Improved Flow Cytometric Method for HLA-B27 Typing

1992 ◽  
Vol 29 (6) ◽  
pp. 663-667 ◽  
Author(s):  
W C M Janssen ◽  
J A C N Rouwen ◽  
J J M L Hoffmann
Keyword(s):  
Monoclonal Antibody ◽  
Flow Cytometer ◽  
Complete Agreement ◽  
Hla B27 ◽  
Cell Marker ◽  
High Association ◽  
Flow Cytometric Method ◽  
Flow Cytometric ◽  
A Cell ◽  
Clinical Interest

HLA-B27 is a cell marker of clinical interest because of its high association with certain diseases. The HLA-B27 antigen was detected on lymphocytes using a monoclonal antibody in an indirect immuno-fluorescence assay using a fluorescence flow cytometer. The considerable crossreaction of the monoclonal antibody with the HLA-B7 antigen was effectively suppressed by masking it by means of human anti-HLA-B7 antiserum. The flow cytometric method was evaluated by comparing the results with those obtained by the standard lymphocytotoxicity test and showed complete agreement in 107 selected patient samples.

scholarly journals COMPARISON BETWEEN FLOW CYTOMETERIC METHOD AND OTHER METHODS FORRETICULOCYTE COUNT IN DIAGNOSIS OF PHENYLHYDRAZINE-INDUCED HEMOLYTIC ANEMIA

2021 ◽  
Vol 9 (02) ◽  
pp. 938-952
Author(s):  
Rania Mohammed Baker ◽  
◽  
Fatma Abdel-Monem Gad ◽  
Khalid Mostafa Fararh ◽  
◽  
...  
Keyword(s):  
Hemolytic Anemia ◽  
Blood Cells ◽  
Reticulocyte Count ◽  
Osmotic Fragility ◽  
Flow Cytometer ◽  
Control Group ◽  
Albino Rats ◽  
Flow Cytometric Method ◽  
Flow Cytometric ◽  
Manual Methods

Reticulocyte count is the salient evidence of the effectiveness of bone marrow to produce red blood cells. Currently, the reticulocyte counting is a challenge for clinical laboratoriesmainly for the ordinary ones, which still use the manual method.This study was designed to evaluate the performance of flow cytometer for reticulocytes counting comparing to traditional and optimized manual methods which helpful in diagnosis of phenylhydazine-induced anemia.For that 45 male white Albino rats were divided into 5 groups, control group,phenylhydrazine group (PHZ) which injected by phz(20 mg/kg b.w, I/P),quercetin+phz group (quercetin, 50 mg/kgb.w per os), silymarin+phz group (silymarin, 100 mg/kgb.w per os) and quercetin group. Whole blood samples of these groups were collected at day 3, 5 and 10 after 1st injection of phz which used for reticulocyte counts by flow cytometeric method and other manual methods in addition to measurement ofCBC and osmotic fragility. Analysis of the results showed that phenylhydrazine injection induced hemolytic anemia with significant reticulocytosis and using of flow cytometer in reticulocyte count more precise, easy and fast than traditional and optimized manual methods. Furthermore, degree of hemolysis was significantly increases in phz group comparing to other groups. Therefore, we concluded that flow cytometric method for reticulocyte counts was simple, fast and highly reliable comparable to traditional and optimized manual methods. Also optimized manual showed that more perfect than traditional manual method and nearly to accuracy of flow cytometeric method.

scholarly journals A novel flow cytometric method to study cytotoxic activity in whole blood samples

2020 ◽  
Author(s):  
Laura G. Rico ◽  
Mike D. Ward ◽  
Jolene A. Bradford ◽  
Jordi Petriz
Keyword(s):  
Cytotoxic Activity ◽  
Whole Blood ◽  
Low Frequency ◽  
Target Cells ◽  
Cytotoxic Cells ◽  
Minimal Sample ◽  
Flow Cytometric Method ◽  
Flow Cytometric ◽  
Cytotoxicity Studies ◽  
A Cell

We have developed a quantitative method for cell-mediated cytotoxicity studies, preserving cellular function with minimal sample manipulation. Cytotoxic activity is simply and reproducibly measured as the ability of cytotoxic cells to lyse K562 target cells previously loaded with Calcein-AM vital stain. After spiking a known number of fluorescent viable K562 target cells into whole blood, cell mixtures are incubated for 2 hours in a cell incubator and the remaining spiked cells are counted by flow cytometry. In order to discriminate nucleated cells, erythrocytes and debris, unlysed whole blood is stained with a cell permeable DNA vital fluorescent dye. Cell-mediated lysis is measured by comparing target counts for different effector-to-target ratios. Since the cytotoxicity of these dyes is relatively low, this method can be broadly applied to studies of innate immune response to tumors and infections, especially where target-killing activity might be compromised by small volume samples or low frequency of cytotoxic cells.

scholarly journals Immunophenotyping of Posttraumatic Neutrophils on a Routine Haematology Analyser

2012 ◽  
Vol 2012 ◽  
pp. 1-6 ◽  
Author(s):  
Kathelijne Maaike Groeneveld ◽  
Marjolein Heeres ◽  
Loek Petrus Hendrikus Leenen ◽  
Albert Huisman ◽  
Leo Koenderman
Keyword(s):  
Active Form ◽  
Flow Cytometric Analysis ◽  
Flow Cytometer ◽  
Clinical Diagnostics ◽  
Trauma Research ◽  
Haematology Analyser ◽  
Flow Cytometric ◽  
Healthy Donors ◽  
A Cell ◽  
High Level

Introduction.Flow cytometry markers have been proposed as useful predictors for the occurrence of posttraumatic inflammatory complications. However, currently the need for a dedicated laboratory and the labour-intensive analytical procedures make these markers less suitable for clinical practice. We tested an approach to overcome these limitations.Material and Methods.Neutrophils of healthy donors were incubated with antibodies commonly used in trauma research: CD11b (MAC-1), L-selectin (CD62L), FcγRIII (CD16), and FcγRII (CD32) in active form (MoPhab A27). Flow cytometric analysis was performed both on a FACSCalibur, a standard flow cytometer, and on a Cell-Dyn Sapphire, a routine haematology analyser.Results.There was a high level of agreement between the two types of analysers, with 41% for FcγRIII, 80% for L-selectin, 98% for CD11b, and even a 100% agreement for active FcγRII. Moreover, analysis on the routine haematology analyser was possible in less than a quarter of the time in comparison to the flow cytometer.Conclusion.Analysis of neutrophil phenotype on the Cell-Dyn Sapphire leads to the same conclusion compared to a standard flow cytometer. The markedly reduced time necessary for analysis and reduced labour intensity constitutes a step forward in implementation of this type of analysis in clinical diagnostics in trauma research.

Quantitative determination of CD4/CD8 molecules by a cell marker ELISA

1994 ◽  
Vol 40 (1) ◽  
pp. 38-42 ◽  
Author(s):  
L Franke ◽  
E Nugel ◽  
W D Döcke ◽  
T Porstmann
Keyword(s):  
Flow Cytometry ◽  
T Lymphocytes ◽  
Flow Cytometric Analysis ◽  
Sample Volume ◽  
Small Sample ◽  
Cell Marker ◽  
Cell Counts ◽  
Flow Cytometric ◽  
A Cell

Abstract Determination of percentages of CD4+ and CD8+ T cells from patients with human immunodeficiency virus infection is usually done by flow cytometric analysis. We compared a cell marker ELISA with flow cytometry for quantitation of CD4 and CD8 molecules on T lymphocytes, and correlated the values both with the number of CD4+ and CD8+ T lymphocytes and with clinical data. Results by cell marker ELISA (y) correlated well with those by flow cytometric analysis (x); r = 0.69, P < 0.001 (y = 0.01x + 3.9) for CD4; r = 0.81, P < 0.001 (y = 0.03x + 5.4) for CD8; n = 343. The ELISA detected changes in numbers of CD8 molecules on the cells earlier than flow cytometry recognized changes in CD8+ T-cell counts. The advantages of the ELISA are the small sample volume required (0.5 mL of blood), its internal standardization by a CD4+/CD8+ cell line, and its simple and fast performance. The cell marker ELISA appears to be an efficient alternative to flow cytometry.

Expression of Platelet Membrane Glycoprotein V in Human Megakaryocytes and Megakaryocytic Cell Lines: A Study Using a Novel Monoclonal Antibody against GPV

1994 ◽  
Vol 72 (05) ◽  
pp. 762-769 ◽  
Author(s):  
Toshiro Takafuta ◽  
Kingo Fujirmura ◽  
Hironori Kawano ◽  
Masaaki Noda ◽  
Tetsuro Fujimoto ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Cell Lines ◽  
Immunohistochemical Analysis ◽  
Specific Protein ◽  
Platelet Membrane ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Flow Cytometric ◽  
Polymerase Chain ◽  
Megakaryocytic Cell

SummaryGlycoprotein V (GPV) is a platelet membrane protein with a molecular weight of 82 kD, and one of the leucine rich glycoproteins (LRG). By reverse transcription-polymerase chain reaction (RT-PCR), GPV cDNA was amplified from mRNA of platelets and megakaryocytic cell lines. However, since there are few reports indicating whether GPV protein is expressed in megakaryocytes as a lineage and maturation specific protein, we studied the GPV expression at the protein level by using a novel monoclonal antibody (1D9) recognizing GPV. Flow cytometric and immunohistochemical analysis indicated that GPV was detected on the surface and in the cytoplasm of only the megakaryocytes in bone marrow aspirates. In a megakaryocytic cell line UT-7, GPV antigen increased after treatment with phorbol-12-myri-state-13-acetate (PMA). These data indicate that only megakaryocytes specifically express the GPV protein among hematopoietic cells and that the expression of GPV increases with differentiation of the megakaryocyte as GPIb-IX complex.

scholarly journals Cord Blood Extracellular Vesicles Analyzed by Flow Cytometry with Thresholding Using 405 nm or 488 nm Laser Leads to Concurrent Results

Diagnostics ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 1320
Author(s):  
Kristýna Pekárková ◽  
Jakub Soukup ◽  
Marie Kostelanská ◽  
Jan Širc ◽  
Zbyněk Straňák ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cord Blood ◽  
Extracellular Vesicles ◽  
Correlation Coefficients ◽  
Flow Cytometer ◽  
Liquid Biopsies ◽  
Physiological Conditions ◽  
Flow Cytometric ◽  
Term Births ◽  
And Control

Extracellular vesicles (EVs) from liquid biopsies are extensively analyzed by flow cytometry, a technology that is continuously evolving. Thresholding utilizing a violet 405 nm laser side scatter (VSSC) has recently been implemented. Here, we collected set of large EV (lEV) samples from cord blood, which we analyzed using a standard flow cytometer improved via a 405 nm laser side scatter. Samples were analyzed using two distinct thresholding methods—one based on VSSC, and one based on VSSC combined with fluorescence thresholding on stained phosphatidylserine. Through these thresholding methods, we compared lEVs from pre-term births and control cord blood. Double-labeled lEVs with platelet CD36+/CD41+, activated platelet CD41+/CD62P+ and endothelial CD31+/CD105+ antibodies were used. Apart from comparing the two groups together, we also correlated measured lEVs with the thresholding methods. We also correlated the results of this study with data analyzed in our previous study in which we used a conventional 488 nm laser SSC. We did not find any difference between the two cord blood groups. However, we found highly concurrent data via our correlation of the thresholding methods, with correlation coefficients ranging from 0.80 to 0.96 even though the numbers of detected lEVs differed between thresholding methods. In conclusion, our approaches to thresholding provided concurrent data and it seems that improving the cytometer with the use of a VSSC increases its sensitivity, despite not being particularly critical to the validity of flow cytometric studies that compare pathological and physiological conditions in liquid biopsies.

scholarly journals Nuclear oncoprotein expression as a function of lineage, differentiation stage, and proliferative status of normal human hematopoietic cells

Blood ◽  
1989 ◽  
Vol 74 (5) ◽  
pp. 1517-1524 ◽  
Author(s):  
MB Kastan ◽  
KD Stone ◽  
CI Civin
Keyword(s):  
Progenitor Cells ◽  
Control Cell ◽  
Hematopoietic Cells ◽  
Maturation Stage ◽  
Flow Cytometric Assay ◽  
Lineage Differentiation ◽  
Flow Cytometric ◽  
A Cell ◽  
Normal Human ◽  
Differentiation Stage

Abstract Relative levels of the nuclear oncoproteins c-myb, c-myc, and c-fos were determined in selected subpopulations of normal human bone marrow (BM) cells using a flow cytometric assay which simultaneously detects a cell-surface antigen (as a marker of lineage and stage of maturation) and levels of an intracellular protein. At least two monoclonal antibodies directed against each oncoprotein and specific peptide inhibition controls were used for these determinations. Hematopoietic progenitor cells (CD34+) express the highest levels of c-myb and c-myc, whereas c-fos levels in CD34+ progenitor cells are similar to c-fos levels in mature monocytes and granulocytes. Granulocytes are the only hematopoietic cells examined which do not express detectable levels of c-myb and c-myc. The levels of these oncoproteins in these normal, unstimulated BM cell populations were more closely linked to lineage and maturation stage than to the proliferative status of the given population, as determined by either DNA staining or expression of the cell-cycle specific nuclear protein, Ki67. This flow cytometric assay helps in interpreting the significance of oncoprotein levels in leukemia cells by allowing direct comparisons of a leukemia with the phenotypically similar “normal counterpart control” cell population in normal BM.

A sensitive flow cytometric method for measuring the oxidative burst

1997 ◽  
Vol 202 (2) ◽  
pp. 105-111 ◽  
Author(s):  
Michael A Model ◽  
Mark A KuKuruga ◽  
Robert F Todd
Keyword(s):  
Oxidative Burst ◽  
Flow Cytometric Method ◽  
Flow Cytometric
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