Levering Wilfried H B M, van Wieringen Wessel N, Kraan Jaco, van Beers Wil A M, Sintnicolaas Kees, van Rhenen Dick J, Gratama Jan W
Laboratory for Histocompatibility and Immunogenetics, Sanquin Blood Bank South West Region, Rotterdam, The Netherlands.
Cytometry B Clin Cytom. 2008 Mar;74(2):79-90. doi: 10.1002/cyto.b.20370.
A biannual external quality assessment (EQA) scheme for flow cytometric lymphocyte immunophenotyping is operational in the Benelux countries since 1996. We studied the effects of the methods used on assay outcome, and whether or not this EQA exercise was effective in reducing between-laboratory variation. Eighty test samples were distributed in 20 biannual send-outs. Per send-out, 50-71 participants were requested to enumerate CD3+, CD4+, and CD8+ T cells, B cells, and NK cells, and to provide methodological details. Participants received written debriefings with personalized recommendations after each send-out. For this report, data were analyzed using robust multivariate regression. Five variables were associated with significant positive or negative bias of absolute lymphocyte subset counts: (i) platform methodology (i.e., single-platform assays yielded lower CD4+ and CD8+ T-cell counts than did dual-platform assays); (ii) sample preparation technique (i.e., assays based on mononuclear cells isolation yielded lower T-cell counts than those based on red cell lysis); (iii) gating strategies based on CD45 and sideward scatter gating of lymphocytes yielded higher CD4+ T-cell counts than those based on "backgating" of lymphocytes guided by CD45 and CD14); (iv) stabilized samples were generally associated with higher lymphocyte subset counts than nonstabilized samples; and (v) laboratory. Platform methodology, sample stabilization, and laboratory also affected assay variability. With time, assay variability tended to decline; this trend was significant for B-cell counts only. In addition, significant bias and variability of results, independent of the variables tested for in this analysis, were also associated with individual laboratories. In spite of our recommendations, participants tended to standardize their techniques mainly with respect to sample preparation and gating strategies, but less with absolute counting techniques. Failure to fully standardize protocols may have led to only modest reductions in variability of results between laboratories.
自1996年起，一项针对流式细胞术淋巴细胞免疫表型分析的两年一次的外部质量评估（EQA）计划在比荷卢三国开展。我们研究了所使用方法对检测结果的影响，以及这项EQA活动是否有效地减少了实验室间的差异。80个测试样本分20次每两年发放一次。每次发放时，邀请50 - 71名参与者对CD3 +、CD4 +和CD8 + T细胞、B细胞和NK细胞进行计数，并提供方法学细节。每次发放后，参与者都会收到带有个性化建议的书面总结。为撰写本报告，使用稳健多元回归分析数据。有五个变量与淋巴细胞亚群绝对计数的显著正偏差或负偏差相关：（i）平台方法（即单平台检测产生的CD4 +和CD8 + T细胞计数低于双平台检测）；（ii）样本制备技术（即基于单核细胞分离的检测产生的T细胞计数低于基于红细胞裂解的检测）；（iii）基于淋巴细胞CD45和侧向散射门控的门控策略产生的CD4 + T细胞计数高于基于CD45和CD14引导的淋巴细胞“反向门控”的策略；（iv）稳定样本通常比未稳定样本的淋巴细胞亚群计数更高；以及（v）实验室。平台方法、样本稳定性和实验室也影响检测变异性。随着时间推移，检测变异性趋于下降；仅B细胞计数的这一趋势具有显著性。此外，与本分析中测试的变量无关的结果的显著偏差和变异性也与个别实验室有关。尽管我们给出了建议，但参与者倾向于主要在样本制备和门控策略方面规范他们的技术，而在绝对计数技术方面规范较少。未能完全标准化方案可能仅导致实验室间结果变异性的适度降低。
