BACKGROUND:Accuracy of bone marrow (BM) blast count in low-risk myelodysplastic syndromes (MDS) still remains a challenge though it is essential for prognosis. We investigated whether the enumeration of CD34+ myeloid cells by flow cytometry immunophenotyping (FCI) could be used as a consistent parameter for clinical MDS studies. METHODS:Six clinical centers entered the study and information on their FCI protocols was recorded. Sixty-seven flow cytometry listmodes from BM samples of patients with low-risk MDS with <5% BM blasts were exchanged among participants in two different rounds. Interlaboratory variations on the quantification of CD34+ myeloid cells were calculated and strategies to solve differences were evaluated. RESULTS:An overall "very good" agreement on CD34+ cell count among participants (intraclass correlation coefficient = 0.720) was observed, but agreement was "low" in 22 files. No single parameter could fully explain all discrepancies, but 3 technical issues were identified as relevant: the use of the CD34/CD45/CD117/HLA-DR mAb combination, acquisition of ≥50,000 events and a low percentage of debris/aggregates. The frequency of discordant results increased with the accumulation of pitfalls (none, 16%; 1 pitfall, 40%; 2 pitfalls, 83%; P = 0.006). Finally, the use of a common gating strategy for analysis increased the percentage of files with "very good" agreement to 100%. CONCLUSIONS:Prevention of specific technical pitfalls is mandatory to reach a good reproducibility of CD34+ cell count among centers. These recommendations set the basis for laboratory standardization and enable the use of CD34+ cell enumeration as additional information in low-risk MDS patients. © 2017 International Clinical Cytometry Society.

译文

背景:尽管对于预后至关重要,但低危骨髓增生异常综合征(MDS)中骨髓(BM)blast计数的准确性仍然是一个挑战。我们调查通过流式细胞仪免疫表型(FCI)的CD34髓细胞计数是否可以用作临床MDS研究的一致参数。
方法:六个临床中心参加了研究,并记录了有关其FCI方案的信息。在两个不同的阶段中,参与者之间交换了来自BM blast小于5%的低风险MDS患者的BM样本中的67种流式细胞术列表模式。计算了实验室间对CD34髓样细胞定量的变异,并评估了解决差异的策略。
结果:观察到参与者的CD34细胞计数总体“非常好”一致性(类内相关系数= 0.720),但在22个文件中一致性“低”。没有一个单一的参数可以完全解释所有差异,但是确定了3个相关的技术问题:CD34 / CD45 / CD117 / HLA-DR mAb组合的使用,≥50,000个事件的发生和碎片/聚集体的百分比低。结果不一致的频率随着陷阱的累积而增加(无,16%; 1个陷阱,40%; 2个陷阱,83%; P = 0.006)。最后,使用通用门控策略进行分析将“非常好”协议的文件百分比提高到100%。
结论:预防特定技术陷阱是强制性的,以实现中心之间CD34细胞计数的良好再现性。这些建议为实验室标准化奠定了基础,并使CD34细胞计数可作为低危MDS患者的补充信息。 ©2017国际临床细胞计量学会。

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