Janes S L, Wilson D J, Chronos N, Goodall A H
Haemophilia Centre, Royal Free Hospital and School of Medicine, London, UK.
Thromb Haemost. 1993 Oct 18;70(4):659-66.
Activated platelets can be detected by measuring platelet-bound fibrinogen in a whole blood, flow cytometric assay, using a fluorescently-conjugated polyclonal antibody. Fibrinogen binding to unstimulated platelets from normal subjects was low in this assay, as was expression of the CD63 antigen. Single cell counting of samples prepared for flow cytometric analysis showed platelet aggregates do not form during the assay procedure. Immune complexes were not seen, and fibrinogen binding to the platelets was unaffected by the CD32 MAb, IV.3. Artefactual activation of the unfixed samples could be minimised by control of phlebotomy, time and temperature of incubation. Variations in platelet count in the range 140-430 x 10(9) 1(-1) and in plasma fibrinogen in the range 2-6 g 1(-1) did not affect the assay results. Comparison of fibrinogen binding with expression of CD63 antigen on normal platelets, stimulated with agonists in vitro, demonstrated that fibrinogen binding detects an earlier stage of platelet activation. Platelet bound fibrinogen was shown to be sensitive in detecting small numbers of activated platelets in clinical samples in twelve patients on intensive care, four undergoing haemofiltration. The patients had a significantly higher median percentage of circulating platelets with bound fibrinogen (p < 0.005), but fibrinogen binding was significantly lower (p < 0.02) in response to 10(-5) M ADP, compared to twelve age-matched normal controls.
通过使用荧光偶联多克隆抗体的全血流式细胞术检测血小板结合的纤维蛋白原，可以检测活化的血小板。在该检测中，正常受试者未受刺激的血小板与纤维蛋白原的结合较低，CD63抗原的表达也是如此。对用于流式细胞术分析的样品进行单细胞计数显示，在检测过程中不会形成血小板聚集体。未观察到免疫复合物，并且血小板与纤维蛋白原的结合不受CD32单克隆抗体IV.3的影响。通过控制静脉穿刺、孵育时间和温度，可以将未固定样品的人为活化降至最低。血小板计数在140 - 430×10⁹/L范围内以及血浆纤维蛋白原在2 - 6g/L范围内的变化不会影响检测结果。将纤维蛋白原结合与体外经激动剂刺激的正常血小板上CD63抗原的表达进行比较，结果表明纤维蛋白原结合检测到血小板活化的早期阶段。在12名重症监护患者和4名接受血液滤过的患者的临床样本中，血小板结合的纤维蛋白原被证明对检测少量活化血小板很敏感。与12名年龄匹配的正常对照相比，这些患者循环中结合纤维蛋白原的血小板的中位数百分比显著更高（p < 0.005），但对10⁻⁵M ADP的反应中纤维蛋白原结合显著更低（p < 0.02）。
