Department of Hematology, Hospital General Universitario Gregorio Marañón, Instituto de Investigación Sanitaria Gregorio Marañón (IiSGM), Madrid, Spain.
Department of Hematology, Hospital Universitario de Guadalajara, Guadalajara, Spain.
Cytometry B Clin Cytom. 2018 May;94(3):527-535. doi: 10.1002/cyto.b.21538. Epub 2017 Jul 6.
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.
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.
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%.
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.
尽管骨髓（BM）原始细胞计数对于预后至关重要，但在低危骨髓增生异常综合征（MDS）中仍难以准确计数。我们研究了流式细胞免疫表型（FCI）中 CD34+髓细胞的计数是否可作为临床 MDS 研究的一致参数。
六个临床中心参与了该研究，并记录了他们的 FCI 方案信息。在两轮实验中，参与者之间交换了 67 个低危 MDS 患者 BM 样本中＜5%BM 原始细胞的 FCI 列表模式。计算了 CD34+髓细胞定量的实验室间变异性，并评估了解决差异的策略。
观察到参与者之间 CD34+细胞计数总体上具有“非常好”的一致性（组内相关系数=0.720），但在 22 个文件中一致性“低”。没有单个参数可以完全解释所有差异，但确定了 3 个相关技术问题：使用 CD34/CD45/CD117/HLA-DR mAb 组合、获取≥50000 个事件和低比例的碎片/聚集物。不一致结果的频率随着陷阱的积累而增加（无陷阱，16%；1 个陷阱，40%；2 个陷阱，83%；P=0.006）。最后，使用通用的分析门控策略将“非常好”的一致性文件百分比提高到 100%。
为了达到中心间 CD34+细胞计数的良好可重复性，必须预防特定的技术陷阱。这些建议为实验室标准化奠定了基础，并使 CD34+细胞计数能够作为低危 MDS 患者的附加信息使用。©2017 国际临床细胞化学协会。
