双平台流式细胞术检测外周血淋巴细胞亚群生物学变异的研究

Biological variations of peripheral blood lymphocytesusing flow cytometric double-platform method

目的探讨双平台流式细胞术（FCM）检测正常人群淋巴细胞亚群八项指标绝对计数的生物学变异。方法2013年9月在北京协和医院募集20名健康志愿者，于研究的第1天、第3天及第5天的上午8：00、中午12：00及下午16：00采集志愿者静脉血，进行双平台流式细胞术淋巴细胞亚群八项指标绝对计数的检测。采用标准化的分析前处理流程，使用规范校准的血液分析仪和流式细胞仪进行检测，研究期间均使用同一批号试剂进行样本的前处理和检测。采用SPSS13．0软件中的巢式方差分析计算八项指标百分含量和绝对计数的个体内变异、个体间变异，采用Excel2003计算分析变异、个体指数和参考变化值。各指标的日内和日间差异采用One-WayANOVA检验及方差分析进行比较，两组间的比较采用独立样本t检验。结果T细胞（CD3’）、辅助性T细胞（CD3＋CD4＋CD8-）、抑制性T细胞（CD3＋CD4-CD8=）、B细胞（CD3-CD19＋）百分含量的个体内变异和个体间变异与文献报道相近，各指标的个体内变异分别为0．03、0．06、0．05、0．14，个体间变异分别为0．12、0．16、0．23、0．31。以上4项指标百分含量的个体指数和95％的参考变化值与文献差异较大，个体指数分别为0．26、0．40、0．22、0．44，95％的参考变化值分别为8．77、16．86、14．93、39．69。所研究的淋巴细胞亚群八项指标绝对计数的个体内变异、个体间变异及分析变异均比其相应的百分含量变异大。含量低的三类细胞亚群（CD3＋CIM—CD8-、CD3＋CD4＋CD8＋、CD3＋CD16＋CD56＋）的个体内变异、个体间变异及分析变异相对较大，其中CD3＋CD4-CD8-的个体内变异、个体间变异及分析变异分别为0．12、0．49、0．16，CD3＋CD4＋CD8＋的分别为0．40、0．93、0．55，CD3＋CD16＋CD56＋的分别为0．28、1．11、0．16。结论通过研究淋巴细胞亚群绝对计数的个体内变异、个体间变异及分析变异可以计算个体指数和参考变化值，从而能够确定相应指标参考区间的实用性和判断连续检测结果之间的差异是否有显著性。

Objective To evaluate the biological variations of 8 lymphocyte subsets using flow cytometric double-platform method. Methods Twenty healthy adults were recruited from Peking Union Medical College Hospital in September 2013. At 8：00 AM, 12：00 PM, and 4：00 PM on days 1,3, and 5, venous blood was collected from the volunteers. The percent and absolute lymphocyte subset counts were measured using duel-platform method. The sample collection and handling techniques were standardized. Before each batch analysis, the instrument quality controls were performed using the same lots of reagents. The intra-individual coefficient of variation （CV1 ） and inter-individual coefficient of variation （CVG） were calculated by nested ANOVA with SPSS 13.0 software. The analytical coefficient of variation （CVA） ,index of individuality （II） and reference change value （RCV） were calculated by Excel2003. A mean pairwise comparison was determined by one-way ANOVA and variance analysis. The values between groups were analyzed by independent sample t test. Results For T cells （ CD3＋ ）, helper T cells （ CD3 ＋ CD4＋ CD8 - ）, suppressor T cells （ CD3 ＋ CIM - CD8＋ ） and B cells （ CD3 - CD19 ＋ ）, the intra-individual coefficient of variation （ CVI ） and inter-individual coefficient of variation （ CVG ） were O. 03,0. 06,0.05, 0. 14 and 0. 12,0. 16,0. 23,0. 31 respectively,which were all similar to those in previous studies. However, the II and RCV of the four lymphocyte subsets were very different from those in previous studies, which were 0. 26 ,0. 40 ,0. 22 ,0. 44 and 8.77,16. 86,14.93,39.69, respectively. Moreover, variations in absolute count, CVI, CVG, and analysis coefficient of variation （ CVA ） of all 8 lymphocyte subsets were greater than those of relative count. Variations in the percent and absolute counts for the CD3 ＋ CD4 - CD8 - , CD3 ＋ CD4 ＋ CD8 ＋ , and CD3 ＋ CD16＋ CD56＋ cell subsets were relatively high. The CV1, CVG and CVA for the cells of CD3 ＋ CIM- CD8 - were 0. 12,0. 49 and 0. 16. The CVI, CVG and CVA for the cells of CD3 ＋ CD4＋ CD8 ＋ were 0. 40,0. 93 and 0. 55. The CVI, CVG and CVA for the cells of CD3 ＋ CD16 ＋ CD56 ＋ were 0. 28,1.11 and 0. 16. Conclusions Investigation on the CVI, CVG and CVA may allow us to obtain II and RCV, by which we can determine the utility of traditional population based reference ranges. Documentation of the RCV indices may be used as objective delta-check values in quality management and decide whether clinical significance existed in the continuously detected results.