Detection of activated platelets in whole blood using activation- dependent monoclonal antibodies and flow cytometry
Detection of activated platelets in whole blood using activation- dependent monoclonal antibodies and flow cytometry
复制标题
DOI：
10.1182/blood.v70.1.307.bloodjournal701307
复制
发表时间：
1987-07
期刊：
影响因子：
20.3
通讯作者：
S. Shattil;M. Cunningham;J. Hoxie
中科院分区：
文献类型：
--
作者：
S. Shattil;M. Cunningham;J. Hoxie
在许多临床疾病中，血小板可能被激活并参与血栓形成。我们开发了一种使用流式细胞术直接检测全血中活化血小板的方法。将全血与生物素-PAC 1（一种对活化血小板上的纤维蛋白原受体具有特异性的单克隆抗体）或生物素-S12（一种对分泌期间与血小板表面结合的α-颗粒膜蛋白具有特异性的抗体）一起孵育。血小板结合抗体用链霉亲和素与异硫氰酸荧光素（FITC）或藻红蛋白（PE）结合检测。血小板与较大的红细胞和白细胞通过其光散射谱进行区分。或者，可以用FITC-AP 1（一种对血小板膜糖蛋白Ib具有特异性的抗体）鉴定血小板，并进一步分析PAC 1或S12与PE-链霉亲和素的结合。不需要离心或洗涤步骤。对于凝胶过滤血小板，ADP诱导的生物素-PAC 1结合与常规125 I-PAC 1结合试验中测定的结合之间存在直接相关性（r = 0.99; P小于0.001）。此外，当活化血小板与未刺激的血小板混合时，流式细胞术可检测到低至0.8%的活化血小板。在全血中，未刺激的血小板没有显示出PAC 1或S12特异性荧光，这表明它们不结合这些抗体。然而，在用激动剂刺激时，血小板表现出与血浆或缓冲液中血小板所观察到的荧光类似的荧光的剂量依赖性增加。低浓度的ADP和肾上腺素，诱导纤维蛋白原受体，但很少分泌，刺激近最大的PAC 1结合，但很少S12结合。在另一方面，佛波醇肉豆蔻酸酯醋酸酯（TPA），引起充分的血小板聚集和分泌诱导最大的PAC 1和S12结合的浓度。活化的血小板也可以在用多聚甲醛固定的全血样品中进行分析。这些研究表明，活化血小板可以可靠地检测全血中使用活化依赖性单克隆抗体和流式细胞术。这项技术可能是有用的，以评估血小板活化的程度和抗血小板治疗的疗效在临床疾病。
Platelets may become activated in a number of clinical disorders and participate in thrombus formation. We developed a direct test for activated platelets in whole blood using flow cytometry. Whole blood was incubated with either biotin-PAC1, a monoclonal antibody specific for the fibrinogen receptor on activated platelets, or biotin-S12, an antibody specific for an alpha-granule membrane protein that associates with the platelet surface during secretion. Platelet-bound antibodies were detected with streptavidin conjugated with fluorescein isothiocyanate (FITC) or phycoerythrin (PE). Platelets were differentiated from the larger erythrocytes and WBCs by their light- scatter profile. Alternatively, platelets could be identified with FITC- AP1, an antibody specific for platelet membrane glycoprotein Ib, and analyzed further for PAC1 or S12 binding with PE-streptavidin. No centrifugation or washing steps were required. With gel-filtered platelets, there was a direct correlation between ADP-induced biotin- PAC1 binding and binding determined in a conventional 125I-PAC1 binding assay (r = .99; P less than .001). Furthermore, as few as 0.8% activated platelets could be detected by flow cytometry when activated platelets were mixed with unstimulated platelets. In whole blood, unstimulated platelets demonstrated no PAC1- or S12-specific fluorescence, indicating that they did not bind these antibodies. On stimulation with agonists, however, the platelets demonstrated a dose- dependent increase in fluorescence similar to that observed for platelets in plasma or buffer. Low concentrations of ADP and epinephrine, which induce fibrinogen receptors but little secretion, stimulated near-maximal PAC1 binding but little S12 binding. On the other hand, a concentration of phorbol myristate acetate (TPA) that evokes full platelet aggregation and secretion induced maximal PAC1 and S12 binding. Activated platelets could also be analyzed in whole blood samples that had been fixed with paraformaldehyde. These studies demonstrate that activated platelets can be reliably detected in whole blood using activation-dependent monoclonal antibodies and flow cytometry. This technique may be useful to assess the degree of platelet activation and the efficacy of antiplatelet therapy in clinical disorders.