction Trauma-induced changes in the red blood cells ( RBC ) contribute to the reduction of blood flow to distant...The expression of C4d on the surface of RBCs was measured by flow cytometry and expressed as mean fluorescence intensity. (A) Representative data. (B...measured by flow cytometry. Fluorescence levels of RBCs were acquired for 30 seconds using FACScan flow cytometer to establish a baseline for intra- 8

  20. Research on red cell membrane permeability in arterial hypertension.

    PubMed

    Gatina, R; Balta, N; Moisin, C; Burtea, C; Botea, S; Ioan, M; Teleianu, C

    1998-01-01

    Arterial hypertension, including the elucidation of hypertension pathogenic mechanisms involving elements in the composition of the blood, continues to represent a topical research area. Recent work, such as nuclear magnetic resonance studies looking into red cell permeability, illustrates the presence of modifications of red cell permeability to water (RCPW) related to the stage of arterial hypertension. The identification of a significant increase of RCPW compared to that present in the population with normal arterial pressure values can be useful both in early diagnosis and in warning about a possible predisposition for this condition. At the same time, the dynamic investigation of protonic relaxation time of both intra- and extra-erythrocytic water, the assessment of proton exchange time across the red cell and the calculation of permeability to water enable one not only to diagnose arterial hypertension but also to ascertain the evolution of the disease, its complications and the effectiveness of anti-hypertensive medication. Our studies have also proven the existence of a correlation between the values of systolic arterial pressure and red cell permeability to water. The curve describing the interdependence of the two values has the shape of a bell, in the case of males. The peak of the curve is reached for a systolic pressure of 160 mmHg and gets below the values of the control group in the case of systolic pressures above 200 mmHg. The RCPW test can also be considered a valuable indicator in evaluating the risk of stroke in hypertensive patients. In the chronic therapy of arterial hypertension with various types of anti-hypertensive drugs, one can note differences in the RCPW values related to the effectiveness of the respective medication, to the clinical form and stage of the disease, the sex of the patient as well as to the existence of cerebro-vascular complications.

  1. Progress in improving the pathogen safety of red cell concentrates.

    PubMed

    Chapman, J

    2000-01-01

    Current methods of preparing red cell concentrates do not include a process step to decontaminate pathogens potentially present in the transfusion product. Although substantial progress has been made in the reduction of the frequency of transmission of HIV, HCV, HBV and HTLV-I as a result of the implementation of diagnostic screening processes, the need for further reduction in transmission rate of these viruses remains. In addition, there are viruses which are known to be be present in blood but for which no screening test has been implemented to remove contaminated units from the blood supply. These viruses include but are not limited to TTV, HGV and Parvo B19. Finally, the lack of a pathogen inactivation process for red cells maintains the blood supply in a state of vulnerability to new viruses or virus variants as they enter the donor population. Recently, substantial progress has been made in the research and development of a class of chemical compounds designated as INACTINE. These compounds are being investigated for their potential to inactivate viruses in red cells without adversely affecting their physiologic function. One of the INACTINE compounds, designated as PEN110, is now in the clinical trial phase of development.

  2. Thymoma associated with hypogammaglobulinaemia and pure red cell aplasia

    PubMed Central

    Briones, Juan; Iruretagoyena, Mirentxu; Galindo, Héctor; Ortega, Claudia; Zoroquiain, Pablo; Valbuena, José; Acevedo, Francisco; Ocqueteau, Mauricio; Sánchez, Cesar

    2013-01-01

    Thymomas are neoplasias that begin in the thymus and develop in the anterior mediastinum. They are commonly associated with a variety of systemic and autoimmune disorders, such as pure red cell aplasia, hypogammaglobulinaemia, pancytopaenia, collagen diseases, and, most commonly, myasthenia gravis. The presence of inter-current infections, especially diarrhoea and pneumonia, in the presence of lymphocyte B depletion and hypogammaglobulinaemia is known as Good’s syndrome and may affect up to 5% of patients with thymoma. While anaemia is present in 50%–86% of patients with Good’s syndrome, only 41.9% of cases present pure red cell aplasia. Concomitance of these two conditions has only been rarely studied. We report on the case of a 55-year-old man diagnosed with advanced thymoma, who, during the progression of his disease, developed signs and symptoms suggesting Good’s syndrome and pure red cell aplasia. We also performed a brief review of the literature concerning this association, its clinical characteristics, and treatment. PMID:24171048

  3. 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.

  4. Training the next generation analyst using red cell analytics

    NASA Astrophysics Data System (ADS)

    Graham, Meghan N.; Graham, Jacob L.

    2016-05-01

    We have seen significant change in the study and practice of human reasoning in recent years from both a theoretical and methodological perspective. Ubiquitous communication coupled with advances in computing and a plethora of analytic support tools have created a push for instantaneous reporting and analysis. This notion is particularly prevalent in law enforcement, emergency services and the intelligence community (IC), where commanders (and their civilian leadership) expect not only a birds' eye view of operations as they occur, but a play-by-play analysis of operational effectiveness. This paper explores the use of Red Cell Analytics (RCA) as pedagogy to train the next-gen analyst. A group of Penn State students in the College of Information Sciences and Technology at the University Park campus of The Pennsylvania State University have been practicing Red Team Analysis since 2008. RCA draws heavily from the military application of the same concept, except student RCA problems are typically on non-military in nature. RCA students utilize a suite of analytic tools and methods to explore and develop red-cell tactics, techniques and procedures (TTPs), and apply their tradecraft across a broad threat spectrum, from student-life issues to threats to national security. The strength of RCA is not always realized by the solution but by the exploration of the analytic pathway. This paper describes the concept and use of red cell analytics to teach and promote the use of structured analytic techniques, analytic writing and critical thinking in the area of security and risk and intelligence training.

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 660 nm red light-enhanced bone marrow mesenchymal stem cell transplantation for hypoxic-ischemic brain damage treatment.

    PubMed

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

    2014-02-01

    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/cm(2), 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 × 10(6) bone marrow mesenchymal stem cells, followed by irradiation under red light-emitting diodes at 660 nm and 60 mW/cm(2) 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.

  11. Studies on Red Cell Aplasia. V. PRESENCE OF ERYTHROBLAST CYTOTOXICITY IN γG-GLOBULIN FRACTION OF PLASMA

    PubMed Central

    Krantz, Sanford B.; Moore, W. H.; Zaentz, S. Donald

    1973-01-01

    The marrow cells of a patient with pure red cell aplasia markedly increased their rate of heme synthesis when they were freed from the host environment and were incubated in vitro. When the red cell aplasia was treated with cyclophosphamide and prednisone, marrow cell incorporation of 59Fe into heme in vitro increased several weeks before a reticulocytosis was apparent, and was the earliest effect noted. The plasma γG-globulins of this patient inhibited heme synthesis by normal marrow cells or the patient's own marrow cells obtained after remission of the disease. Since the inhibition of heme synthesis could be the result of damage to erythroblasts, the patient's posttreatment marrow cells or normal marrow cells were labeled with 59Fe and were then incubated with the patient's pretreatment, treatment, and posttreatment γG-globulins as well as normal γG-globulins. At the end of this incubation the supernatant and cells were separated and counted. Heme was extracted and also was counted. Treatment of the cells with the patient's pretreatment γG-globulins resulted in a release of 40% of the radioactive heme from the cells. This represented the loss of radioactive hemoglobin and was an index of erythroblast cytotoxicity. A progressive disappearance of the cytotoxic factor in the γG-globulins occurred in the 3 wk period preceding the onset of reticulocytes in the patient's blood. Posttreatment and normal γG-globulins did not produce this effect and increased injury of red cells and lymphocytes was not produced by the patient's pretreatment γG-globulins. These studies demonstrate a method for measuring erythroblast cytoxicity and show that red cell aplasia is associated with γG-globulins that specifically damage erythroblasts. Whether interference with new erythroblast development also occurs and contributes to the inhibition of heme synthesis has not yet been ascertained. Images PMID:4119161

  12. Dealcoholated red wine induces autophagic and apoptotic cell death in an osteosarcoma cell line.

    PubMed

    Tedesco, I; Russo, M; Bilotto, S; Spagnuolo, C; Scognamiglio, A; Palumbo, R; Nappo, A; Iacomino, G; Moio, L; Russo, G L

    2013-10-01

    Until recently, the supposed preventive effects of red wine against cardiovascular diseases, the so-called "French Paradox", has been associated to its antioxidant properties. The interest in the anticancer capacity of polyphenols present in red wine strongly increased consequently to the enormous number of studies on resveratrol. In this study, using lyophilized red wine, we present evidence that its anticancer effect in a cellular model is mediated by apoptotic and autophagic cell death. Using a human osteosarcoma cell line, U2Os, we found that the lyophilized red wine was cytotoxic in a dose-dependent manner with a maximum effect in the range of 100-200 μg/ml equivalents of gallic acid. A mixed phenotype of types I/II cell death was evidenced by means of specific assays following treatment of U2Os with lyophilized red wine, e.g., autophagy and apoptosis. We found that cell death induced by lyophilized red wine proceeded through a mechanism independent from its anti-oxidant activity and involving the inhibition of PI3K/Akt kinase signaling. Considering the relative low concentration of each single bioactive compound in lyophilized red wine, our study suggests the activation of synergistic mechanism able to inhibit growth in malignant cells.

  13. Development of UV-excitable red and near-infrared fluorescent labels and their application for simultaneous multicolor bioimaging by single-wavelength excitation.

    PubMed

    Mizuno, Tetsuya; Umezawa, Keitaro; Shindo, Yutaka; Citterio, Daniel; Oka, Kotaro; Suzuki, Koji

    2013-09-01

    We report a new type of UV-excitable red/NIR-emissive fluorescent dyads (PKF series). Conjugation of a pyrene and a novel bright red/near-infrared (NIR) fluorophore resulted in large quasi-Stokes shift while retaining intense fluorescence emission and sharp spectral bands. Labeling of PKF dyads to biomolecules was performed by means of introduction of a succinimidyl ester. Simultaneous Ca(2+)/albumin dual-color intracellular imaging by PKF in combination with fura-2 (UV-excitable/VIS-emissive Ca(2+) indicator) reveals its usefulness as a new bioimaging tool.

  14. 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

  15. 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.

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

    PubMed Central

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

    2016-01-01

    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

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. Challenges for red blood cell biomarker discovery through proteomics.

    PubMed

    Barasa, Benjamin; Slijper, Monique

    2014-05-01

    Red blood cells are rather unique body cells, since they have lost all organelles when mature, which results in lack of potential to replace proteins that have lost their function. They maintain only a few pathways for obtaining energy and reducing power for the key functions they need to fulfill. This makes RBCs highly sensitive to any aberration. If so, these RBCs are quickly removed from circulation, but if the RBC levels reduce extremely fast, this results in hemolytic anemia. Several causes of HA exist, and proteome analysis is the most straightforward way to obtain deeper insight into RBC functioning under the stress of disease. This should result in discovery of biomarkers, typical for each source of anemia. In this review, several challenges to generate in-depth RBC proteomes are described, like to obtain pure RBCs, to overcome the wide dynamic range in protein expression, and to establish which of the identified/quantified proteins are active in RBCs. The final challenge is to acquire and validate suited biomarkers unique for the changes that occur for each of the clinical questions; in red blood cell aging (also important for transfusion medicine), for thalassemias or sickle cell disease. Biomarkers for other hemolytic anemias that are caused by dysfunction of RBC membrane proteins (the RBC membrane defects) or RBC cytosolic proteins (the enzymopathies) are sometimes even harder to discover, in particular for the patients with RBC rare diseases with unknown cause. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge.

  3. Traceless affinity labeling of endogenous proteins for functional analysis in living cells.

    PubMed

    Hayashi, Takahiro; Hamachi, Itaru

    2012-09-18

    Protein labeling and imaging techniques have provided tremendous opportunities to study the structure, function, dynamics, and localization of individual proteins in the complex environment of living cells. Molecular biology-based approaches, such as GFP-fusion tags and monoclonal antibodies, have served as important tools for the visualization of individual proteins in cells. Although these techniques continue to be valuable for live cell imaging, they have a number of limitations that have only been addressed by recent progress in chemistry-based approaches. These chemical approaches benefit greatly from the smaller probe sizes that should result in fewer perturbations to proteins and to biological systems as a whole. Despite the research in this area, so far none of these labeling techniques permit labeling and imaging of selected endogenous proteins in living cells. Researchers have widely used affinity labeling, in which the protein of interest is labeled by a reactive group attached to a ligand, to identify and characterize proteins. Since the first report of affinity labeling in the early 1960s, efforts to fine-tune the chemical structures of both the reactive group and ligand have led to protein labeling with excellent target selectivity in the whole proteome of living cells. Although the chemical probes used for affinity labeling generally inactivate target proteins, this strategy holds promise as a valuable tool for the labeling and imaging of endogenous proteins in living cells and by extension in living animals. In this Account, we summarize traceless affinity labeling, a technique explored mainly in our laboratory. In our overview of the different labeling techniques, we emphasize the challenge of designing chemical probes that allow for dissociation of the affinity module (often a ligand) after the labeling reaction so that the labeled protein retains its native function. This feature distinguishes the traceless labeling approach from the traditional

  4. Imidazolium-tagged glycan probes for non-covalent labeling of live cells.

    PubMed

    Benito-Alifonso, David; Tremell, Shirley; Sadler, Joanna C; Berry, Monica; Galan, M Carmen

    2016-04-07

    Selective, bioorthogonal and fast labeling of glycoconjugates in living cells is a major challenge for synthetic and cellular biology. Here we report the use imidazolium tagged-mannosamine derivative (ITag-Man) for the non-covalent, rapid and site-specific labeling of sialic acid containing glycoproteins using commercial N-nitrilotriacetate fluorescent reagents in a range of cell lines.

  5. Methods for Quantitative Detection of Antibody-induced Complement Activation on Red Blood Cells

    PubMed Central

    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 - fatal1. 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 lysis1-4. 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

  6. /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.

  7. 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.

  8. Aquaporin 2-labeled cells differentiate to intercalated cells in response to potassium depletion.

    PubMed

    Kim, Wan-Young; Nam, Sun Ah; Choi, Arum; Kim, Yu-Mi; Park, Sang Hee; Kim, Yong Kyun; Kim, Jin

    2016-01-01

    The mammalian renal collecting duct consists of principal cells (PCs) and intercalated cells (ICs). Both PCs and ICs are involved in potassium (K(+)) homeostasis, PCs through their role in K(+) secretion and ICs through their ability to facilitate K(+) resorption. We previously hypothesized that PCs may differentiate into ICs upon K(+) depletion. However, no direct evidence has yet been obtained to conclusively demonstrate that PCs differentiate into ICs in response to K(+) depletion. Here, we present direct evidence for the differentiation of PCs into ICs by cell lineage tracing using aquaporin 2 (AQP2)-Cre mice and R26R-EYFP transgenic mice. In control mice, AQP2-EYFP(+) cells exhibited mainly a PC phenotype (AQP2-positive/H(+)-ATPase-negative). Interestingly, some AQP2-EYFP(+) cells exhibited an IC phenotype (H(+)-ATPase-positive/AQP2-negative); these cells accounted for 1.7 %. After K(+) depletion, the proportion of AQP2-EYFP(+) cells with an IC phenotype was increased to 4.1 %. Furthermore, some AQP2-EYFP(+) cells exhibited a "null cell" phenotype (AQP2-negative/H(+)-ATPase-negative) after K(+) depletion. Collectively, our data demonstrate that AQP2-labeled cells can differentiate into ICs, as well as null cells, in response to K(+) depletion. This finding indicates that some of AQP2-labeled cells possess properties of progenitor cells and that they can differentiate into ICs in the adult mouse kidney.

  9. 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.

  10. Fourier transform infra-red spectroscopic signatures for lung cells' epithelial mesenchymal transition: A preliminary report

    NASA Astrophysics Data System (ADS)

    Sarkar, Atasi; Sengupta, Sanghamitra; Mukherjee, Anirban; Chatterjee, Jyotirmoy

    2017-02-01

    Infra red (IR) spectral characterization can provide label-free cellular metabolic signatures of normal and diseased circumstances in a rapid and non-invasive manner. Present study endeavoured to enlist Fourier transform infra red (FTIR) spectroscopic signatures for lung normal and cancer cells during chemically induced epithelial mesenchymal transition (EMT) for which global metabolic dimension is not well reported yet. Occurrence of EMT was validated with morphological and immunocytochemical confirmation. Pre-processed spectral data was analyzed using ANOVA and principal component analysis-linear discriminant analysis (PCA-LDA). Significant differences observed in peak area corresponding to biochemical fingerprint (900-1800 cm- 1) and high wave-number (2800-3800 cm- 1) regions contributed to adequate PCA-LDA segregation of cells undergoing EMT. The findings were validated by re-analysis of data using another in-house built binary classifier namely vector valued regularized kernel approximation (VVRKFA), in order to understand EMT progression. To improve the classification accuracy, forward feature selection (FFS) tool was employed in extracting potent spectral signatures by eliminating undesirable noise. Gradual increase in classification accuracy with EMT progression of both cell types indicated prominence of the biochemical alterations. Rapid changes in cellular metabolome noted in cancer cells within first 24 h of EMT induction along with higher classification accuracy for cancer cell groups in comparison to normal cells might be attributed to inherent differences between them. Spectral features were suggestive of EMT triggered changes in nucleic acid, protein, lipid and bound water contents which can emerge as the useful markers to capture EMT related cellular characteristics.

  11. Water-Soluble Lipophilic MR Contrast Agents for Cell Membrane Labeling

    PubMed Central

    Carney, Christiane E.; MacRenaris, Keith W.; Meade, Thomas J.

    2015-01-01

    Long-term cell tracking with MR imaging necessitates the development of contrast agents that both label and are retained by cells. One promising strategy for long-term cell labeling is the development of lipophilic Gd(III)-based contrast agents that anchor into the cell membrane. We have previously reported the efficacy of monomeric and multimeric lipophilic agents and showed that the monomeric agents have improved labeling and contrast enhancement of cell populations. Here, we report on the synthesis, characterization, and in vitro testing of a series of monomeric lipophilic contrast agents with varied alkyl chain compositions. We show that these agents disperse in water, localize to the cell membrane, and label HeLa and MCF7 cells effectively. Additionally, these agents have up to 10-fold improved retention in cells compared to clinically available ProHance®. PMID:26215869

  12. SNAP-, CLIP- and Halo-tag labelling of budding yeast cells.

    PubMed

    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.

  13. Measurement of red blood cell mechanics during morphological changes.

    PubMed

    Park, YongKeun; Best, Catherine A; Badizadegan, Kamran; Dasari, Ramachandra R; Feld, Michael S; Kuriabova, Tatiana; Henle, Mark L; Levine, Alex J; Popescu, Gabriel

    2010-04-13

    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, area, 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. 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).

  16. Structural analysis of red blood cell aggregates under shear flow.

    PubMed

    Chesnutt, J K W; Marshall, J S

    2010-03-01

    A set of measures of red blood cell (RBC) aggregates are developed and applied to examine the aggregate structure under plane shear and channel flows. Some of these measures are based on averages over the set of red blood cells which are in contact with each other at a given time. Other measures are developed by first fitting an ellipse to the planar projection of the aggregate, and then examining the area and aspect ratio of the fit ellipse as well as the orientations of constituent RBCs with respect to the fit ellipse axes. The aggregate structural measures are illustrated using a new mesoscale computational model for blood cell transport, collision and adhesion. The sensitivity of this model to change in adhesive surface energy density and shear rate on the aggregate structure is examined. It is found that the mesoscale model predictions exhibit reasonable agreement with experimental and theoretical data for blood flow in plane shear and channel flows. The new structural measures are used to examine the differences between predictions of two- and three-dimensional computations of the aggregate formation, showing that two-dimensional computations retain some of the important aspects of three-dimensional computations.

  17. 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).

  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. Red blood cell adhesion on a solid/liquid interface

    PubMed Central

    Lavalle, Ph.; Stoltz, J.-F.; Senger, B.; Voegel, J.-C.; Schaaf, P.

    1996-01-01

    Red blood cells (RBCs), previously fixed with glutaraldehyde, adhere to glass slides coated with fibrinogen. The RBC deposition process on the horizontal glass surface is investigated by analyzing the relative surface covered by the RBCs, as well as the variance of this surface coverage, as a function of the concentration of particles. This study is performed by optical microscopy and image analysis. A model, derived from the classical random sequential adsorption model, has been developed to account for the experimental results. This model highlights the strong influence of the hydrodynamic interactions during the deposition process. PMID:8986776

  20. Aplastic anemia and red cell aplasia due to pentachlorophenol

    SciTech Connect

    Roberts, H.J.

    1983-01-01

    Repeated exposure to commercial (technical grade) pentachlorophenol (PCP) preceded aplastic anemia in four patients and pure red cell aplasia in two. Two patients developed concomitant or subsequent Hodgkin's disease and acute leukemia. The hematologic, mutagenic, and carcinogenic effect of PCP and its chemical contaminants have been documented in other clinical and experimental reports. In view of the widespread contamination of our environment by PCP, clinicians and public health investigators must seek out such exposure in these and related disorders and initiate measures to reduce it.

  1. 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

  2. Cytoskeleton confinement and tension of red blood cell membranes.

    PubMed

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

    2003-06-06

    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.

  3. Effect of asbestos on lipid peroxidation in the red cells.

    PubMed Central

    Gabor, S; Anca, Z

    1975-01-01

    In vitro exposure of red cells to vie International Union against Cancer (UICC) standard reference asbestos samples resulted in an increase of thiobarbituric acid substances. Chrysotiles developed the largest amounts of lipid peroxides, followed by anthophyllite, amosite, and crocidolite in decreasing order. Compared with the control samples erythrocytes free of dusts, all types of the asbestos examined disclosed significant differences. The results obtained provide support for the cytotoxic potential of amosite and crocidolite and, on the other hand, suggest that a lipid peroxidation of unsaturated fatty acids may be involved in the mechanisms(s) of membrane-damaging effects of asbestos dusts. PMID:1125126

  4. [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.

  5. Utilization and quality of cryopreserved red blood cells in transfusion medicine.

    PubMed

    Henkelman, S; Noorman, F; Badloe, J F; Lagerberg, J W M

    2015-02-01

    Cryopreserved (frozen) red blood cells have been used in transfusion medicine since the Vietnam war. The main method to freeze the red blood cells is by usage of glycerol. Although the usage of cryopreserved red blood cells was promising due to the prolonged storage time and the limited cellular deterioration at subzero temperatures, its usage have been hampered due to the more complex and labour intensive procedure and the limited shelf life of thawed products. Since the FDA approval of a closed (de) glycerolization procedure in 2002, allowing a prolonged postthaw storage of red blood cells up to 21 days at 2-6°C, cryopreserved red blood cells have become a more utilized blood product. Currently, cryopreserved red blood cells are mainly used in military operations and to stock red blood cells with rare phenotypes. Yet, cryopreserved red blood cells could also be useful to replenish temporary blood shortages, to prolong storage time before autologous transfusion and for IgA-deficient patients. This review describes the main methods to cryopreserve red blood cells, explores the quality of this blood product and highlights clinical settings in which cryopreserved red blood cells are or could be utilized.

  6. Cell labeling and tracking method without distorted signals by phagocytosis of macrophages.

    PubMed

    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.

  7. Bio-inspired cryo-ink preserves red blood cell phenotype and function during nanoliter vitrification.

    PubMed

    El Assal, Rami; Guven, Sinan; Gurkan, Umut Atakan; Gozen, Irep; Shafiee, Hadi; Dalbeyler, 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

    2014-09-03

    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 bioprinting approach.

  8. 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

  9. Developments in red cell rheology at the Institut de Pathologie Cellulaire.

    PubMed

    Evans, E; Mohandas, N

    1986-01-01

    The present day rheological approximation, which has been used successfully to quantitate the deformability properties of red cells, is based on the view that the cell has a liquid interior encapsulated by a viscoelastic solid membrane shell. A review of historical developments in this field shows that determination of intrinsic red cell membrane properties has not come from simple mathematical analysis of experiments. On the contrary, considerable insight has been required to bring together physical and biological methods to rationalize the unique deformability characteristics of the red blood cell. Key developments at the Institut de Pathologie Cellulaire (IPC) in the early 1970s played a role in our improved understanding of red cell rheology. In this article, we describe the material concepts of the red cell membrane held before 1970, discuss the seminal developments at Bicetre, and, finally, outline the contemporary view of red cell deformability.

  10. 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.

  11. Geometric localization of thermal fluctuations in red blood cells.

    PubMed

    Evans, Arthur A; Bhaduri, Basanta; Popescu, Gabriel; Levine, Alex J

    2017-02-27

    The thermal fluctuations of membranes and nanoscale shells affect their mechanical characteristics. Whereas these fluctuations are well understood for flat membranes, curved shells show anomalous behavior due to the geometric coupling between in-plane elasticity and out-of-plane bending. Using conventional shallow shell theory in combination with equilibrium statistical physics we theoretically demonstrate that thermalized shells containing regions of negative Gaussian curvature naturally develop anomalously large fluctuations. Moreover, the existence of special curves, "singular lines," leads to a breakdown of linear membrane theory. As a result, these geometric curves effectively partition the cell into regions whose fluctuations are only weakly coupled. We validate these predictions using high-resolution microscopy of human red blood cells (RBCs) as a case study. Our observations show geometry-dependent localization of thermal fluctuations consistent with our theoretical modeling, demonstrating the efficacy in combining shell theory with equilibrium statistical physics for describing the thermalized morphology of cellular membranes.

  12. A new material concept for the red cell membrane.

    PubMed

    Evans, E A

    1973-09-01

    The proposition is made that the red cell membrane is a two-dimensional, incompressible material and a general stress-strain law is developed for finite deformations. In the linear form, the character of such a material is analogous to a two-dimensional Mooney material (e.g., rubber), indicating that the molecular structure in the plane of the membrane would consist of long chains, randomly kinked and cross-linked in the natural state. The loose network could be provided by the protein component and the lipid phase could exist interstitially as a liquid bilayer, giving the membrane its two-dimensional incompressibility. The material provides the capability of large deformations exhibited by the discocyte and yet the rigidity associated with the osmotic spherocyte state. It is demonstrated that a membrane of this type can form a sphere at constant area. An illustrative example of the application to single cell discocyte-to-osmotic spherocyte transformations is presented.

  13. Geometric localization of thermal fluctuations in red blood cells

    PubMed Central

    Evans, Arthur A.; Bhaduri, Basanta; Popescu, Gabriel; Levine, Alex J.

    2017-01-01

    The thermal fluctuations of membranes and nanoscale shells affect their mechanical characteristics. Whereas these fluctuations are well understood for flat membranes, curved shells show anomalous behavior due to the geometric coupling between in-plane elasticity and out-of-plane bending. Using conventional shallow shell theory in combination with equilibrium statistical physics we theoretically demonstrate that thermalized shells containing regions of negative Gaussian curvature naturally develop anomalously large fluctuations. Moreover, the existence of special curves, “singular lines,” leads to a breakdown of linear membrane theory. As a result, these geometric curves effectively partition the cell into regions whose fluctuations are only weakly coupled. We validate these predictions using high-resolution microscopy of human red blood cells (RBCs) as a case study. Our observations show geometry-dependent localization of thermal fluctuations consistent with our theoretical modeling, demonstrating the efficacy in combining shell theory with equilibrium statistical physics for describing the thermalized morphology of cellular membranes. PMID:28242681

  14. 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.

  15. 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

  16. Silicon phthalocyanine Pc 4 and red light causes apoptosis in HIV-infected cells.

    PubMed

    Ben-Hur, E; Oetjen, J; Horowitz, B

    1997-03-01

    The silicon phthalocyanine HOSiPcOSi(CH3)2(CH2)3 N(CH3)2 (Pc 4), is being studied as a photosensitizer for virus inactivation in red blood cell concentrates (RBCC). The RBCC spiked with cell-free human immunodeficiency virus (HIV) or with HIV actively replicating in the T-lymphocytic cell line CEM can be successfully inactivated (> or = 6 log10) when exposed to 2 microM Pc 4 and 90 J/cm2 red light (600-800 nm). Inactivation of > or = 6 log10 inducible HIV in the latently infected promonocytic cell line U1 occurred at 22.5 J/cm2 (H. Margolis-Nunno et al., Transfusion 36, 743-750, 1996). In order to understand the reason for the increased susceptibility of U1 to photosensitized inactivation we looked for induction of apoptosis by photodynamic treatment (PDT). Agarose gel electrophoresis was used to observe the appearance of a characteristic 180-200 base pair DNA ladder, which can indicate apoptosis. Using this assay it is shown that Pc 4 treatment induced apoptosis in U1 cells in a light dose-dependent manner, starting 30 min after light exposure. Using the ApopTag Plus kit (which attaches a fluorescent label to the 3'-OH ends of the degraded DNA) and flow cytometry, the percentage of cells undergoing apoptosis was quantitated. At 10.5 J/cm2, 3 h after light exposure, about 92.5% of the cells were apoptotic. Under these conditions 99% of the cells eventually die. The CEM cells similarly treated underwent apoptosis at slower kinetics and required higher light doses. Other cell lines latently infected with HIV (ACH-2 and OM 10.1) were as sensitive as U1 to HIV inactivation by Pc 4-PDT (H. Margolis-Nunno et al., Transfusion 36, 743-750, 1996) and underwent apoptosis at a similar kinetic. These results suggest that the enhanced inactivation of HIV in latently infected cells compared to CEM cells by Pc 4-PDT may be due, at least in part, to apoptosis in the former.

  17. From stem cell to erythroblast: regulation of red cell production at multiple levels by multiple hormones.

    PubMed

    Lodish, Harvey; Flygare, Johan; Chou, Song

    2010-07-01

    This article reviews the regulation of production of red blood cells at several levels: (1) the ability of erythropoietin and adhesion to a fibronectin matrix to stimulate the rapid production of red cells by inducing terminal proliferation and differentiation of committed erythroid CFU-E progenitors; (2) the regulated expansion of the pool of earlier BFU-E erythroid progenitors by glucocorticoids and other factors that occurs during chronic anemia or inflammation; and (3) the expansion of thehematopoietic cell pool to produce more progenitors of all hematopoietic lineages.

  18. In Vivo Quantum Dot Labeling of Mammalian Stem and Progenitor Cells

    PubMed Central

    Slotkin, Jonathan R.; Chakrabarti, Lina; Dai, Hai Ning; Carney, Rosalind S.E.; Hirata, Tsutomu; Bregman, Barbara S.; Gallicano, G. Ian; Corbin, Joshua G.; Haydar, Tarik F.

    2009-01-01

    Fluorescent semiconductor nanocrystal quantum dots (QDs) are a class of multifunctional inorganic fluorophores that hold great promise for clinical applications and biomedical research. Because no methods currently exist for directed QD-labeling of mammalian cells in the nervous system in vivo, we developed novel in utero electroporation and ultrasound-guided in vivo delivery techniques to efficiently and directly label neural stem and progenitor cells (NSPCs) of the developing mammalian central nervous system with QDs. Our initial safety and proof of concept studies of one and two-cell QD-labeled mouse embryos reveal that QDs are compatible with early mammalian embryonic development. Our in vivo experiments further show that in utero labeled NSPCs continue to develop in an apparent normal manner. These studies reveal that QDs can be effectively used to label mammalian NSPCs in vivo and will be useful for studies of in vivo fate mapping, cellular migration, and NSPC differentiation during mammalian development. PMID:17626285

  19. 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.

  20. A new class of macrocyclic lanthanide complexes for cell labeling and magnetic resonance imaging applications.

    PubMed

    Zheng, Quan; Dai, Houquan; Merritt, Matthew E; Malloy, Craig; Pan, Cai Yuan; Li, Wen-Hong

    2005-11-23

    Lanthanide complexes have wide applications in biochemical research and biomedical imaging. We have designed and synthesized a new class of macrocyclic lanthanide chelates, Ln/DTPA-PDA-C(n), for cell labeling and magnetic resonance imaging (MRI) applications. Two lipophilic Gd3+ complexes, Gd/DTPA-PDA-C(n) (n = 10, 12), labeled a number of cultured mammalian cells noninvasively at concentrations as low as a few micromolar. Cells took up these agents rapidly and showed robust intensity increases in T1-weighed MR images. Labeled cells showed normal morphology and doubling time as control cells. In addition to cultured cells, these agents also labeled primary cells in tissues such as dissected pancreatic islets. To study the mechanism of cellular uptake, we applied the technique of diffusion enhanced fluorescence resonance energy transfer (DEFRET) to determine the cellular localization of these lipophilic lanthanide complexes. After loading cells with a luminescent complex, Tb/DTPA-PDA-C10, we observed DEFRET between the Tb3+ complex and extracellular, but not intracellular, calcein. We concluded that these cyclic lanthanide complexes label cells by inserting two hydrophobic alkyl chains into cell membranes with the hydrophilic metal binding site facing the extracellular medium. As the first imaging application of these macrocyclic lanthanide chelates, we labeled insulin secreting beta-cells with Gd/DTPA-PDA-C12. Labeled cells were encapsulated in hollow fibers and were implanted in a nude mouse. MR imaging of implanted beta-cells showed that these cells could be followed in vivo for up to two weeks. The combined advantages of this new class of macrocyclic contrast agents ensure future imaging applications to track cell movement and localization in different biological systems.

  1. Site-directed spin labeling of proteins for distance measurements in vitro and in cells.

    PubMed

    Roser, P; Schmidt, M J; Drescher, M; Summerer, D

    2016-06-15

    Site-directed spin labeling (SDSL) in combination with electron paramagnetic resonance (EPR) spectroscopy allows studying the structure, dynamics, and interactions of proteins via distance measurements in the nanometer range. We here give an overview of available spin labels, the strategies for their introduction into proteins, and the associated potentials for protein structural studies in vitro and in the context of living cells.

  2. Characterization of red blood cell deformability change during blood storage.

    PubMed

    Zheng, Yi; Chen, Jun; Cui, Tony; Shehata, Nadine; Wang, Chen; Sun, Yu

    2014-02-07

    Stored red blood cells (RBCs) show progressive deformability changes during blood banking/storage. Their deformability changes over an 8 weeks' storage period were measured using a microfluidic device. Hydrodynamic focusing controls the orientation and position of individual RBCs within the microchannel. High-speed imaging (5000 frames s(-1)) captures the dynamic deformation behavior of the cells, and together with automated image analysis, enables the characterization of over 1000 RBCs within 3 minutes. Multiple parameters including deformation index (DI), time constant (shape recovery rate), and RBC circularity were quantified. Compared to previous studies on stored RBC deformability, our results include a significantly higher number of cells (>1000 cells per sample vs. a few to tens of cells per sample) and, for the first time, reveal deformation changes of stored RBCs when traveling through human-capillary-like microchannels. Contrary to existing knowledge, our results demonstrate that the deformation index of RBCs under folding does not change significantly over blood storage. However, significant differences exist in time constants and circularity distribution widths, which can be used to quantify stored RBC quality or age.

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

    PubMed

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

    2016-06-01

    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.

  4. Skeleton deformation of red blood cells during tank treading motions

    NASA Astrophysics Data System (ADS)

    Zhu, Qiang; Peng, Zhangli

    2012-11-01

    By coupling a fluid-structure interaction algorithm with a three-level multiscale structural model, we simulate the tank treading responses of erythrocytes (red blood cells, or RBC) in shear flows. The fluid motion is depicted within the Stokes-flow framework, and is mathematically formulated with the boundary integral equations. The structural model takes into account the flexible connectivity between the lipid bilayer and the protein skeleton as well as the viscoelastic responses. The concentration of this study is on the transient process involving the development of the local area deformation of the protein skeleton. Under the assumption that the protein skeleton is stress-free in the natural biconcave configuration, our simulations indicate the following properties: (1) During tank treading motions it takes long time for significant area deformations to establish. For cells with diminished connectivity between the lipid bilayer and the protein skeleton (e.g. cells with mutations or defects), the relaxation time will be greatly reduced; (2) Deformations of the skeleton depend on the initial orientation of the cell with respect to the incoming flow; (3) The maximum area expansion occurs around the regions corresponding to the dimples in the original biconcave state; (4) Oscillations in cell geometry (breathing) and orientation (e.g. swinging) are observed. This work was supported by the National Heart, Lung, and Blood Institute under award number R01HL092793.

  5. Imaging red blood cell dynamics by quantitative phase microscopy.

    PubMed

    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.

  6. 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)

  7. 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.

  8. 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.

  9. New Developments in Red Blood Cell Preservation Using Liquid and Freezing Procedures.

    DTIC Science & Technology

    1982-04-02

    phosphate ion exchange resin.30 ൪ Solutions of bicarbonate, adenine, glucose and mannitol (BAGM), saline, adenine and RED BLOOD CELL PRESERVATION 10...glucose (SAO), and adenine, glucose, sodium chloride and mannitol (ADSOL), have been used to maintain or increase the red cell organic phosphate...compounds, ATP which influences posttransfusion survival, and 2,3 DPG which influences red cell oxygen transport function. Studies have shown that mannitol

  10. 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.

  11. Specific chemotaxis of magnetically labeled mesenchymal stem cells: Implications for MRI of glioma

    PubMed Central

    Bennewitz, Margaret F.; Tang, Kevin S.; Markakis, Eleni A.; Shapiro, Erik M.

    2012-01-01

    Purpose Glioblastoma multiforme (GBM) is a lethal disease, marked by infiltration of cancerous cells into the surrounding normal brain. The dire outcome of GBM patients stems in part from the limitations of current neuroimaging methods. Notably, early cancer detection methodologies are lacking, without the ability to identify aggressive, metastatic tumor cells. We propose a novel approach for tumor detection using MRI, based on imaging specific tumor tropism of mesenchymal stem cells (MSCs) labeled with micron-sized iron oxide particles (MPIOs). Procedures MPIO labeled and unlabeled MSCs were compared for viability, multi-lineage differentiation, and migration, where both chemotactic and chemokinetic movement were assessed in the presence of serum free medium, serum containing medium, and glioma conditioned medium. MRI was performed on agarose samples, consisting of MPIO labeled single MSCs, to confirm the capability to detect single cells. Results We determined that MPIO labeled MSCs exhibit specific and significant chemotactic migration towards glioma conditioned medium in vitro. Confocal fluorescence microscopy confirmed that MPIOs are internalized and do not impact important cell processes of MSCs. Lastly, MPIO labeled MSCs appear as single distinct, dark spots on T2* weighted MRI, supporting the robustness of this contrast agent for cell tracking. Conclusions This is the first study to show that MPIO labeled MSCs exhibit specific tropism toward tumor-secreted factors in vitro. The potential for detecting single MPIO labeled MSCs provides rationale for in vivo extension of this methodology to visualize GBM in animal models. PMID:22418788

  12. Sucralose sweetener in vivo effects on blood constituents radiolabeling, red blood cell morphology and radiopharmaceutical biodistribution in rats.

    PubMed

    Rocha, G S; Pereira, M O; Benarroz, M O; Frydman, J N G; Rocha, V C; Pereira, M J; Fonseca, A S; Medeiros, A C; Bernardo-Filho, M

    2011-01-01

    Effects of sucralose sweetener on blood constituents labelled with technetium-99m ((99m)Tc) on red blood cell (RBC) morphology, sodium pertechnetate (Na(99m)TcO(4)) and diethylenetriaminepentaacetic acid labeled with (99m)Tc ((99m)Tc-DTPA) biodistribution in rats were evaluated. Radiolabeling on blood constituents from Wistar rats was undertaken for determining the activity percentage (%ATI) on blood constituents. RBC morphology was also evaluated. Na(99m)TcO(4) and (99m)Tc-DTPA biodistribution was used to determine %ATI/g in organs. There was no alteration on RBC blood constituents and morphology %ATI. Sucralose sweetener was capable of altering %ATI/g of the radiopharmaceuticals in different organs. These findings are associated to the sucralose sweetener in specific organs.

  13. 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.

  14. Surface-Enhanced Raman Scattering Nanoparticles as Optical Labels for Imaging Cell Surface Proteins

    NASA Astrophysics Data System (ADS)

    MacLaughlin, Christina M.

    Assaying the expression of cell surface proteins has widespread application for characterizing cell type, developmental stage, and monitoring disease transformation. Immunophenotyping is conducted by treating cells with labelled targeting moieties that have high affinity for relevant surface protein(s). The sensitivity and specificity of immunophenotyping is defined by the choice of contrast agent and therefore, the number of resolvable signals that can be used to simultaneously label cells. Narrow band width surface-enhanced Raman scattering (SERS) nanoparticles are proposed as optical labels for multiplexed immunophenotying. Two types of surface coatings were investigated to passivate the gold nanoparticles, incorporate SERS functionality, and to facilitate attachment of targeting antibodies. Thiolated poly(ethylene glycol) forms dative bonds with the gold surface and is compatible with multiple physisorbed Raman-active reporter molecules. Ternary lipid bilayers are used to encapsulate the gold nanoparticles particles, and incorporate three different classes of Raman reporters. TEM, UV-Visible absorbance spectroscopy, DLS, and electrophoretic light scattering were used characterize the particle coating. Colourimetric protein assay, and secondary antibody labelling were used to quantify the antibody conjugation. Three different in vitromodels were used to investigate the binding efficacy and specificity of SERS labels for their biomarker targets. Primary human CLL cells, LY10 B lymphoma, and A549 adenocarcinoma lines were targeted. Dark field imaging was used to visualize the colocalization of SERS labels with cells, and evidence of receptor clustering was obtained based on colour shifts of the particles' Rayleigh scattering. Widefield, and spatially-resolved Raman spectra were used to detect labels singly, and in combination from labelled cells. Fluorescence flow cytometry was used to test the particles' binding specificity, and SERS from labelled cells was also

  15. Rheology of red blood cell aggregation by computer simulation

    NASA Astrophysics Data System (ADS)

    Liu, Yaling; Liu, Wing Kam

    2006-12-01

    The aggregation of red blood cells (RBC) induced by the interactions between RBCs is a dominant factor of the in vitro rheological properties of blood, and existing models of blood do not contain full cellular information. In this work, we introduce a new three-dimensional model that couples Navier-Stokes equations with cell interactions to investigate RBC aggregation and its effect on blood rheology. It consists of a depletion mediated aggregation model to describe the interactions of RBCs and an immersed continuum model to track the deformation/motion of RBCs in blood plasma. To overcome the large deformation of RBCs, the meshfree method is used to model the RBCs. Three important phenomena in blood rheology are successfully captured and studied via this approach: the shear rate dependence of blood viscosity, the influence of cell rigidity on blood viscosity, and the Fahraeus-Lindqvist effect. As a microscopic illustration of the shear-rate dependence of the blood's viscoelasticity, the disaggregation of an RBC rouleau at shear rates varying between 0.125 and 24 s -1 is modeled. Lower RBC deformability and higher shear rates above 0.5 s -1 are found to facilitate disaggregation. The effective viscosities at different shear rates and for cells with different deformabilities are simulated. The numerical results are shown to agree with the reported experimental measurements. The Fahraeus-Lindqvist effect is, for the first time, studied through three-dimensional numerical simulations of blood flow through tubes with different diameters and is shown to be directly linked to axial-migration of deformable cells. This study shows that cell-cell interaction and cell deformability have significant effects on blood rheology in capillaries.

  16. Storage time of red blood cells and mortality of transfusion recipients.

    PubMed

    Middelburg, Rutger A; van de Watering, Leo M G; Briët, Ernest; van der Bom, Johanna G

    2013-01-01

    Storage of red cells and the associated storage lesion have been suggested to contribute to adverse clinical outcomes. The aim of this study was to investigate whether increasing storage time of red cells is associated with mortality of recipients. From all patients who received red cell transfusions between January 2005 and May 2009, in the Leiden University Medical Center, we selected those who received only-young or only-old red cells, defined as below or above the median storage time. Mortality was compared in a Cox regression model. Subsequently, similar comparisons were made between subgroups with increasing contrast between old and young red cells. Among adult patients, after correction for potential confounders, the hazard ratio of death within 1 year after receiving red cells stored for more than 17 days compared with 17 days or less was 0.98 (95% confidence interval, 0.83-1.2). With increasing contrast, the hazard ratio decreased to 0.56 (95% confidence interval, 0.32-0.97) for red cells stored for more than 24 days compared with less than 10 days. In contrast to what has previously been suggested, we find an almost 2-fold increase in mortality rate after the transfusion of fresh red cells compared with old red cells. Results dependent on analyses chosen and previous studies may not have used optimal analyses. The tendency to demand ever-fresher blood could actually be detrimental for at least some patient groups.

  17. The deformation behavior of multiple red blood cells in a capillary vessel.

    PubMed

    Gong, Xiaobo; Sugiyama, Kazuyasu; Takagi, Shu; Matsumoto, Yoichiro

    2009-07-01

    The deformation of multiple red blood cells in a capillary flow was studied numerically. The immersed boundary method was used for the fluid red blood cells interaction. The membrane of the red blood cell was modeled as a hyperelastic thin shell. The numerical results show that the apparent viscosity in the capillary flow is more sensitive to the change of shear coefficient of the membrane than the bending coefficient and surface dilation coefficient, and the increase in the shear coefficient results in an increase in the pressure drop in the blood flow in capillary vessels in order to sustain the same flux rate of red blood cells.

  18. The novel role of peroxiredoxin-2 in red cell membrane protein homeostasis and senescence.

    PubMed

    Matté, Alessandro; Pantaleo, Antonella; Ferru, Emanuela; Turrini, Franco; Bertoldi, Mariarita; Lupo, Francesca; Siciliano, Angela; Ho Zoon, Chae; De Franceschi, Lucia

    2014-11-01

    Peroxiredoxin-2 (Prx2), a typical two-cysteine peroxiredoxin, is the third most abundant protein in red cells. Although progress has been made in the functional characterization of Prx2, its role in red cell membrane protein homeostasis is still under investigation. Here, we studied Prx2(-/-) mouse red cells. The absence of Prx2 promotes (i) activation of the oxidative-induced Syk pathway; (ii) increased band 3 Tyr phosphorylation, with clustered band 3; and (iii) increased heat shock protein (HSP27 and HSP70) membrane translocation. This was associated with enhanced in vitro erythrophagocytosis of Prx2(-/-) red cells and reduced Prx2(-/-) red cell survival, indicating the possible role of Prx2 membrane recruitment in red cell aging and in the clearance of oxidized hemoglobin and damaged proteins through microparticles. Indeed, we observed an increased release of microparticles from Prx2(-/-) mouse red cells. The mass spectrometric analysis of erythroid microparticles found hemoglobin chains, membrane proteins, and HSPs. To test these findings, we treated Prx2(-/-) mice with antioxidants in vivo. We observed that N-acetylcysteine reduced (i) Syk activation, (ii) band 3 clusterization, (iii) HSP27 membrane association, and (iv) erythroid microparticle release, resulting in increased Prx2(-/-) mouse red cell survival. Thus, we propose that Prx2 may play a cytoprotective role in red cell membrane protein homeostasis and senescence.

  19. Red cell or serum folate: what to do in clinical practice?

    PubMed

    Farrell, Christopher-John L; Kirsch, Susanne H; Herrmann, Markus

    2013-03-01

    Folate deficiency has been linked to diverse clinical manifestations and despite the importance of accurate assessment of folate status, the best test for routine use is uncertain. Both serum and red cell folate assays are widely available in clinical laboratories; however, red cell folate is the more time-consuming and costly test. This review sought to evaluate whether the red cell assay demonstrated superior performance characteristics to justify these disadvantages. Red cell folate, but not serum folate, measurements demonstrated analytical variation due to sample pre-treatment parameters, oxygen saturation of haemoglobin and haematocrit. Neither marker was clearly superior in characterising deficiency but serum folate more frequently showed the higher correlation with homocysteine, a sensitive marker of deficiency. Similarly, both serum and red cell folate were shown to increase in response to folic acid supplementation. However, serum folate generally gave the greater response and was able to distinguish different supplementation doses. The C677T polymorphism of methylenetetrahydrofolate reductase alters the distribution of folate forms in red cells and may thereby cause further analytical variability in routine red cell folate assays. Overall, serum folate is cheaper and faster to perform than red cell folate, is influenced by fewer analytical variables and provides an assessment of folate status that may be superior to red cell folate.

  20. Siloxane nanoprobes for labeling and dual modality imaging of neural stem cells

    PubMed Central

    Addington, Caroline P.; Cusick, Alex; Shankar, Rohini Vidya; Agarwal, Shubhangi; Stabenfeldt, Sarah E.; Kodibagkar, Vikram D.

    2015-01-01

    Cell therapy represents a promising therapeutic for a myriad of medical conditions, including cancer, traumatic brain injury, and cardiovascular disease among others. A thorough understanding of the efficacy and cellular dynamics of these therapies necessitates the ability to non-invasively track cells in vivo. Magnetic resonance imaging (MRI) provides a platform to track cells as a non-invasive modality with superior resolution and soft tissue contrast. We recently reported a new nanoprobe platform for cell labeling and imaging using fluorophore doped siloxane core nanoemulsions as dual modality (1H MRI/Fluorescence), dual-functional (oximetry/detection) nanoprobes. Here, we successfully demonstrate the labeling, dual-modality imaging, and oximetry of neural progenitor/stem cells (NPSCs) in vitro using this platform. Labeling at a concentration of 10 μl/104 cells with a 40%v/v polydimethylsiloxane core nanoemulsion, doped with rhodamine, had minimal effect on viability, no effect on migration, proliferation and differentiation of NPSCs and allowed for unambiguous visualization of labeled NPSCs by 1H MR and fluorescence and local pO2 reporting by labeled NPSCs. This new approach for cell labeling with a positive contrast 1H MR probe has the potential to improve mechanistic knowledge of current therapies, and guide the design of future cell therapies due to its clinical translatability. PMID:26597417

  1. The application of super paramagnetic iron oxide-labeled mesenchymal stem cells in cell-based therapy.

    PubMed

    Qi, Yiying; Feng, Gang; Huang, Zhongming; Yan, Weiqi

    2013-03-01

    Mesenchymal stem cell (MSC)-based therapy has great potential for tissue regeneration. However, being able to monitor the in vivo behavior of implanted MSCs and understand the fate of these cells is necessary for further development of successful therapies and requires an effective, non-invasive and non-toxic technique for cell tracking. Super paramagnetic iron oxide (SPIO) is an idea label and tracer of MSCs. MRI can be used to follow SPIO-labeled MSCs and has been proposed as a gold standard for monitoring the in vivo biodistribution and migration of implanted SPIO-labeled MSCs. This review discusses the biological effects of SPIO labeling on MSCs and the therapeutic applications of local or systemic delivery of these labeled cells.

  2. 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.

  3. Scattering pulse of label free fine structure cells to determine the size scale of scattering structures.

    PubMed

    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.

  4. Dynamics of Red Cells in Spleen: How Does Vesiculation Happen?

    NASA Astrophysics Data System (ADS)

    Zhu, Qiang; Salehyar, Sara; Cabrales, Pedro; Asaro, Robert

    2016-11-01

    Vesiculation of red blood cells as a result of local separation between lipid bilayer and cytoskeleton is known to happen in vivo, most likely inside spleen where they sustain large mechanical loads during the passage through venus slits. There is, however, little knowledge about the detailed scenario and condition. We address this question via a fluid-cell interaction model by coupling a multiscale model of the cell membrane (including molecular details) with a fluid dynamics model based on boundary-integral equations. A numerical flow channel is created where the cell is driven through a narrow slit by pressure (imitating the transit through venus slits in spleen). The concentration is the occurrence of large dissociation (negative) pressure between the skeleton/membrane connection that promotes separation, a precursor of vesicle formation. Critical levels for the negative pressure are estimated using published data. By following the maximum range of pressure, we conclude that for vesiculation to happen there must be biochemical influences (e.g. binding of degraded haemoglobin) that significantly reduce effective attachment density. This is consistent with reported trends in vesiculation that are believed to occur in cases of various hereditary anemias and during blood storage. Our findings also suggest the criticality of understanding the biochemical phenomena involved with cytoskeleton/membrane attachment.

  5. Axial dispersion in flowing red blood cell suspensions

    NASA Astrophysics Data System (ADS)

    Podgorski, Thomas; Losserand, Sylvain; Coupier, Gwennou

    2016-11-01

    A key parameter in blood microcirculation is the transit time of red blood cells (RBCs) through an organ, which can influence the efficiency of gas exchange and oxygen availability. A large dispersion of this transit time is observed in vivo and is partly due to the axial dispersion in the flowing suspension. In the classic Taylor-Aris example of a solute flowing in a tube, the combination of molecular diffusion and parabolic velocity profile leads to enhanced axial dispersion. In suspensions of non-Brownian deformable bodies such as RBCs, axial dispersion is governed by a combination of shear induced migration and shear-induced diffusion arising from hydrodynamic interactions. We revisit this problem in the case of RBC pulses flowing in a microchannel and show that the axial dispersion of the pulse eventually saturates with a final extension that depends directly on RBC mechanical properties. The result is especially interesting in the dilute limit since the final pulse length depends only on the channel width, exponent of the migration law and dimensionless migration velocity. In continuous flow, the dispersion of transit times is the result of complex cell-cell and cell-wall interactions and is strongy influenced by the polydispersity of the blood sample. The authors acknowledge support from LabEx TEC21 and CNES.

  6. 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.

  7. 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

  8. 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.

  9. 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.

  10. 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

  11. Vesicles, capsules and red blood cells under flow

    NASA Astrophysics Data System (ADS)

    Misbah, Chaouqi

    2012-12-01

    Blood flow is dictated by the dynamics of red blood cells (RBCs), which constitute by far the major component. RBCs are made of a a two dimensional fluid bilayer of phospholipids, having underneath a network of proteins conferring to them shear elasticity, and they possess many membrane and transmembrane proteins (like ion channels). Simplified systems, like vesicles (made of a pure bilayer of phospholipid) and capsules (made of an extensible polymer shell) are used as models for RBCs. Both systems reproduce several features known for RBCs under flow. Their interest lies, besides some simplicity, in the fact that they can be fabricated in the laboratory, and their properties (size, stiffness, internal content....) can be varied in a wide range allowing thus to explore a quite significant parameter space that is essential to test predictions and discriminate between different models. We shall review the main recent achievement in this field, both for a single entity, collective effects and the impact on rheology.

  12. 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.

  13. Stretching Behavior of Red Blood Cells at High Strain Rates

    NASA Astrophysics Data System (ADS)

    Mancuso, Jordan; Ristenpart, William

    2016-11-01

    Most work on the mechanical behavior of red blood cells (RBCs) has focused on simple shear flows. Relatively little work has examined RBC deformations in the physiologically important extensional flow that occurs at the entrance to a constriction. In particular, previous work suggests that RBCs rapidly stretch out and then retract upon entering the constriction, but to date no model predicts this behavior for the extremely high strain rates typically experienced there. In this work, we use high speed video to perform systematic measurements of the dynamic stretching behavior of RBCs as they enter a microfluidic constriction. We demonstrate that a simple viscoelastic model captures the observed stretching dynamics, up to strain rates as high as 1000 s-1. The results indicate that the effective elastic modulus of the RBC membrane at these strain rates is an order of magnitude larger than moduli measured by micropipette aspiration or other low strain rate techniques.

  14. 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.

  15. Simulation of red blood cell aggregation in shear flow.

    PubMed

    Lim, B; Bascom, P A; Cobbold, R S

    1997-01-01

    A simulation model has been developed for red blood cell (RBC) aggregation in shear flow. It is based on a description of the collision rates of RBC, the probability of particles sticking together, and the breakage of aggregates by shear forces. The influence of shear rate, hematocrit, aggregate fractal dimension, and binding strength on aggregation kinetics were investigated and compared to other theoretical and experimental results. The model was used to simulate blood flow in a long large diameter tube under steady flow conditions at low Reynolds numbers. The time and spatial distribution of the state of aggregation are shown to be in qualitative agreement with previous B-mode ultrasound studies in which a central region of low echogenicity was noted. It is suggested that the model can provide a basis for interpreting prior measurements of ultrasound echogenicity and may help relate them to the local state of aggregation.

  16. Duration of red blood cell storage and inflammatory marker generation

    PubMed Central

    Sut, Caroline; Tariket, Sofiane; Chou, Ming Li; Garraud, Olivier; Laradi, Sandrine; Hamzeh-Cognasse, Hind; Seghatchian, Jerard; Burnouf, Thierry; Cognasse, Fabrice

    2017-01-01

    Red blood cell (RBC) transfusion is a life-saving treatment for several pathologies. RBCs for transfusion are stored refrigerated in a preservative solution, which extends their shelf-life for up to 42 days. During storage, the RBCs endure abundant physicochemical changes, named RBC storage lesions, which affect the overall quality standard, the functional integrity and in vivo survival of the transfused RBCs. Some of the changes occurring in the early stages of the storage period (for approximately two weeks) are reversible but become irreversible later on as the storage is extended. In this review, we aim to decipher the duration of RBC storage and inflammatory marker generation. This phenomenon is included as one of the causes of transfusion-related immunomodulation (TRIM), an emerging concept developed to potentially elucidate numerous clinical observations that suggest that RBC transfusion is associated with increased inflammatory events or effects with clinical consequence. PMID:28263172

  17. Pressure and temperature effects on human red cell cation transport.

    PubMed

    Hall, A C; Ellory, J C; Klein, R A

    1982-01-01

    The effects of hydrostatic pressure and temperature on the three components of K+ uptake in human red cells have been investigated, using ouabain and bumetanide to distinguish between the pump, passive diffusion and cotransport. The pressure sensitivity for passive diffusion has been shown to depend on the counter-ion present. The order of this effect, Cl- greater than Br- greater than NO3- greater than I-, is the same as for the ionic partial molal volumes and the Hofmeister series. We have analyzed our experimental results thermodynamically, and propose a model for the activated transition-state complex of the potassium ion which involves the loss of water molecules from the secondary hydration shell, cosphere II.

  18. Autoimmune Hemolytic Anemia and Red Blood Cell Autoantibodies.

    PubMed

    Quist, Erin; Koepsell, Scott

    2015-11-01

    Autoimmune hemolytic anemia is a rare disorder caused by autoreactive red blood cell (RBC) antibodies that destroy RBCs. Although autoimmune hemolytic anemia is rare, RBC autoantibodies are encountered frequently and can complicate transfusion workups, impede RBC alloantibody identification, delay distribution of compatible units, have variable clinical significance that ranges from benign to life-threatening, and may signal an underlying disease or disorder. In this review, we discuss the common presenting features of RBC autoantibodies, laboratory findings, ancillary studies that help the pathologist investigate the clinical significance of autoantibodies, and how to provide appropriate patient care and consultation for clinical colleagues. Pathologists must be mindful of, and knowledgeable about, this entity because it not only allows for direct clinical management but also can afford an opportunity to preemptively treat an otherwise silent malignancy or disorder.

  19. Pure red cell aplasia following autoimmune hemolytic anemia: an enigma.

    PubMed

    Saha, M; Ray, S; Kundu, S; Chakrabarti, P

    2013-01-01

    A 26-year-old previously healthy female presented with a 6-month history of anemia. The laboratory findings revealed hemolytic anemia and direct antiglobulin test was positive. With a diagnosis of autoimmune hemolytic anemia (AIHA), prednisolone was started but was ineffective after 1 month of therapy. A bone marrow trephine biopsy revealed pure red cell aplasia (PRCA) showing severe erythroid hypoplasia. The case was considered PRCA following AIHA. This combination without clear underlying disease is rare. Human parvovirus B19 infection was not detected in the marrow aspirate during reticulocytopenia. The patient received azathioprine, and PRCA improved but significant hemolysis was once again documented with a high reticulocyte count. The short time interval between AIHA and PRCA phase suggested an increased possibility of the evolution of a single disease.

  20. P2X and P2Y receptor signaling in red blood cells.

    PubMed

    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.

  1. Label-free isolation of circulating tumor cells in microfluidic devices: Current research and perspectives

    PubMed Central

    Cima, Igor; Wen Yee, Chay; Iliescu, Florina S.; Min Phyo, Wai; Hon Lim, Kiat; Iliescu, Ciprian; Han Tan, Min

    2013-01-01

    This review will cover the recent advances in label-free approaches to isolate and manipulate circulating tumor cells (CTCs). In essence, label-free approaches do not rely on antibodies or biological markers for labeling the cells of interest, but enrich them using the differential physical properties intrinsic to cancer and blood cells. We will discuss technologies that isolate cells based on their biomechanical and electrical properties. Label-free approaches to analyze CTCs have been recently invoked as a valid alternative to “marker-based” techniques, because classical epithelial and tumor markers are lost on some CTC populations and there is no comprehensive phenotypic definition for CTCs. We will highlight the advantages and drawbacks of these technologies and the status on their implementation in the clinics. PMID:24403992

  2. Importance of Mean Red Cell Distribution Width in Hypertensive Patients

    PubMed Central

    Bilal, Ahmed; Farooq, Junaid H; Assad, Salman; Ghazanfar, Haider; Ahmed, Imran

    2016-01-01

    Purpose Red cell distribution width (RDW), expressed in femtoliters (fl), is a measure of the variation in the size of circulating erythrocytes and is often expressed as a direct measurement of the width of the distribution. We aim to observe the mean value of red cell distribution width (RDW) in hypertensive patients. Increased RDW can be used as a tool for early diagnosis, as an inflammatory marker, and a mortality indicator in hypertensive patients due to its close relation to inflammation. Materials and methodology Hypertensive patients who had the condition for more than one year duration, diagnosed according to the Joint National Committee (JNC 7) criteria were subjected to complete blood count and RDW measurement. One hundred patients, aged between 12 years and 65 years were enrolled from the outpatient department of medicine at the Military Hospital Rawalpindi. Results The mean age (± SD) of the patients was 51.48 ± 10.08 years. Out of 100 patients 69% were males whereas 31% were females. The overall frequency of hypertension more than five years was 55% subjects whereas 45% individuals had duration of hypertension less than five years. Mean RDW in females was found to be 49.35±8.42 fl while mean RDW in males was 44.78±7.11 fl. An independent sample t-test was applied to assess if there was any significant difference between age and gender. No significant difference between age and gender was found (p<0.05). The Mann-Whitney test was used to assess any association of RDW with gender. RDW values in females was found to be statistically significantly higher than in males (U=603, p=0.01). Linear regression showed that mean RDW value increased with increasing age (P <0.001). Conclusions A significant number of patients with hypertension have increased levels of RDW. Therefore, it is recommended that serum RDW should be checked regularly in patients with hypertension. PMID:28070471

  3. Platelet and red blood cell indices in Harris platelet syndrome.

    PubMed

    Naina, Harris V K; Harris, Samar

    2010-01-01

    Inherited thrombocytopenias, including inherited giant platelet disorders (IGPD) or macro thrombocytopenias are relatively rare, but their prevalence is likely underestimated from complexities of diagnosis and a spectrum of subclinical phenotypes. Harris platelet syndrome (HPS) is the most common IGPD reported from the Indian subcontinent. Of note there are an increased number of hemoglobinopathies reported from the geographic location. We analysed red blood cell and platelet indices of blood donors with HPS from the north eastern part of India and compared them with blood indices of blood donors of south India. We found a statistically significant lower platelet count in blood donors with HPS (median, range) 132 (71-267) vs. 252 (160-478) as compared to donors from south India (P < 0.001). Mean platelet volume (MPV) was higher in donors with HPS 13.1, (range 12-21.9 fl) as compared to donors from south India 7.35 (range 6-9.2 fl) (P < 0.001). This study showed that blood donors with HPS had a low median platelet bio-mass 0.17 (0.10-0.38%) vs. 0.19 (0.13-0.28%) in donors from south India. The platelet distribution width (PDW) was 17.4 (14.9-19.6) in donors with HPS vs. 16.38 (15.2-18.5) in south Indian blood donors (P < 0.001). Thirty-three donors with HPS had a normal platelet count with MPV more than 12 fL. Only donors with HPS had giant platelets and thrombocytopenia on peripheral blood smear examination. None of these donors had Dohle body inclusion in their leukocytes. Compared to donors from south India, donors with HPS had a significantly lower hemoglobin 13.8 (12-16.3 gm/dL) vs. 14.8 (12-18) respectively (P < 0.001) while red distribution width (RDW) was higher in HPS 13.6 (11.5-16.7) vs. 12.8 (11.4-15.1). However we did not find any statistically significant difference in MCV, MCH, MCHC between the two groups. Peripheral blood smear did not show any obvious abnormal red blood cell morphology. In the blood donors with HPS we found a statistically higher MPV

  4. 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.

  5. The influence of nanodiamond on the oxygenation states and micro rheological properties of human red blood cells in vitro.

    PubMed

    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.

  6. Clinical utility of flow cytometry in the study of erythropoiesis and nonclonal red cell disorders.

    PubMed

    Chesney, Alden; Good, David; Reis, Marciano

    2011-01-01

    Erythropoiesis involves proliferation and differentiation of small population of hematopoietic stem cells resident in the bone marrow into mature red blood cells. The determination of the cellular composition of the blood is a valuable tool in the diagnosis of diseases and monitoring of therapy. Flow cytometric analysis is increasingly being used to characterize the heterogeneous cell populations present in the blood and the hematopoietic cell differentiation and maturation pathways of the bone marrow. Here we discuss the role of flow cytometry in the study of erythropoiesis and nonclonal red blood cell disorders. First, we discuss flow cytometric analysis of reticulocytes. Next, we review salient quantitative methods that can be used for detection of fetal-maternal hemorrhage (FMH). We also discuss flow cytometric analysis of high hemoglobin F (HbF) in Sickle Cell Disease (SCD), hereditary spherocytosis (HS), red cell survival and red cell volume. We conclude by discussing cell cycle of erythroid cells.

  7. High-resolution X-ray imaging of Plasmodium falciparum-infected red blood cells.

    PubMed

    Williams, Garth J; Hanssen, Eric; Peele, Andrew G; Pfeifer, Mark A; Clark, Jesse; Abbey, Brian; Cadenazzi, Guido; de Jonge, Martin D; Vogt, Stefan; Tilley, Leann; Nugent, Keith A

    2008-10-01

    Methods for imaging cellular architecture and ultimately macromolecular complexes and individual proteins, within a cellular environment, are an important goal for cell and molecular biology. Coherent diffractive imaging (CDI) is a method of lensless imaging that can be applied to any individual finite object. A diffraction pattern from a single biological structure is recorded and an iterative Fourier transform between real space and reciprocal space is used to reconstruct information about the architecture of the sample to high resolution. As a test system for cellular imaging, we have applied CDI to an important human pathogen, the malaria parasite, Plasmodium falciparum. We have employed a novel CDI approach, known as Fresnel CDI, which uses illumination with a curved incident wavefront, to image red blood cells infected with malaria parasites. We have examined the intrinsic X-ray absorption contrast of these cells and compared them with cells contrasted with heavy metal stains or immunogold labeling. We compare CDI images with data obtained from the same cells using scanning electron microscopy, light microscopy, and scanning X-ray fluorescence microscopy. We show that CDI can offer new information both within and at the surface of complex biological specimens at a spatial resolution of better than 40 nm. and we demonstrate an imaging modality that conveniently combines scanning X-ray fluorescence microscopy with CDI. The data provide independent confirmation of the validity of the coherent diffractive image and demonstrate that CDI offers the potential to become an important and reliable new high-resolution imaging modality for cell biology. CDI can detect features at high resolution within unsectioned cells.

  8. 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.

  9. 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.

  10. Red blood cell as an adaptive optofluidic microlens.

    PubMed

    Miccio, L; Memmolo, P; Merola, F; Netti, P A; Ferraro, P

    2015-03-11

    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.

  11. Fibrinogen and red blood cells in venous thrombosis.

    PubMed

    Aleman, Maria M; Walton, Bethany L; Byrnes, James R; Wolberg, Alisa S

    2014-05-01

    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.

  12. Pure red cell aplasia and lymphoproliferative disorders: an infrequent association.

    PubMed

    Vlachaki, Efthymia; Diamantidis, Michael D; Klonizakis, Philippos; Haralambidou-Vranitsa, Styliani; Ioannidou-Papagiannaki, Elizabeth; Klonizakis, Ioannis

    2012-01-01

    Pure red cell aplasia (PRCA) is a rare bone marrow failure syndrome defined by a progressive normocytic anaemia and reticulocytopenia without leukocytopenia and thrombocytopenia. Secondary PRCA can be associated with various haematological disorders, such as chronic lymphocytic leukaemia (CLL) or non-Hodgkin lymphoma (NHL). The aim of the present review is to investigate the infrequent association between PRCA and lymphoproliferative disorders. PRCA might precede the appearance of lymphoma, may present simultaneously with the lymphoid neoplastic disease, or might appear following the lymphomatic disorder. Possible pathophysiological molecular mechanisms to explain the rare association between PRCA and lymphoproliferative disorders are reported. Most cases of PRCA are presumed to be autoimmune mediated by antibodies against either erythroblasts or erythropoietin, by T-cells secreting factors selectively inhibiting erythroid colonies in the bone marrow or by NK cells directly lysing erythroblasts. Finally, focus is given to the therapeutical approach, as several treatment regimens have failed for PRCA. Immunosuppressive therapy and/or chemotherapy are effective for improving anaemia in the majority of patients with lymphoma-associated PRCA. Further investigation is required to define the pathophysiology of PRCA at a molecular level and to provide convincing evidence why it might appear as a rare complication of lymphoproliferative disorders.

  13. Interactions of chrysotile and crocidolite asbestos with red blood cell membranes. Chrysotile binds to sialic acid.

    PubMed

    Brody, A R; George, G; Hill, L H

    1983-10-01

    Chrysotile and crocidolite are commonly used forms of asbestos. Hemolysis has been widely used as a test of membrane injury, and it has been shown previously that chrysotile causes rapid breakdown of red blood cells (RBCs), whereas crocidolite is only weakly hemolytic. A reasonable hypothesis set forth to explain the cytotoxic effects of chrysotile maintains that positively charged chrysotile fibers bind to negatively charged sialic acid residues on RBC membranes causing clustering of membrane proteins and increased cell permeability to Na and K ions. Our studies presented here provide two lines of evidence in direct support of this hypothesis. (a) Morphologic--Ultrastructural techniques showed that both chrysotile and crocidolite asbestos bind to and distort more than 85% of RBCs treated for 15 minutes. The distorting effects of chrysotile, but not crocidolite, were almost totally ablated by pretreating the cells with neuraminidase. In addition, gold-conjugated wheat germ agglutinin was used to label the distribution of sialic acid groups on RBC membranes. Pretreatment of the RBCs with chrysotile, but not crocidolite, reduced the number of gold-conjugated wheat germ agglutinin-labeled sites to less than 30% of the control level. (b) Biochemical--The thiobarbituric acid assay was used to determine the percentage of sialic acid that remained with the cell pellet after neuraminidase and/or asbestos treatment. Asbestos treatment alone caused no release of sialic acid from the cells. Neuraminidase treatment for 3.5 hours removed more than 80% of the sialic acid from cell surfaces. Chrysotile, but not crocidolite, asbestos prevented neuraminidase-mediated removal of sialic acid from RBCs. In addition, x-ray energy spectrometry of freeze-dried cells showed that RBCs distorted by chrysotile, but not by crocidolite, exhibited significant alterations in intracellular Na:K ratios. The morphologic and biochemical data strongly support the hypothesis that chrysotile asbestos

  14. Endowing carbon nanotubes with superparamagnetic properties: applications for cell labeling, MRI cell tracking and magnetic manipulations.

    PubMed

    Lamanna, Giuseppe; Garofalo, Antonio; Popa, Gabriela; Wilhelm, Claire; Bégin-Colin, Sylvie; Felder-Flesch, Delphine; Bianco, Alberto; Gazeau, Florence; Ménard-Moyon, Cécilia

    2013-05-21

    Coating of carbon nanotubes (CNTs) with magnetic nanoparticles (NPs) imparts novel magnetic, optical, and thermal properties with potential applications in the biomedical domain. Multi-walled CNTs have been decorated with iron oxide superparamagnetic NPs. Two different approaches have been investigated based on ligand exchange or "click chemistry". The presence of the NPs on the nanotube surface allows conferring magnetic properties to CNTs. We have evaluated the potential of the NP/CNT hybrids as a contrast agent for magnetic resonance imaging (MRI) and their interactions with cells. The capacity of the hybrids to magnetically monitor and manipulate cells has also been investigated. The NP/CNTs can be manipulated by a remote magnetic field with enhanced contrast in MRI. They are internalized into tumor cells without showing cytotoxicity. The labeled cells can be magnetically manipulated as they display magnetic mobility and are detected at a single cell level through high resolution MRI.

  15. Variations in labeling protocol influence incorporation, distribution and retention of iron oxide nanoparticles into human umbilical vein endothelial cells.

    PubMed

    van Tiel, Sandra T; Wielopolski, Piotr A; Houston, Gavin C; Krestin, Gabriel P; Bernsen, Monique R

    2010-01-01

    Various studies have shown that various cell types can be labeled with iron oxide particles and visualized by magnetic resonance imaging (MRI). However, reported protocols for cell labeling show a large variation in terms of labeling dose and incubation time. It is therefore not clear how different labeling protocols may influence labeling efficiency. Systematic assessment of the effects of various labeling protocols on labeling efficiency of human umbilical vein endothelial cells (HUVEC) using two different types of iron oxide nanoparticles, i.e. super paramagnetic iron oxide particles (SPIOs) and microparticles of iron oxide (MPIOs), demonstrated that probe concentration, incubation time and particle characteristics all influence the efficiency of label incorporation, label distribution, label retention and cell behavior. For SPIO the optimal labeling protocol consisted of a dose of 12.5 µg iron/2 ml/9.5 cm(2) and an incubation time of 24 h, resulting in an average iron load of 12.0 pg iron/per cell (uptake efficiency of 9.6%). At 4 h many SPIOs are seen sticking to the outside of the cell instead of being taken up by the cell. For MPIO optimal labeling was obtained with a dose of 50 µg iron/2 ml/9.5 cm(2). Incubation time was of less importance since most of the particles were already incorporated within 4 h with a 100% labeling efficiency, resulting in an intracellular iron load of 626 pg/cell. MPIO were taken up more efficiently than SPIO and were also better tolerated. HUVEC could be exposed to and contain higher amounts of iron without causing significant cell death, even though MPIO had a much more pronounced effect on cell appearance. Using optimal labeling conditions as found for HUVEC on other cell lines, we observed that different cell types react differently to identical labeling conditions. Consequently, for each cell type separately an optimal protocol has to be established.

  16. 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.

  17. 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

    2016-03-02

    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.

  18. Integrating cell phone imaging with magnetic levitation (i-LEV) for label-free blood analysis at the point-of-living

    PubMed Central

    Durmus, Naside Gozde; Davis, Ronald W.; Steinmetz, Lars M.; Demirci, Utkan

    2016-01-01

    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, we present a magnetic levitation-based diagnosis system 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, we introduce an easy-to-use, smartphone incorporated levitation system for cell analysis. Using our portable imaging magnetic levitation (i-LEV) system, we show 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

  19. 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.

  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. Supernova: A Versatile Vector System for Single-Cell Labeling and Gene Function Studies in vivo

    PubMed Central

    Luo, Wenshu; Mizuno, Hidenobu; Iwata, Ryohei; Nakazawa, Shingo; Yasuda, Kosuke; Itohara, Shigeyoshi; Iwasato, Takuji

    2016-01-01

    Here we describe “Supernova” series of vector systems that enable single-cell labeling and labeled cell-specific gene manipulation, when introduced by in utero electroporation (IUE) or adeno-associated virus (AAV)-mediated gene delivery. In Supernova, sparse labeling relies on low TRE leakage. In a small population of cells with over-threshold leakage, initial tTA-independent weak expression is enhanced by tTA/TRE-positive feedback along with a site-specific recombination system (e.g., Cre/loxP, Flpe/FRT). Sparse and bright labeling by Supernova with little background enables the visualization of the morphological details of individual neurons in densely packed brain areas such as the cortex and hippocampus, both during development and in adulthood. Sparseness levels are adjustable. Labeled cell-specific gene knockout was accomplished by introducing Cre/loxP-based Supernova vectors into floxed mice. Furthermore, by combining with RNAi, TALEN, and CRISPR/Cas9 technologies, IUE-based Supernova achieved labeled cell-specific gene knockdown and editing/knockout without requiring genetically altered mice. Thus, Supernova system is highly extensible and widely applicable for single-cell analyses in complex organs, such as the mammalian brain. PMID:27775045

  2. Supernova: A Versatile Vector System for Single-Cell Labeling and Gene Function Studies in vivo.

    PubMed

    Luo, Wenshu; Mizuno, Hidenobu; Iwata, Ryohei; Nakazawa, Shingo; Yasuda, Kosuke; Itohara, Shigeyoshi; Iwasato, Takuji

    2016-10-24

    Here we describe "Supernova" series of vector systems that enable single-cell labeling and labeled cell-specific gene manipulation, when introduced by in utero electroporation (IUE) or adeno-associated virus (AAV)-mediated gene delivery. In Supernova, sparse labeling relies on low TRE leakage. In a small population of cells with over-threshold leakage, initial tTA-independent weak expression is enhanced by tTA/TRE-positive feedback along with a site-specific recombination system (e.g., Cre/loxP, Flpe/FRT). Sparse and bright labeling by Supernova with little background enables the visualization of the morphological details of individual neurons in densely packed brain areas such as the cortex and hippocampus, both during development and in adulthood. Sparseness levels are adjustable. Labeled cell-specific gene knockout was accomplished by introducing Cre/loxP-based Supernova vectors into floxed mice. Furthermore, by combining with RNAi, TALEN, and CRISPR/Cas9 technologies, IUE-based Supernova achieved labeled cell-specific gene knockdown and editing/knockout without requiring genetically altered mice. Thus, Supernova system is highly extensible and widely applicable for single-cell analyses in complex organs, such as the mammalian brain.

  3. 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

    2016-05-06

    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.

  4. Endowing carbon nanotubes with superparamagnetic properties: applications for cell labeling, MRI cell tracking and magnetic manipulations

    NASA Astrophysics Data System (ADS)

    Lamanna, Giuseppe; Garofalo, Antonio; Popa, Gabriela; Wilhelm, Claire; Bégin-Colin, Sylvie; Felder-Flesch, Delphine; Bianco, Alberto; Gazeau, Florence; Ménard-Moyon, Cécilia

    2013-05-01

    Coating of carbon nanotubes (CNTs) with magnetic nanoparticles (NPs) imparts novel magnetic, optical, and thermal properties with potential applications in the biomedical domain. Multi-walled CNTs have been decorated with iron oxide superparamagnetic NPs. Two different approaches have been investigated based on ligand exchange or ``click chemistry''. The presence of the NPs on the nanotube surface allows conferring magnetic properties to CNTs. We have evaluated the potential of the NP/CNT hybrids as a contrast agent for magnetic resonance imaging (MRI) and their interactions with cells. The capacity of the hybrids to magnetically monitor and manipulate cells has also been investigated. The NP/CNTs can be manipulated by a remote magnetic field with enhanced contrast in MRI. They are internalized into tumor cells without showing cytotoxicity. The labeled cells can be magnetically manipulated as they display magnetic mobility and are detected at a single cell level through high resolution MRI.Coating of carbon nanotubes (CNTs) with magnetic nanoparticles (NPs) imparts novel magnetic, optical, and thermal properties with potential applications in the biomedical domain. Multi-walled CNTs have been decorated with iron oxide superparamagnetic NPs. Two different approaches have been investigated based on ligand exchange or ``click chemistry''. The presence of the NPs on the nanotube surface allows conferring magnetic properties to CNTs. We have evaluated the potential of the NP/CNT hybrids as a contrast agent for magnetic resonance imaging (MRI) and their interactions with cells. The capacity of the hybrids to magnetically monitor and manipulate cells has also been investigated. The NP/CNTs can be manipulated by a remote magnetic field with enhanced contrast in MRI. They are internalized into tumor cells without showing cytotoxicity. The labeled cells can be magnetically manipulated as they display magnetic mobility and are detected at a single cell level through high

  5. Extensive labeling with [3H]ethanolamine of a hydrophilic protein of animal cells.

    PubMed

    Tisdale, E J; Tartakoff, A M

    1988-06-15

    Murine T-lymphomas and Thy-1- mutants were labeled overnight with [3H]ethanolamine to detect proteins which possess a glycophospholipid anchor. When labeled cells were treated with 10% trichloroacetic acid and then analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography, both Thy-1 and a second intensely labeled protein (46 kDa) were observed. The presence of the radiolabeled 46-kDa protein in wild type and class E Thy-1 negative cells (cells in which Thy-1 is synthesized but cannot be labeled with [3H]ethanolamine) suggested incorporation into a distinct moiety. Labeling of the 46-kDa protein with [3H]ethanolamine is rapidly inhibited by cycloheximide. Further characterization of the 46-kDa protein by subcellular fractionation and Triton X-114 partitioning indicated that the protein is located in the cytosol. The protein is basic and does not bind to either concanavalin A or wheat germ agglutinin. Labeling of a 46-kDa protein has also been demonstrated in Chinese hamster ovary, COS, rat myeloma, cloned human T-lymphocytes, and HeLa cells. Pronase digestion of the [3H]ethanolamine-labeled 46-kDa protein of wild type lymphoma cells generated a nonbasic and polar labeled fragment which is labile to strong acid and base ([3H]ethanolamine is liberated), insensitive to periodate oxidation and alkaline phosphatase, and does not bind to concanavalin A or wheat germ agglutinin. Judging from methylation studies, the labeled ethanolamine residue does not contain a free amino group. Based on these results, we report a novel post-translational modification of selected protein(s) by the covalent addition of [3H]ethanolamine.

  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. 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

  8. Chronic kidney disease predicts impaired membrane microviscosity of red blood cells in hypertensive and normotensive subjects.

    PubMed

    Tsuda, Kazushi

    2013-01-01

    Current evidence indicates that abnormalities in physical properties of the cell membranes may be strongly linked to hypertension and other circulatory disorders. Recent studies have shown that chronic kidney disease (CKD) might be a risk factor for cardiovascular and cerebrovascular outcomes. The purpose of the present study was to examine the possible relationship between kidney function and membrane fluidity (a reciprocal value of membrane microviscosity) of red blood cells (RBCs) in hypertensive and normotensive subjects using an electron spin resonance (ESR) and spin-labeling method. The order parameter (S) for the ESR spin-label agent (5-nitroxide stearate) in RBC membranes was significantly higher in hypertensive subjects than in normotensive subjects, indicating that membrane fluidity was decreased in hypertension. The order parameter (S) of RBCs was inversely correlated with estimated glomerular filtration rate (eGFR), suggesting that a decreased eGFR value might be associated with reduced membrane fluidity of RBCs. Multivariate regression analysis also demonstrated that, after adjustment for general risk factors, eGFR might be a significant predictor of membrane fluidity of RBCs. The reduced levels of both membrane fluidity of RBCs and eGFR were associated with increased plasma 8-iso-prostaglandin F2α (an index of oxidative stress) and decreased plasma nitric oxide (NO)-metabolites, suggesting that kidney function could be a determinant of membrane microviscosity of RBCs, at least in part, via oxidative stress- and NO-dependent mechanisms. The ESR study suggests that CKD might have a close correlation with impaired rheologic behavior of RBCs and microcirculatory disorders in hypertensive subjects.

  9. Detection of microparticles from human red blood cells by multiparametric flow cytometry

    PubMed Central

    Grisendi, Giulia; Finetti, Elena; Manganaro, Daniele; Cordova, Nicoletta; Montagnani, Giuliano; Spano, Carlotta; Prapa, Malvina; Guarneri, Valentina; Otsuru, Satoru; Horwitz, Edwin M.; Mari, Giorgio; Dominici, Massimo

    2015-01-01

    Background During storage, red blood cells (RBC) undergo chemical and biochemical changes referred to as “storage lesions”. These events determine the loss of RBC integrity, resulting in lysis and release of microparticles. There is growing evidence of the clinical importance of microparticles and their role in blood transfusion-related side effects and pathogen transmission. Flow cytometry is currently one of the most common techniques used to quantify and characterise microparticles. Here we propose multiparametric staining to monitor and quantify the dynamic release of microparticles by stored human RBC. Material and methods RBC units (n=10) were stored under blood bank conditions for up to 42 days. Samples were tested at different time points to detect microparticles and determine the haemolysis rate (HR%). Microparticles were identified by flow cytometry combining carboxyfluorescein diacetate succinimidyl ester (CFSE) dye, annexin V and anti-glycophorin A antibody. Results We demonstrated that CFSE can be successfully used to label closed vesicles with an intact membrane. The combination of CFSE and glycophorin A antibody was effective for monitoring and quantifying the dynamic release of microparticles from RBC during storage. Double staining with CFSE/glycophorin A was a more precise approach, increasing vesicle detection up to 4.7-fold vs the use of glycophorin A/annexin V alone. Moreover, at all the time points tested, we found a robust correlation (R=0.625; p=0.0001) between HR% and number of microparticles detected. Discussion Multiparametric staining, based on a combination of CFSE, glycophorin A antibody and annexin V, was able to detect, characterise and monitor the release of microparticles from RBC units during storage, providing a sensitive approach to labelling and identifying microparticles for transfusion medicine and, more broadly, for cell-based therapies. PMID:25369588

  10. Red blood cells and thrombin generation in sickle cell disease.

    PubMed

    Whelihan, Matthew F; Lim, Ming Y; Key, Nigel S

    2014-05-01

    The prothrombotic nature of sickle cell disease (SCD) is evidenced by the chronically elevated levels of almost all coagulation activation biomarkers, and an increased incidence of certain thrombotic events, including venous thromboembolism. Numerous studies have attempted to define the extent and elucidate the mechanism of the observed increase in thrombin generation in SCD patients in vivo. In general, these studies were performed using thrombin generation assays in platelet poor or platelet rich plasma and showed little difference in endogenous thrombin potential between the SCD cohort and healthy matched controls. In SCD, erythrocytes and monocytes have been demonstrated to exhibit procoagulant characteristics. Thus, the absence of these cellular components in standard thrombin generation assays may fail to reflect global hypercoagulability in the whole blood of patients with SCD. We were therefore surprised to see no difference in net thrombin generation in tissue factor-initiated initiated clotting of whole blood from patients with SCD. However, we are continuing to reconcile these seemingly disparate observations by slight modifications of the whole blood model that include alternative coagulation triggers and a re-examination of the net thrombin generation when the protein/protein S system is simultaneously interrogated.

  11. 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

  12. Magnetic Resonance Imaging of Iron Oxide-Labeled Human Embryonic Stem Cell-Derived Cardiac Progenitors

    PubMed Central

    Skelton, Rhys J.P.; Khoja, Suhail; Almeida, Shone; Rapacchi, Stanislas; Han, Fei; Engel, James; Zhao, Peng; Hu, Peng; Stanley, Edouard G.; Elefanty, Andrew G.; Kwon, Murray

    2016-01-01

    Given the limited regenerative capacity of the heart, cellular therapy with stem cell-derived cardiac cells could be a potential treatment for patients with heart disease. However, reliable imaging techniques to longitudinally assess engraftment of the transplanted cells are scant. To address this issue, we used ferumoxytol as a labeling agent of human embryonic stem cell-derived cardiac progenitor cells (hESC-CPCs) to facilitate tracking by magnetic resonance imaging (MRI) in a large animal model. Differentiating hESCs were exposed to ferumoxytol at different time points and varying concentrations. We determined that treatment with ferumoxytol at 300 μg/ml on day 0 of cardiac differentiation offered adequate cell viability and signal intensity for MRI detection without compromising further differentiation into definitive cardiac lineages. Labeled hESC-CPCs were transplanted by open surgical methods into the left ventricular free wall of uninjured pig hearts and imaged both ex vivo and in vivo. Comprehensive T2*-weighted images were obtained immediately after transplantation and 40 days later before termination. The localization and dispersion of labeled cells could be effectively imaged and tracked at days 0 and 40 by MRI. Thus, under the described conditions, ferumoxytol can be used as a long-term, differentiation-neutral cell-labeling agent to track transplanted hESC-CPCs in vivo using MRI. Significance The development of a safe and reproducible in vivo imaging technique to track the fate of transplanted human embryonic stem cell-derived cardiac progenitor cells (hESC-CPCs) is a necessary step to clinical translation. An iron oxide nanoparticle (ferumoxytol)-based approach was used for cell labeling and subsequent in vivo magnetic resonance imaging monitoring of hESC-CPCs transplanted into uninjured pig hearts. The present results demonstrate the use of ferumoxytol labeling and imaging techniques in tracking the location and dispersion of cell grafts

  13. 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.

  14. 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...

  15. 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.

  16. A Teenage Girl with Acute Dyspnea and Hypoxemia during Red Blood Cell Transfusion

    PubMed Central

    Tanpowpong, P.; Thongpo, P.

    2016-01-01

    Transfusion-related acute lung injury (TRALI) can cause morbidity and mortality. We present the case of teenager who developed dyspnea and hypoxemia few hours after red cell transfusion. After being admitted for close monitoring and oxygen therapy, her symptoms spontaneously resolved. Message: dyspnea during red cell transfusion should raise the suspicion of TRALI. PMID:27891282

  17. Cryopreserved packed red blood cells in surgical patients: past, present, and future.

    PubMed

    Chang, Alex; Kim, Young; Hoehn, Richard; Jernigan, Peter; Pritts, Timothy

    2016-09-08

    Since the advent of anticoagulation and component storage of human blood products, allogeneic red blood cell transfusion has been one of the most common practices in modern medicine. Efforts to reduce the biochemical effects of storage, collectively known as the red blood cell storage lesion, and prolong the storage duration have led to numerous advancements in erythrocyte storage solutions. Cryopreservation and frozen storage of red blood cells in glycerol have been successfully utilised by many civilian and military institutions worldwide. Through progressive improvements in liquid storage of erythrocytes in novel storage solutions, the logistical need for cryopreserved red blood cells in the civilian setting has diminished. A growing body of current literature is focused on the clinical consequences of packed red blood cell age. Modern cryopreservation techniques show promise as a cost-effective method to ameliorate the negative effect of the red blood cell storage lesion, while meeting the technical and logistical needs of both civilian and military medicine. This review outlines the history of red blood cell cryopreservation, the clinical impact of red cell storage, and highlights the current literature on frozen blood and its impact on modern transfusion.

  18. Nucleated red blood cell count in term and preterm newborns: reference values at birth.

    PubMed

    Perrone, S; Vezzosi, P; Longini, M; Marzocchi, B; Tanganelli, D; Testa, M; Santilli, T; Buonocore, G

    2005-03-01

    The prognostic value of nucleated red blood cell count at birth in relation to neonatal outcome has been established. However, reference values were needed to usefully interpret this variable. The normal range of reference values for absolute nucleated red blood cell count in 695 preterm and term newborns is reported.

  19. Fate of 3H-thymidine labelled myogenic cells in regeneration of muscle isografts.

    PubMed

    Gutmann, E; Mares, V; Stichová, J

    1976-03-05

    Intact and denervated extensor digitorum longus (EDL) muscles of 20-day-old inbred Lewis-Wistar rats were labelled with 3H-thymidine. Ninety minutes after the injection of the isotope 4.0% of the nuclei were labelled in the intact (i.e. innervated) and 9.6% in the muscles, denervated 3 days before administration of the isotope. The labelled EDL muscles were grafted into the bed of the previously removed EDL muscles of inbred animals and these isografts were studied 30 days later. In the EDL muscles, regenerated from innervated isografts only occasionally labelled endothelial cells were found whereas in the muscles regenerated from denervated isografts also parenchymal muscle nuclei were regularly labelled. The incidence of labelled nuclei in the regenerated EDL muscles was, however, about 20 times lower than in the donor EDL muscles. The presen experiments provide a direct proof of utilization of donor satelite cell nuclei for regeneration in grafted muscle tissue. With respect to the low incidence of labelled nuclei in regenerated EDL muscles, other sources of cells apparently also contribute to the regeneration process.

  20. 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.

  1. Targeted erythropoietin selectively stimulates red blood cell expansion in vivo

    PubMed Central

    Burrill, Devin R.; Vernet, Andyna; Collins, James J.; Silver, Pamela A.; Way, Jeffrey C.

    2016-01-01

    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

  2. Abcg2-Labeled Cells Contribute to Different Cell Populations in the Embryonic and Adult Heart.

    PubMed

    Doyle, Michelle J; Maher, Travis J; Li, Qinglu; Garry, Mary G; Sorrentino, Brian P; Martin, Cindy M

    2016-02-01

    ATP-binding cassette transporter subfamily G member 2 (Abcg2)-expressing cardiac-side population cells have been identified in the developing and adult heart, although the role they play in mammalian heart growth and regeneration remains unclear. In this study, we use genetic lineage tracing to follow the cell fate of Abcg2-expressing cells in the embryonic and adult heart. During cardiac embryogenesis, the Abcg2 lineage gives rise to multiple cardiovascular cell types, including cardiomyocytes, endothelial cells, and vascular smooth muscle cells. This capacity for Abcg2-expressing cells to contribute to cardiomyocytes decreases rapidly during the postnatal period. We further tested the role of the Abcg2 lineage following myocardial injury. One month following ischemia reperfusion injury, Abcg2-expressing cells contributed significantly to the endothelial cell lineage, however, there was no contribution to regenerated cardiomyocytes. Furthermore, consistent with previous results showing that Abcg2 plays an important cytoprotective role during oxidative stress, we show an increase in Abcg2 labeling of the vasculature, a decrease in the scar area, and a moderate improvement in cardiac function following myocardial injury. We have uncovered a difference in the capacity of Abcg2-expressing cells to generate the cardiovascular lineages during embryogenesis, postnatal growth, and cardiac regeneration.

  3. Dynamics of Red Blood Cells through submicronic splenic slits

    NASA Astrophysics Data System (ADS)

    Helfer, Emmanuele; Gambhire, Priya; Atwell, Scott; Bedu, Frederic; Ozerov, Igor; Viallat, Annie; Charrier, Anne; Badens, Catherine; Centre de reference Thalassemie, Badens Team; Physics; Engineering of Living Systems Team

    2016-11-01

    Red Blood Cells (RBCs) are periodically monitored for changes in their deformability by the spleen, and are entrapped and destroyed if unable to pass through the splenic interendothelial slits (IESs). In particular, in sickle cell disease (SCD), where hemoglobin form fibers inside the RBCs, and in hereditary spherocytosis (HS), where RBCs are more spherical and membrane-cytoskekeleton bonds are weakened, the loss of RBC deformability leads to spleen dysfunction. By combining photolithography and anisotropic wet etching techniques, we developed a new on-chip PDMS device with channels replicating the submicronic physiological dimensions of IESs to study the mechanisms of deformation of the RBCs during their passage through these biomimetic slits. For the first time, with HS RBCs, we show the disruption of the links between the RBC membrane and the underlying spectrin network. In the case of SCD RBCs we show the appearance of a tip at the front of the RBC with a longer time relaxation due to the increased cytoplasmic viscosity. This work has been carried out thanks to the support of the A*MIDEX project (n° ANR-11-IDEX-0001-02) funded by the «Investissements d'Avenir». French Government program, managed by ANR.

  4. 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.

  5. 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.

  6. 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

  7. Premature red blood cells have decreased aggregation and enhanced aggregability.

    PubMed

    Arbell, D; Orkin, B; Bar-Oz, B; Barshtein, G; Yedgar, S

    2008-06-01

    Preterm infants are highly susceptible to ischemic damage. This damage is most obvious in the brain, retina, and gastrointestinal tract. Studies focusing on the rheological properties of premature red blood cells (pRBCs) have consistently shown minimal or no RBC aggregation. Previously, measurements of pRBC aggregation kinetics indicated that specific plasma properties are responsible for the decreased RBC aggregation observed in the neonates, but that their specific RBC properties do not affect it. However, the strength of interaction in the pRBC aggregates as a function of medium composition has not been tested. In our previous research, we described clinically relevant parameters, that is, the aggregate resistance to disaggregation by flow. With the help of a cell flow property analyzer (CFA), we can monitor RBC aggregation by direct visualization of its dynamics during flow. We used the CFA to examine pRBC (from 9 premature babies) in the natural plasma and in PBS buffer supplemented with dextran (500 kDa) to distinguish between RBC intrinsic-cellular and plasma factors. pRBCs suspended in the native plasma showed minimal or no aggregation in comparison to normal adult RBC. When we transferred pRBCs from the same sample to the dextran solution, enhanced resistance to disaggregation by flow was apparent.

  8. Invasive Thymoma with Pure Red Cell Aplasia and Amegakaryocytic Thrombocytopenia

    PubMed Central

    Kiyoki, Yusuke; Ueda, Sho; Yamaoka, Masatoshi; Shimizu, Seiich; Inagaki, Masaharu

    2016-01-01

    We here describe a case involving a 67-yearold female patient who was referred to our hospital due to severe anemia (hemoglobin, 5.0 g/dL), thrombocytopenia (platelet count, 0.6 × 104/μL), and a mediastinal shadow with calcification noted on X-ray. On admission, an anterior mediastinal tumor was detected, and bone marrow biopsy revealed few megakaryocytes and severely reduced numbers of erythroid cells. The diagnosis was thymoma with pure red cell aplasia (PRCA) and acquired amegakaryocytic thrombocytopenia (AAMT). On Day 8 of admission, the patient received immunosuppressive therapy together with cyclosporine for the 2 severe hematologic diseases, which were stabilized within 2 months. Subsequently, total thymectomy was performed. The diagnosis of the tumor invading the left lung was invasive thymoma, Masaokakoga stage III. The histological diagnosis was World Health Organization type AB. Thymoma accompanied with PRCA and AAMT is very rare, and, based on our case, immunotherapeutic therapy for the hematologic disorders should precede surgical intervention. PMID:28053696

  9. Safety Evaluation of Hemoglobin-Albumin Cluster "HemoAct" as a Red Blood Cell Substitute.

    PubMed

    Haruki, Risa; Kimura, Takuya; Iwasaki, Hitomi; Yamada, Kana; Kamiyama, Ikuo; Kohno, Mitsutomo; Taguchi, Kazuaki; Nagao, Saori; Maruyama, Toru; Otagiri, Masaki; Komatsu, Teruyuki

    2015-07-29

    A hemoglobin (Hb) wrapped covalently by human serum albumins (HSAs), a core-shell structured hemoglobin-albumin cluster designated as "HemoAct", is an O2-carrier designed for use as a red blood cell (RBC) substitute. This report describes the blood compatibility, hemodynamic response, and pharmacokinetic properties of HemoAct, and then explains its preclinical safety. Viscosity and blood cell counting measurements revealed that HemoAct has good compatibility with whole blood. Intravenous administration of HemoAct into anesthetized rats elicited no unfavorable increase in systemic blood pressure by vasoconstriction. The half-life of (125)I-labeled HemoAct in circulating blood is markedly longer than that of HSA. Serum biochemical tests conducted 7 days after HemoAct infusion yielded equivalent values to those observed in the control group with HSA. Histopathologic inspections of the vital organs revealed no marked abnormality in their tissues. All results indicate that HemoAct has sufficient preclinical safety as an alternative material for RBC transfusion.

  10. 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

    2015-10-28

    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.

  11. Rapid Single-Cell Electroporation for Labeling Organotypic Cultures

    DTIC Science & Technology

    2010-06-01

    in Patients Affected with Mental Retardation : Lentiviral Injection in Organotypic Brain Slice Cultures. Methods in Enzymology 439, 255-266 (2008). 19...introducing a labeling agent, protein , or genetic element. It has only been in the past ten years that successful, repeatable, and efficient electroporation...34Screen Control", used during operation of the system is shown in Figure 5 below and is detailed in the caption. Due to the fragility of many of elements of

  12. 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.

  13. Numerical modelling of label-structured cell population growth using CFSE distribution data

    PubMed Central

    Luzyanina, Tatyana; Roose, Dirk; Schenkel, Tim; Sester, Martina; Ehl, Stephan; Meyerhans, Andreas; Bocharov, Gennady

    2007-01-01

    Background The flow cytometry analysis of CFSE-labelled cells is currently one of the most informative experimental techniques for studying cell proliferation in immunology. The quantitative interpretation and understanding of such heterogenous cell population data requires the development of distributed parameter mathematical models and computational techniques for data assimilation. Methods and Results The mathematical modelling of label-structured cell population dynamics leads to a hyperbolic partial differential equation in one space variable. The model contains fundamental parameters of cell turnover and label dilution that need to be estimated from the flow cytometry data on the kinetics of the CFSE label distribution. To this end a maximum likelihood approach is used. The Lax-Wendroff method is used to solve the corresponding initial-boundary value problem for the model equation. By fitting two original experimental data sets with the model we show its biological consistency and potential for quantitative characterization of the cell division and death rates, treated as continuous functions of the CFSE expression level. Conclusion Once the initial distribution of the proliferating cell population with respect to the CFSE intensity is given, the distributed parameter modelling allows one to work directly with the histograms of the CFSE fluorescence without the need to specify the marker ranges. The label-structured model and the elaborated computational approach establish a quantitative basis for more informative interpretation of the flow cytometry CFSE systems. PMID:17650320

  14. Noninvasive imaging of protein metabolic labeling in single human cells using stable isotopes and Raman microscopy.

    PubMed

    van Manen, Henk-Jan; Lenferink, Aufried; Otto, Cees

    2008-12-15

    We have combined nonresonant Raman microspectroscopy and spectral imaging with stable isotope labeling by amino acids in cell culture (SILAC) to selectively detect the incorporation of deuterium-labeled phenylalanine, tyrosine, and methionine into proteins in intact, single HeLa cells. The C-D stretching vibrational bands in these amino acids are observed in the 2100-2300 cm(-1) spectral region that is devoid of vibrational contributions from other, nondeuterated intracellular constituents. We found that incubation with deuterated amino acids for 8 h in cell culture already led to clearly detectable isotope-related signals in Raman spectra of HeLa cells. As expected, the level of isotope incorporation into proteins increased with incubation time, reaching 55% for deuterated phenylalanine after 28 h. Raman spectral imaging of HeLa cells incubated with deuterium-labeled amino acids showed similar spatial distributions for both isotope-labeled and unlabeled proteins, as evidenced by Raman ratio imaging. The SILAC-Raman methodology presented here combines the strengths of stable isotopic labeling of cells with the nondestructive and quantitative nature of Raman chemical imaging and is likely to become a powerful tool in both cell biology applications and research on tissues or whole organisms.

  15. Differential Labeling of Cell-surface and Internalized Proteins after Antibody Feeding of Live Cultured Neurons

    PubMed Central

    Munro, Kathryn M.; Kennedy, Matthew J.; Gunnersen, Jenny M.

    2014-01-01

    In order to demonstrate the cell-surface localization of a putative transmembrane receptor in cultured neurons, we labeled the protein on the surface of live neurons with a specific primary antibody raised against an extracellular portion of the protein. Given that receptors are trafficked to and from the surface, if cells are permeabilized after fixation then both cell-surface and internal protein will be detected by the same labeled secondary antibody. Here, we adapted a method used to study protein trafficking (“antibody feeding”) to differentially label protein that had been internalized by endocytosis during the antibody incubation step and protein that either remained on the cell surface or was trafficked to the surface during this period. The ability to distinguish these two pools of protein was made possible through the incorporation of an overnight blocking step with highly-concentrated unlabeled secondary antibody after an initial incubation of unpermeabilized neurons with a fluorescently-labeled secondary antibody. After the blocking step, permeabilization of the neurons allowed detection of the internalized pool with a fluorescent secondary antibody labeled with a different fluorophore. Using this technique we were able to obtain important information about the subcellular location of this putative receptor, revealing that it was, indeed, trafficked to the cell-surface in neurons. This technique is broadly applicable to a range of cell types and cell-surface proteins, providing a suitable antibody to an extracellular epitope is available. PMID:24561550

  16. [In vitro generation of blood red cells from stem cells: a sketch of the future].

    PubMed

    Mazurier, Christelle; Douay, Luc

    2016-01-01

    Human adult pluripotent stem cells, stem cells of embryonic origin and induced pluripotent stem cells (iPS) provide cellular sources for new promising regenerative medicine approaches. Because these cells can be patient-specific, they allow considering a personalized medicine appropriate to the diagnosis of each. The generation of cultured red blood cells (cRBC) derived from stem cells is emblematic of personalized medicine. Indeed, these cells have the advantage of being selected according to a blood phenotype of interest and they may provide treatments to patients in situation of impossible transfusion (alloimmunized patients, rare phenotypes). Essential progresses have established proof of concept for this approach, still a concept some years ago. From adult stem cells, all steps of upstream research were successfully achieved, including the demonstration of the feasibility of injection into human. This leads us to believe that Red Blood Cells generated in vitro from stem cells will be the future players of blood transfusion. However, although theoretically ideal, these stem cells raise many biological challenges to overcome, although some tracks are identified.

  17. Self-Assembled Superparamagnetic Iron Oxide Nanoclusters for Universal Cell Labeling and MRI

    NASA Astrophysics Data System (ADS)

    Chen, Shuzhen; Zhang, Jun; Jiang, Shengwei; Lin, Gan; Luo, Bing; Yao, Huan; Lin, Yuchun; He, Chengyong; Liu, Gang; Lin, Zhongning

    2016-05-01

    Superparamagnetic iron oxide (SPIO) nanoparticles have been widely used in a variety of biomedical applications, especially as contrast agents for magnetic resonance imaging (MRI) and cell labeling. In this study, SPIO nanoparticles were stabilized with amphiphilic low molecular weight polyethylenimine (PEI) in an aqueous phase to form monodispersed nanocomposites with a controlled clustering structure. The iron-based nanoclusters with a size of 115.3 ± 40.23 nm showed excellent performance on cellular uptake and cell labeling in different types of cells, moreover, which could be tracked by MRI with high sensitivity. The SPIO nanoclusters presented negligible cytotoxicity in various types of cells as detected using MTS, LDH, and flow cytometry assays. Significantly, we found that ferritin protein played an essential role in protecting stress from SPIO nanoclusters. Taken together, the self-assembly of SPIO nanoclusters with good magnetic properties provides a safe and efficient method for universal cell labeling with noninvasive MRI monitoring capability.

  18. Detecting Pyronin Y labeled RNA transcripts in live cell microenvironments by phasor-FLIM analysis.

    PubMed

    Andrews, Laura M; Jones, Mark R; Digman, Michelle A; Gratton, Enrico

    2013-03-01

    Pyronin Y is an environment-sensitive probe which labels all double-stranded RNA in live cells. Methods to determine which RNA species Pyronin Y may be labeling are limited due to the lack of studies aimed at determining whether this probe has different spectroscopic properties when bound to specific transcripts. A major issue is that transcripts are difficult to isolate and study individually. We detected transcripts directly in their biological environment allowing us to identify RNA species on the basis of their location in the cell. We show that the phasor approach to lifetime analysis has the sensitivity to determine at least six different RNA species in live fibroblast cells. The detected lifetime differences were consistent among cells. To our knowledge this is the first application of a spectroscopic technique aimed at identifying Pyronin Y labeled RNA subtypes in living cells.

  19. Detecting Pyronin Y labeled RNA transcripts in live cell microenvironments by phasor-FLIM analysis

    NASA Astrophysics Data System (ADS)

    Andrews, Laura M.; Jones, Mark R.; Digman, Michelle A.; Gratton, Enrico

    2013-03-01

    Pyronin Y is an environment-sensitive probe which labels all double-stranded RNA in live cells. Methods to determine which RNA species Pyronin Y may be labeling are limited due to the lack of studies aimed at determining whether this probe has different spectroscopic properties when bound to specific transcripts. A major issue is that transcripts are difficult to isolate and study individually. We detected transcripts directly in their biological environment allowing us to identify RNA species on the basis of their location in the cell. We show that the phasor approach to lifetime analysis has the sensitivity to determine at least six different RNA species in live fibroblast cells. The detected lifetime differences were consistent among cells. To our knowledge this is the first application of a spectroscopic technique aimed at identifying Pyronin Y labeled RNA subtypes in living cells.

  20. Single cell systems biology by super-resolution imaging and combinatorial labeling

    PubMed Central

    Lubeck, Eric; Cai, Long

    2012-01-01

    Fluorescence microscopy is a powerful quantitative tool for exploring regulatory networks in single cells. However, the number of molecular species that can be measured simultaneously is limited by the spectral separability of fluorophores. Here we demonstrate a simple but general strategy to drastically increase the capacity for multiplex detection of molecules in single cells by using optical super-resolution microscopy (SRM) and combinatorial labeling. As a proof of principle, we labeled mRNAs with unique combinations of fluorophores using Fluorescence in situ Hybridization (FISH), and resolved the sequences and combinations of fluorophores with SRM. We measured the mRNA levels of 32 genes simultaneously in single S. cerevisiae cells. These experiments demonstrate that combinatorial labeling and super-resolution imaging of single cells provides a natural approach to bring systems biology into single cells. PMID:22660740

  1. Fluorine-19 Labeling of Stromal Vascular Fraction Cells for Clinical Imaging Applications

    PubMed Central

    Rose, Laura C.; Kadayakkara, Deepak K.; Wang, Guan; Bar-Shir, Amnon; Helfer, Brooke M.; O’Hanlon, Charles F.; Kraitchman, Dara L.; Rodriguez, Ricardo L.

    2015-01-01

    Stromal vascular fraction (SVF) cells are used clinically for various therapeutic targets. The location and persistence of engrafted SVF cells are important parameters for determining treatment failure versus success. We used the GID SVF-1 platform and a clinical protocol to harvest and label SVF cells with the fluorinated (19F) agent CS-1000 as part of a first-in-human phase I trial (clinicaltrials.gov identifier NCT02035085) to track SVF cells with magnetic resonance imaging during treatment of radiation-induced fibrosis in breast cancer patients. Flow cytometry revealed that SVF cells consisted of 25.0% ± 15.8% CD45+, 24.6% ± 12.5% CD34+, and 7.5% ± 3.3% CD31+ cells, with 2.1 ± 0.7 × 105 cells per cubic centimeter of adipose tissue obtained. Fluorescent CS-1000 (CS-ATM DM Green) labeled 87.0% ± 13.5% of CD34+ progenitor cells compared with 47.8% ± 18.5% of hematopoietic CD45+ cells, with an average of 2.8 ± 2.0 × 1012 19F atoms per cell, determined using nuclear magnetic resonance spectroscopy. The vast majority (92.7% ± 5.0%) of CD31+ cells were also labeled, although most coexpressed CD34. Only 16% ± 22.3% of CD45−/CD31−/CD34− (triple-negative) cells were labeled with CS-ATM DM Green. After induction of cell death by either apoptosis or necrosis, >95% of 19F was released from the cells, indicating that fluorine retention can be used as a surrogate marker for cell survival. Labeled-SVF cells engrafted in a silicone breast phantom could be visualized with a clinical 3-Tesla magnetic resonance imaging scanner at a sensitivity of approximately 2 × 106 cells at a depth of 5 mm. The current protocol can be used to image transplanted SVF cells at clinically relevant cell concentrations in patients. Significance Stromal vascular fraction (SVF) cells harvested from adipose tissue offer great promise in regenerative medicine, but methods to track such cell therapies are needed to ensure correct administration and monitor survival. A clinical protocol

  2. 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

  3. Autophagic vesicles on mature human reticulocytes explain phosphatidylserine-positive red cells in sickle cell disease.

    PubMed

    Mankelow, Tosti J; Griffiths, Rebecca E; Trompeter, Sara; Flatt, Joanna F; Cogan, Nicola M; Massey, Edwin J; Anstee, David J

    2015-10-08

    During maturation to an erythrocyte, a reticulocyte must eliminate any residual organelles and reduce its surface area and volume. Here we show this involves a novel process whereby large, intact, inside-out phosphatidylserine (PS)-exposed autophagic vesicles are extruded. Cell surface PS is a well-characterized apoptotic signal initiating phagocytosis. In peripheral blood from patients after splenectomy or in patients with sickle cell disease (SCD), the number of circulating red cells exposing PS on their surface is elevated. We show that in these patients PS is present on the cell surface of red cells in large (∼1.4 µm) discrete areas corresponding to autophagic vesicles. The autophagic vesicles found on reticulocytes are identical to those observed on red cells from splenectomized individuals and patients with SCD. Our data suggest the increased thrombotic risk associated with splenectomy, and patients with hemoglobinopathies is a possible consequence of increased levels of circulating mature reticulocytes expressing inside-out PS-exposed autophagic vesicles because of asplenia.

  4. Fluorophore Conjugated Iron Oxide Nanoparticle Labeling and Analysis of Engrafting Human Hematopoietic Stem Cells

    PubMed Central

    Maxwell, Dustin J.; Bonde, Jesper; Hess, David A.; Hohm, Sarah A.; Lahey, Ryan; Creer, Michael H.; Piwnica-Worms, David; Nolta, Jan A.

    2010-01-01

    The use of nanometer-sized iron oxide particles combined with molecular imaging techniques enable dynamic studies of homing and trafficking of human hematopoietic stem cells (HSC). Identifying clinically applicable strategies for loading nanoparticles into primitive HSC requires strictly defined culture conditions to maintain viability without inducing terminal differentiation. In the current study, fluorescent molecules were covalently linked to dextran-coated iron oxide nanoparticles (Feridex) to characterize human HSC labeling to monitor the engraftment process. Conjugating fluorophores to the dextran coat for FACS purification eliminated spurious signals from non-sequestered nanoparticle contaminants. A short-term defined incubation strategy was developed which allowed efficient labeling of both quiescent and cycling HSC, with no discernable toxicity in vitro or in vivo. Transplantation of purified primary human cord blood lineage-depleted and CD34+ cells into immunodeficient mice allowed detection of labeled human HSC in the recipient bones. Flow cytometry was used to precisely quantitate the cell populations that had sequestered the nanoparticles, and to follow their fate post-transplantation. Flow cytometry endpoint analysis confirmed the presence of nanoparticle-labeled human stem cells in the marrow. The use of fluorophore-labeled iron oxide nanoparticles for fluorescence imaging in combination with flow cytometry allows evaluation of labeling efficiencies and homing capabilities of defined human HSC subsets. PMID:18055451

  5. Elastic thickness compressibilty of the red cell membrane.

    PubMed

    Heinrich, V; Ritchie, K; Mohandas, N; Evans, E

    2001-09-01

    We have used an ultrasensitive force probe and optical interferometry to examine the thickness compressibility of the red cell membrane in situ. Pushed into the centers of washed-white red cell ghosts lying on a coverglass, the height of the microsphere-probe tip relative to its closest approach on the adjacent glass surface revealed the apparent material thickness, which began at approximately 90 nm per membrane upon detection of contact (force approximately 1-2 pN). With further impingement, the apparent thickness per membrane diminished over a soft compliant regime that spanned approximately 40 nm and stiffened on approach to approximately 50 nm under forces of approximately 100 pN. The same force-thickness response was obtained on recompression after retraction of the probe, which demonstrated elastic recoverability. Scaled by circumferences of the microspheres, the forces yielded energies of compression per area which exhibited an inverse distance dependence resembling that expected for flexible polymers. Attributed to the spectrin component of the membrane cytoskeleton, the energy density only reached one thermal energy unit (k(B)T) per spectrin tetramer near maximum compression. Hence, we hypothesized that the soft compliant regime probed in the experiments represented the compressibility of the outer region of spectrin loops and that the stiff regime < 50 nm was the response of a compact mesh of spectrin backed by a hardcore structure. To evaluate this hypothesis, we used a random flight theory for the entropic elasticity of polymer loops to model the spectrin network. We also examined the possibility that additional steric repulsion and apparent thickening could arise from membrane thermal-bending excitations. Fixing the energy scale to k(B)T/spectrin tetramer, the combined elastic response of a network of ideal polymer loops plus the membrane steric interaction correlated well with the measured dependence of energy density on distance for a statistical

  6. 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.

  7. Method for labelling leucocytes with indium In-111 oxine

    SciTech Connect

    Kaminsky, D.

    1992-03-03

    This patent describes an improved method for radio-labelling leucocytes with Indium In-111 oxine. It comprises separating the leucocytes from whole blood for obtaining separated leucocytes mixed with residual red blood cells; and then labelling the separated leucocytes with Indium In-111 oxine; wherein the improvement comprises the following further step: depleting residual red blood cells from the separated leucocytes by resuspending the leucocytes in an isotonic saline solution, then rocking the resuspended leucocytes for causing the leucocytes to preferentially settle out, and then removing residual red blood cells which remain suspended within the supernatant isotonic saline solution.

  8. Concepts, labeling procedures, and design of cell proliferation studies relating to carcinogenesis.

    PubMed Central

    Goldsworthy, T L; Butterworth, B E; Maronpot, R R

    1993-01-01

    Chemicals may induce cell proliferation directly as mitogens or indirectly via cell death with subsequent proliferation to replace lost cells. Chemically induced proliferation has been demonstrated to play a role in the carcinogenic process. A wide range of procedures and techniques are currently being used to define the quantitative relationship between the extent and duration of chemically induced cell proliferation and carcinogenic potential in different species and target organs. However, a limited database and nonstandard protocols and procedures for measuring cell proliferation have made it difficult to compare results between laboratories. Comparison of frequencies of S phase between control and treated animals is the most commonly used end point in cell proliferation studies and may be regarded as an indirect indication of a proliferative response. This response can be ascertained as labeling indexes (LI; percentage of cells in S phase) after the administration of the DNA precursor labels (tritiated thymidine; 3H-TdR; bromodeoxyuridine, BrdU) or through immunostaining of the endogenous cell replication marker, proliferating cell nuclear antigen (PCNA). Both approaches are applicable to tissue sections. An important issue in the design of experimental studies for measuring LI is determining how and when to investigate proliferative responses in relation to the chemical treatment regimen. Variables to consider when designing cell proliferation studies include the animal's age, chemical dose and method of treatment, choice and dose of label, time and length that the label is administered, and methods of quantitation.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7912190

  9. Biocompatible nanoparticle labeling of stem cells and their distribution in brain.

    PubMed

    Rehni, Ashish K; Singh, Thakur Gurjeet; Chitkara, Mansi; Sandhu, I S

    2012-01-01

    Nanolabeling is an invaluable novel technique in biology to detect and characterize different parts of biological systems including microscopic entities, viz., cells inside the living systems. Stem cells (SCs) are multipotent cells with the potential to differentiate into bone, cartilage, fat, muscle cells, and neurons and are being investigated for their utility in cell-based transplantation therapy. Yet, adequate methods to track transplanted SCs in vivo are limited, precluding functional studies. Nanoparticles (quantum dots) offer an alternative to organic dyes and fluorescent proteins to label and track cells in vitro and in vivo. These nanoparticles are resistant to chemical and metabolic degradation, demonstrating long-term photo stability. Here, we describe the technology of labeling the stem cells with silver nitrate nanoparticles in an in vitro coculture model. This is followed by defining the procedure of administering these cells in vivo and studying the distribution pattern and resultant regenerative effects of the "tagged" stem cells.

  10. Mathematical modelling of adult hippocampal neurogenesis: effects of altered stem cell dynamics on cell counts and bromodeoxyuridine-labelled cells

    PubMed Central

    Ziebell, Frederik; Martin-Villalba, Ana; Marciniak-Czochra, Anna

    2014-01-01

    In the adult hippocampus, neurogenesis—the process of generating mature granule cells from adult neural stem cells—occurs throughout the entire lifetime. In order to investigate the involved regulatory mechanisms, knockout (KO) experiments, which modify the dynamic behaviour of this process, were conducted in the past. Evaluating these KOs is a non-trivial task owing to the complicated nature of the hippocampal neurogenic niche. In this study, we model neurogenesis as a multicompartmental system of ordinary differential equations based on experimental data. To analyse the results of KO experiments, we investigate how changes of cell properties, reflected by model parameters, influence the dynamics of cell counts and of the experimentally observed counts of cells labelled by the cell division marker bromodeoxyuridine (BrdU). We find that changing cell proliferation rates or the fraction of self-renewal, reflecting the balance between symmetric and asymmetric cell divisions, may result in multiple time phases in the response of the system, such as an initial increase in cell counts followed by a decrease. Furthermore, these phases may be qualitatively different in cells at different differentiation stages and even between mitotically labelled cells and all cells existing in the system. PMID:24598209

  11. Intracellular localization of radioactively labeled misonidazole in EMT-6 tumor cells in vitro

    SciTech Connect

    Miller, G.G.; Ngan-Lee, J.; Chapman, J.D.

    1982-03-01

    Misonidazole, as an electron-affinic drug, is capable of accepting electrons from ascorbate, NADPH and a number of enzymes in anoxic reaction mixtures such as cellular homogenates and microsomal fractions. Although the intracellular location of many of the above electron donors has been established, (usually in microsomes and mitochondria), the sites of covalent binding of metabolically activated misonidazle and its catabolites to cellular macromolecules remain to be elucidated. The biological effects of misonidazle differ quantitatively and possibly qualitatively when cells are incubated in air vis-a-vis hypoxic conditions. Difference in uptake of labeled misonidazole by hypoxic or aerobic cells may render this radiosensitizing agent of value in the clinical management of certain tumors. We are currently attempting to establish the intracellular sites of covalent binding of radioactively labeled misonidazole and its catabolites in EMT-6 tumor cells. Single cell suspensions of EMT-6 tumor cells labeled with /sup 14/C-Misonidazole in hypoxic conditions were homogenized to yield nuclear and cytoplasmic fractions, which were assayed for acid-precipitable radioactivity. The distributon of label bound to various intracellular molecular classes, in both the acid soluble and macromolecular fraction, was determined for EMT-6 cells labeled in air and hypoxia.

  12. 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.

  13. In vivo circulation, clearance, and biodistribution of polyglycerol grafted functional red blood cells.

    PubMed

    Chapanian, Rafi; Constantinescu, Iren; Brooks, Donald E; Scott, Mark D; Kizhakkedathu, Jayachandran N

    2012-04-01

    The in vivo circulation of hyperbranched polyglycerol (HPG) grafted red blood cells (RBCs) was investigated in mice. The number of HPG molecules grafted per RBC was measured using tritium labeled HPGs ((3)H-HPG) of different molecular weights; the values ranged from 1 × 10(5) to 2 × 10(6) molecules per RBC. HPG-grafted RBCs were characterized in vitro by measuring the electrophoretic mobility, complement mediated lysis, and osmotic fragility. Our results show that RBCs grafted with 1.5 × 10(5) HPG molecules per RBC having molecular weights 20 and 60 kDa have similar characteristics as that of control RBCs. The in vivo circulation of HPG-grafted RBCs was measured by a tail vain injection of (3)H-HPG60K-RBC in mice. The radioactivity of isolated RBCs, whole blood, plasma, different organs, urine and feces was evaluated at different time intervals. The portion of (3)H-HPG60K-RBC that survived the first day in mice (52%) remained in circulation for 50 days. Minimal accumulation radioactivity in organs other than liver and spleen was observed suggesting the normal clearance mechanism of modified RBCs. Animals gained normal weights and no abnormalities observed in necropsy analysis. The stability of the ester-amide linker between the RBC and HPG was evaluated by comparing the clearance rate of (3)H-HPG60K-RBC and PKH-26 lipid fluorescent membrane marker labeled HPG60K-RBCs. HPG modified RBCs combine the many advantages of a dendritic polymer and RBCs, and hold great promise in systemic drug delivery and other applications of functional RBC.

  14. [Role of protein kinases of human red cell membrane in deformability and aggregation changes].

    PubMed

    Murav'ev, A V; Maĭmistova, A A; Tikhomirova, I A; Bulaeva, S V; Mikhaĭlov, P V; Murav'ev, A A

    2012-01-01

    The proteomic analysis has showed that red cell membrane contains several kinases and phosphatases. Therefore the aim of this study was to investigate the role of protein kinases of human red cell membrane in deformability and aggregation changes. Exposure of red blood cells (RBCs) to some chemical compounds led to change in the RBC microrheological properties. When forskolin (10 microM), an adenylyl cyclase (AC) and a protein kinase A (PKA) stimulator was added to RBC suspension, the RBC deformability (RBCD) was increased by 20% (p < 0.05). Somewhat more significant deformability rise appeared after RBC incubation with dB-AMP (by 26%; p < 0.01). Red cell aggregation (RBCA) was significantly decreased under these conditions (p < 0.01). Markedly less changes of deformability was found after RBC incubation with protein kinase stimulator C (PKC)--phorbol 12-myristate 13-acetate (PMA). This drug reduced red cell aggregation only slightly. It was inhibited red cell tyrosine phosphotase activity by N-vanadat and was obtained a significant RBCD rise and RBCA lowering. The similar effect was found when cells were incubated with cisplatin as a tyrosine protein kinase (TPK) activator. It is important to note that a selective TPK inhibitor--lavendustin eliminated the above mention effects. On the whole the total data clearly show that the red cell aggregation and deformation changes were connected with an activation of the different intracellular signaling pathways.

  15. Magnetic labeling of non-phagocytic adherent cells with iron oxide nanoparticles: a comprehensive study.

    PubMed

    Boutry, Sébastien; Brunin, Stéphanie; Mahieu, Isabelle; Laurent, Sophie; Vander Elst, Luce; Muller, Robert N

    2008-01-01

    Small particles of iron oxide (SPIO) and ultrasmall particles of iron oxide (USPIO), inducing a strong negative contrast on T(2) and T(2)*-weighted MR images, are the most commonly used systems for the magnetic labeling of cultured cells and their subsequent detection by magnetic resonance imaging (MRI). The purpose of this work is to study the influence of iron incubation concentration, nanoparticle size and nanoparticle coating on the magnetic labeling and the viability of non-phagocytic adherent cells in culture. The magnetic labeling of 3T6 fibroblasts was studied by T(2)-weighted MRI at 4.7 T and by dosing-or cytochemical revealing-of iron through methods based on Perl's Prussian blue staining. Cells were incubated for 48 h with increasing iron concentrations of SPIO (25-1000 microg Fe/ml Endorem. Sinerem, a USPIO (20-40 nm) coated with neutral dextran, and Resovist (65 nm), a SPIO bearing an anionic carboxydextran coating, were compared with Endorem (dextran-coated, 80-150 nm) as magnetic tags. The iron loading of marrow stromal cell primary cultures (MSCs) isolated from rat femurs was compared with that of 3T6 fibroblasts. The SPIO-labeling of cells with Endorem was found to be dependent on the iron incubation concentration. MSCs, more sparsely distributed in the culture, exhibited higher iron contents than more densely populated 3T6 fibroblast cultures. A larger iron loading was achieved with Resovist than with Endorem, which in turn was more efficient than Sinerem as a magnetic tag. The magnetic labeling of cultured non-phagocytic adherent cells with iron oxide nanoparticles was thus found to be dependent on the relative concentration of the magnetic tag and of the cells in culture, on the nanoparticle size, and on the coating type. The viability of cells, estimated by methods assessing cell membrane permeability, was not affected by magnetic labeling in the conditions used in this work.

  16. Covalent affinity labeling, radioautography, and immunocytochemistry localize the glucocorticoid receptor in rat testicular Leydig cells

    SciTech Connect

    Stalker, A.; Hermo, L.; Antakly, T. )

    1989-12-01

    The presence and distribution of glucocorticoid receptors in the rat testis were examined by using 2 approaches: in vivo quantitative radioautography and immunocytochemistry. Radioautographic localization was made possible through the availability of a glucocorticoid receptor affinity label, dexamethasone 21-mesylate, which binds covalently to the glucocorticoid receptor, thereby preventing dissociation of the steroid-receptor complex. Adrenalectomized adult rats were injected with a tritiated (3H) form of this steroid into the testis and the tissue was processed for light-microscope radioautography. Silver grains were observed primarily over the Leydig cells of the interstitial space and to a lesser extent, over the cellular layers which make up the seminiferous epithelium, with no one cell type showing preferential labeling. To determine the specificity of the labeling, a 25- or 50-fold excess of unlabeled dexamethasone was injected simultaneously with the same dose of (3H)-dexamethasone 21-mesylate. In these control experiments, a marked reduction in label intensity was noted over the Leydig as well as tubular cells. Endocytic macrophages of the interstitium were non-specifically labeled, indicating uptake of the ligand possibly by fluid-phase endocytosis. A quantitative analysis of the label confirmed the presence of statistically significant numbers of specific binding sites for glucocorticoids in both Leydig cells and the cellular layers of the seminiferous epithelium; 86% of the label was found over Leydig cells, and only 14% over the cells of the seminiferous epithelium. These binding data were confirmed by light-microscope immunocytochemistry using a monoclonal antibody to the glucocorticoid receptor.

  17. 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

  18. Diamond Blackfan anemia: a disorder of red blood cell development.

    PubMed

    Ellis, Steven R; Lipton, Jeffrey M

    2008-01-01

    Diamond Blackfan anemia (DBA) is an inherited hypoplastic anemia that typically presents in the first year of life. The genes identified to date that are mutated in DBA encode ribosomal proteins, and in these cases ribosomal protein haploinsufficiency gives rise to the disease. The developmental timing of DBA presentation suggests that the changes in red blood cell production that occur around the time of birth trigger a pathophysiological mechanism, likely linked to defective ribosome synthesis, which precipitates the hematopoietic phenotype. Variable presentation of other clinical phenotypes in DBA patients indicates that other developmental pathways may also be affected by ribosomal protein haploinsufficiency and that the involvement of these pathways is influenced by modifier genes. Understanding the molecular basis for the developmental timing of DBA presentation promises to shed light on a number of baffling features of this disease. This chapter also attempts to demonstrate how the marriage of laboratory and clinical science may enhance each and permit insights into human disease that neither alone can accomplish.

  19. Red blood cell lifespan, erythropoiesis and hemoglobin control.

    PubMed

    Kruse, Anja; Uehlinger, Dominik E; Gotch, Frank; Kotanko, Peter; Levin, Nathan W

    2008-01-01

    Erythropoietin (EPO) and iron deficiency as causes of anemia in patients with limited renal function or end-stage renal disease are well addressed. The concomitant impairment of red blood cell (RBC) survival has been largely neglected. Properties of the uremic environment like inflammation, increased oxidative stress and uremic toxins seem to be responsible for the premature changes in RBC membrane and cytoskeleton. The exposure of antigenic sites and breakdown of the phosphatidylserine asymmetry promote RBC phagocytosis. While the individual response to treatment with EPO-stimulating agents (ESA) depends on both the RBC's lifespan and the production rate, uniform dosing algorithms do not meet that demand. The clinical use of mathematical models predicting ESA-induced changes in hematocrit might be greatly improved once independent estimates of RBC production rate and/or lifespan become available, thus making the concomitant estimation of both parameters unnecessary. Since heme breakdown by the hemoxygenase pathway results in carbon monoxide (CO) which is exhaled, a simple CO breath test has been used to calculate hemoglobin turnover and therefore RBC survival and lifespan. Future research will have to be done to validate and implement this method in patients with kidney failure. This will result in new insights into RBC kinetics in renal patients. Eventually, these findings are expected to improve our understanding of the hemoglobin variability in response to ESA.

  20. 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.