建立九色流式细胞术定量检测血浆中不同来源微粒

Development of Quantitatively Determining the Microparticles from Different Sources in Human Blood Plasma by Nine-color Flow Cytometry

目的建立九色流式细胞术定量检测人血浆中不同来源微粒的实验方法。 方法用CD235a、CD41a、CD45、CD34、CD66b、CD20、CD3、CD14等8种抗体和Annexin-V建立定量检测人血浆中不同来源微粒九色流式染色方案。用乏血小板血浆标本分别进行抗体的单色染色和缺一色染色实验，确定补偿和电压后，2014年12月-2015年1月对10例正常成人的血浆标本进行检测分析，并进行重复试验及稀释试验。 结果缺一色染色中，阳性微粒群较单色染色变化均＜15%。系列稀释血浆中的血小板及红细胞来源微粒具有良好的直线相关性。重复试验中，红细胞、血小板、粒细胞来源微粒的变异系数均＜10%。检测的10例正常成人乏血小板血浆标本，能分别检测到血小板、红细胞、内皮细胞、单核细胞、粒细胞、B淋巴细胞及T淋巴细胞来源微粒，其平均浓度及范围分别为132.6个/μL（60.6～288.9个/μL）、35.4个/μL（22.0～99.7个/μL）、21.6个/μL（3.3～45.5个/μL）、13.9个/μL（7.3～35.1个/μL）、60.0个/μL（22.5～101.2个/μL）、21.9个/μL（6.0～33.4个/μL）、1.2个/μL（0.7～2.8个/μL）。 结论建立了九色流式细胞术定量检测人血浆中不同来源微粒的实验方案，该方法简单实用，适合临床推广应用。

ObjectiveTo develop a method to quantitatively determine the microparticles （MP） from different sources in plasma by nine-color flow cytometry. MethodsAnnexin-V and 8 antibodies including CD235a, CD41a, CD45, CD34, CD66b, CD20, CD3 and CD14 were used to establish nine-color flow cytometric panel.Platelet poor plasma samples were single-stained and stained with 1 of 8 antibodies lacking respectively, and then we determined the detector voltages and compensations.From December 2014 to January 2015, we detected and analyzed 10 plasma samples from normal adults, and repeatability test and dilution tests were done. ResultsIn staining lacking 1 of 8 antibodies, the percentage of positive MP populations change was all less than 15% based on the population number in single-stained experiment.In dilution tests, there were good linear correlations between MPs from platelets and erythrocytes.In repeatability test, the coefficient of variation of MP from erythrocytes, platelets and granulocytes was all less than 10%.In the platelet poor plasma samples from normal adults, MP from platelets, erythrocytes, endotheliocytes, monocytes, granulocytes, B and T lymphocytes could be detected, and the average concentration of them were respectively 132.6/μL[（60.6-288.9）/μL], 35.4/μL[（22.0-99.7）/μL], 21.6/μL[（3.3-45.5）/μL], 13.9/μL[（7.3-35.1）/μL], 60.0/μL[（22.5-101.2）/μL], 21.9/μL[（6.0-33.4）/μL]and 1.2/μL[（0.7-2.8）/μL]. ConclusionsQuantitatively determining MP from different sources in plasma by nine-color flow cytometry has been successfully developed.This method is simple and fast, and can be applied in clinical detection.