Augustine Tanya N, van der Spuy Wendy J, Kaberry Lindsay L, Shayi Millicent
School of Anatomical Sciences, Faculty of Health Sciences,University of the Witwatersrand,Johannesburg,South Africa.
Microsc Microanal. 2016 Jun;22(3):630-9. doi: 10.1017/S1431927616000854.
Platelet ultrastructural alterations representing spurious activation have been identified in pathological conditions. A limitation of platelet studies is that sample preparation may lead to artifactual activation processes which may confound results, impacting the use of scanning electron microscopy as a supplemental diagnostic tool. We used scanning electron microscopy and flow cytometry to analyze platelet activation in platelet-rich plasma (PRP) and whole blood (WB) samples. PRP generated using a single high g force centrifugation, and WB samples treated with a red blood cell lysis buffer, were exposed to increasing concentrations of the agonist thrombin. Platelets in lysed WB samples responded to thrombin by elevating the activation marker CD62p definitively, with corresponding ultrastructural changes indicating activation. Conversely, CD62p expression in PRP preparations remained static. Ultrastructural analysis revealed fully activated platelets even under low concentration thrombin stimulation, with considerable fibrin deposition. It is proposed that the method for PRP production induced premature platelet activation, preventable by using an inhibitor of platelet aggregation and fibrin polymerization. Nevertheless, our results show a definitive correspondence between flow cytometry and scanning electron microscopy in platelet activation studies, highlighting the potential of the latter technique as a supplemental diagnostic tool.
在病理状态下已发现代表假性激活的血小板超微结构改变。血小板研究的一个局限性在于样本制备可能会导致人为的激活过程，这可能会混淆结果，影响扫描电子显微镜作为辅助诊断工具的应用。我们使用扫描电子显微镜和流式细胞术来分析富血小板血浆（PRP）和全血（WB）样本中的血小板激活情况。使用单次高重力离心产生的PRP以及用红细胞裂解缓冲液处理的WB样本，分别暴露于浓度不断增加的激动剂凝血酶中。裂解的WB样本中的血小板通过明确升高激活标志物CD62p来响应凝血酶，同时相应的超微结构变化表明发生了激活。相反，PRP制剂中CD62p的表达保持不变。超微结构分析显示，即使在低浓度凝血酶刺激下也有完全激活的血小板，并有大量纤维蛋白沉积。有人提出，PRP生产方法会导致血小板过早激活，使用血小板聚集和纤维蛋白聚合抑制剂可预防这种情况。尽管如此，我们的结果表明在血小板激活研究中流式细胞术和扫描电子显微镜之间存在明确的对应关系，突出了后一种技术作为辅助诊断工具的潜力。
