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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。
Objective To establish a nine-color flow cytometry quantitative detection of human plasma derived from different sources of particles. Methods The nine-color flow cytometry (FCM) was used to quantitatively detect nine different color particles in human plasma using 8 kinds of antibodies such as CD235a, CD41a, CD45, CD34, CD66b, CD20, CD3 and CD14 and Annexin-V. Antibody monochromatic staining and single-stained staining were performed on platelet-deficient plasma samples. After compensation and voltage were determined, plasma samples of 10 normal adults were tested and analyzed from December 2014 to January 2015 Dilution test. Results The lack of color staining, the positive particle groups than single-color staining were <15%. Serial dilution plasma platelets and erythrocyte-derived particles have a good linear correlation. Repeated test, the coefficient of variation of erythrocytes, platelets, granulocytes derived particles <10%. The plasma concentrations of platelets, erythrocytes, endothelial cells, monocytes, granulocytes, B lymphocytes and T lymphocytes were all detected in 10 normal plasma samples of human platelets with the average concentration and range of 132.6 cells / μL (60.6 to 288.9 cells / μL), 35.4 cells / μL (22.0 to 99.7 cells / μL), 21.6 cells / μL (3.3 to 45.5 cells / μL), 13.9 cells / μL /L?(22.5~101.2 pcs / μL), 21.9 pL / μL (6.0~33.4 pL / μL), 1.2 pL / μL (0.7~2.8 pL / μL). Conclusion Nine-color flow cytometry was established for quantitative detection of human plasma derived from different sources of the experimental program, the method is simple and practical, suitable for clinical application.