摘要
目的 :探索流式细胞术检测血小板膜抗原表达的最佳标本制备方法。方法 :48例正常成人资料随机进入由不同标本类型、抗凝剂、标本放置时间和标本处理顺序组成的正交实验表 ,用流式细胞仪分别检测外周血血小板膜CD41a、CD42b和CD6 2P的表达情况。结果 :CD41a在PRP、肝素抗凝、2小时放置或先染色再固定的标本中的表达强度分别高于全血、ACD抗凝、放置 15分钟或先固定后染色的标本。CD42b强度除了放置时间对其无影响外 ,其余与CD41a类似。CD6 2P阳性率在PRP、肝素抗凝或 2小时放置的标本中分别高于全血、ACD抗凝或放置 15分钟的标本 ,而先染色后固定的血小板CD6 2P阳性率低于先固定后染色的标本。另外 ,标本放置 2小时后CD6 2P荧光强度增强 ,而先固定后染色的强度低于先染色的标本。结论 :不同标本类型、抗凝剂、标本放置时间和标本处理顺序对血小板膜抗原的表达均有不同程度的影响。最佳的标本制备方法为 :ACD抗凝全血标本在短时间内用 1%多聚甲醛固定后再行免疫染色 ,此法可将血小板自身活化减到最低水平。
Objective: To search for a best specimen handling methods in measurement of platelet antigens by flow cytometry.Methods: 48 cases of healthy volunteers were randomly fitted in the orthogonal design table grouped by different sample types,anticoagulants,standing-time and handling-sequence. The membrane antigens of platelet in peripheral blood such as CD41a,CD42b and CD62P were measured by flow cytometry.Results: The fluorescent intensity of CD41a was higher in sample of PRP,heparin-anticoagulated,standing for 2 hour and fixing after dying than whole blood,ACD,standing within 15 minute and dying after fixing[(705.38±194.98),(721.40±158.16),(684.60±174.20) and (709.97±163.65) channel vs (598.94±120.57),(582.92±153.11),(619.73±161.01) and (594.35±157.19) channel]. Except that standing-time had no effect on fluorescent intensity of CD42b,the other 3 factors had similar effect as on CD41a. The positive rate of CD62P was higher in sample of PRP,heparin-anticoagulated and 2-hour standing than in sample of whole blood,ACD and 15-minute standing [(10.14±4.33),(10.17±3.99) and (10.18±4.37) channel vs (8.01±4.83),(7.99±5.10),and (7.97±4.78) channel]. Besides,it was lower in fixing after dying than dying after fixing(6.77±4.92 channel vs 11.38±3.02 channel).Conclusion: Each experimental factor such as sample type,anticoagulant,standing-time or handling-sequence has diverse effects on the expression of the platelet membrane antigens. The best specimen handling method which may have less effect on self-activated platelet in flow cytometry is dying by antibodies after fixing in whole blood sample anticoagulated by ACD within 15 minutes.
出处
《温州医学院学报》
CAS
2002年第1期16-19,共4页
Journal of Wenzhou Medical College
基金
温州市科学技术委员会科研基金项目 (KW990 0 3)
浙江省教育委员会科研基金项目 (SJ990 12 )
