Huskens Dana, Sang Yaqiu, Konings Joke, van der Vorm Lisa, de Laat Bas, Kelchtermans Hilde, Roest Mark
Cardiovascular Research Institute Maastricht, Maastricht University Medical Centre, Maastricht, the Netherlands.
Synapse Research Institute, Maastricht, the Netherlands.
PLoS One. 2018 Feb 1;13(2):e0192079. doi: 10.1371/journal.pone.0192079. eCollection 2018.
Platelet function testing with flow cytometry has additional value to existing platelet function testing for diagnosing bleeding disorders, monitoring anti-platelet therapy, transfusion medicine and prediction of thrombosis. The major challenge is to use this technique as a diagnostic test. The aim of this study is to standardize preparation, optimization and validation of the test kit and to determine reference values in a population of 129 healthy individuals.
Platelet function tests with 3 agonists and antibodies against P-selectin, activated αIIbβ3 and glycoprotein Ib (GPIb), were prepared and stored at -20°C until used. Diluted whole blood was added and platelet activation was quantified by the density of activation markers, using flow cytometry. Anti-mouse Ig κ particles were included to validate stability of the test and to standardize results. Reference intervals were determined.
Blood stored at room temperature (RT) for up to 4h after blood donation and preheated/tested at 37°C resulted in stable results (%CV<10%), in contrast to measuring at RT. The intra-assay %CV was <5%. Incubation of anti-mouse Ig κ particles with antibodies stored for up to 12 months proved to give a stable fluorescence. The inter-individual variation measured in the 129 individuals varied between 23% and 37% for P-selectin expression and αIIbβ3 activation, respectively.
The current study contributes to the translation of flow cytometry based platelet function testing from a scientific tool to a diagnostic test. Platelet function measurements, using prepared and stored platelet activation kits, are reproducible if executed at 37°C. The reference ranges can be validated in clinical laboratories and ongoing studies are investigating if reduced platelet reactivity in patients with bleeding complications can be detected.
流式细胞术血小板功能检测对于现有血小板功能检测在诊断出血性疾病、监测抗血小板治疗、输血医学及血栓形成预测方面具有附加价值。主要挑战在于将该技术用作诊断测试。本研究的目的是对测试试剂盒的制备、优化及验证进行标准化，并确定129名健康个体的参考值。
制备了针对P-选择素、活化的αIIbβ3和糖蛋白Ib（GPIb）的3种激动剂及抗体的血小板功能检测试剂，并储存于-20°C直至使用。加入稀释的全血，使用流式细胞术通过活化标志物密度对血小板活化进行定量。加入抗小鼠Ig κ颗粒以验证检测的稳定性并使结果标准化。确定参考区间。
献血后在室温（RT）储存长达4小时并在37°C预热/检测的血液产生了稳定的结果（变异系数%CV<10%），与在RT下测量相反。批内变异系数%CV<5%。抗小鼠Ig κ颗粒与储存长达12个月的抗体孵育证明可产生稳定的荧光。在129名个体中测量的个体间变异对于P-选择素表达和αIIbβ3活化分别在23%至37%之间。
本研究有助于将基于流式细胞术的血小板功能检测从科学工具转化为诊断测试。使用制备并储存的血小板活化试剂盒进行血小板功能测量，如果在37°C下执行则具有可重复性。参考范围可在临床实验室中得到验证，并且正在进行的研究正在调查是否能够检测出有出血并发症患者的血小板反应性降低。
