SummaryPlatelet function is dependent upon membrane receptors and their interaction with other proteins. Platelet activation appears to cause a structural change of the glycoprotein IIb/IIIa complex that exposes the fibrinogen binding site, which subsequently binds fibrinogen. Fluorescence-activated flow cytometry (FACS) is an efficient method for studying membrane proteins. Flow cytometry gives single-cell data, allowing the detection of only a small proportion of labelled platelets in whole blood without any washing steps. One problem with this method is that the labelled antibodies and the antigen, if present in plasma, form an immune complex, which may cause false positive reactions due to interaction between mammalian IgG and Fcγ receptors on the platelets.We show that immune complexes with chicken IgG do not activate human platelets. We have developed a method for measuring platelet-bound fibrinogen in whole blood and platelet-rich plasma utilising fluorescein isothiocyanate (FITC)-conjugated chicken antibodies directed towards human fibrinogen. As low as 1% activated platelets could be detected without interference from Fc-interactions.
We used two-color and three-color flow cytometric analysis to study phenotypical activation and functional subsets of T and natural killer cells in the blood and liver tissue of patients with primary biliary cirrhosis, other chronic liver diseases and the blood of healthy subjects. The changes in blood lymphocyte phenotype in patients with primary biliary cirrhosis and other chronic liver diseases were similar and comprised elevated relative or absolute numbers of activated human leukocyte antigen-DR + T subset (CD4+ and CD8+) cells and DR+ natural killer-like (CD16+) cells. B cell (CD19+) numbers were normal. In primary biliary cirrhosis a selective reduction in T cells of suppressor-inducer (CD45RA + CD4 + ) type was registered. The human leukocyte antigen-DR expression among CD4+ T cell subsets was investigated further in primary biliary cirrhosis and healthy controls using triple antibody flow cytometric analysis. Phenotypical cell activation was confined to helper T cells of the primed, memory (CD45RO + CD4+) type. The decrease in suppressor-inducer T cells in primary biliary cirrhosis was paralleled by a reciprocal increase in primed memory T cells. Several significant differences were observed when blood and liver-infiltrating cells from primary biliary cirrhosis patients were compared. In the liver tissue, the CD4/CD8 ratio was decreased, the relative activation of T-subset cells and NK cells was further increased, the suppressor-inducer T subset was further depressed and the primed memory T subset was increased. The cytotoxic T-cell subset (CD11b-) dominated within the CD8+ population. In liver tissue from other chronic liver disease subjects, a lower CD4/CD8 ratio was found compared with primary biliary cirrhosis.(ABSTRACT TRUNCATED AT 250 WORDS)
An investigation of proliferation and activation events i n subsets of human CD4+ cells, delhed by their expression of CD45RA and CD45R0, Is reported. A single-laser based assay for the study of multiple surface antigens and two-parameter cell cycle analysis was used for sorting of and subsequent Key terms: BrdUrd-PI, CD45 isoforms, activation/ adhesion markers, phenotype switching, three-colour immunophenotyping, multiparameter sorting, mitogenic stimulation Flow cytometry (FCM) has evolved into an extremely powerful tool in research as well as clinical environments, with the major fields of application being immunophenotyping and cell cycle analysis. However, the potentials of FCM are not being fully utilized until these two fields can be combined, that is, cell surface antigen staining assayed simultaneously with cell cycle analysis. Numerous such methods have been described, yielding information on cycling patterns of different subsets (8,12,17,24) or phenotypic characteristics of cells at different stages in the cell cycle (26). Such investigations have commonly required dual laser instrumentation (12,31) while still being limited to one proliferation parameter. As the complexity of phenotypic interrelationships increases and the cell cycle phases are further dissected (IS), these limitations prove to be major shortcomings.The aim of this study was to investigate subset-specific differences in proliferation-and activation-related parameters among subsets of human CD4 cells, defined by their expression of CD45 isotypes. The expression of these antigens is the result of alternative splicing of the mRNA generated from the gene for the leukocyte common antigen (LCA,CD45) ( 2 3 ) .
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