Department of Laboratory Medicine, Ghent University Hospital, Ghent, Belgium.
Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Leiden, Netherlands.
Front Immunol. 2020 Nov 2;11:584646. doi: 10.3389/fimmu.2020.584646. eCollection 2020.
Multiparameter flow cytometry (FC) is essential in the diagnostic work-up and classification of primary immunodeficiency (PIDs). The EuroFlow PID Orientation tube (PIDOT) allows identification of all main lymphocyte subpopulations in blood. To standardize data analysis, tools for Automated Gating and Identification (AG&I) of the informative cell populations, were developed by EuroFlow. Here, we evaluated the contribution of these innovative AG&I tools to the standardization of FC in the diagnostic work-up of PID, by comparing AG&I against expert-based (EuroFlow-standardized) Manual Gating (MG) strategy, and its impact on the reproducibility and clinical interpretation of results.
FC data files from 44 patients (13 CVID, 12 PID, 19 non-PID) and 26 healthy donor (HD) blood samples stained with PIDOT were analyzed in parallel by MG and AG&I, using Infinicyt™ software (Cytognos). For comparison, percentage differences in absolute cell counts/µL were calculated for each lymphocyte subpopulation. Data files showing differences >20% were checked for their potential clinical relevance, based on age-matched percentile (p5-p95) reference ranges. In parallel, intra- and inter-observer reproducibility of MG vs AG&I were evaluated in a subset of 12 samples.
The AG&I approach was able to identify the vast majority of lymphoid events (>99%), associated with a significantly higher intra- and inter-observer reproducibility compared to MG. For most HD (83%) and patient (68%) samples, a high degree of agreement (<20% numerical differences in absolute cell counts/µL) was obtained between MG and the AG&I module. This translated into a minimal impact (<5% of observations) on the final clinical interpretation. In all except three samples, extended expert revision of the AG&I approach revealed no error. In the three remaining samples aberrant maturation and/or abnormal marker expression profiles were seen leading in all three cases to numerical alarms by AG&I.
Altogether, our results indicate that replacement of MG by the AG&I module would be associated with a greater reproducibility and robustness of results in the diagnostic work-up of patients suspected of PID. However, expert revision of the results of AG&I of PIDOT data still remains necessary in samples with numerical alterations and aberrant B- and T-cell maturation and/or marker expression profiles.
多参数流式细胞术（FC）是原发性免疫缺陷（PID）诊断和分类的重要手段。EuroFlow PID 定向管（PIDOT）可用于识别血液中的所有主要淋巴细胞亚群。为了标准化数据分析，EuroFlow 开发了用于自动门控和信息细胞群识别（AG&I）的工具。在这里，我们通过比较基于专家的（EuroFlow 标准化）手动门控（MG）策略，以及其对结果的可重复性和临床解释的影响，评估了这些创新的 AG&I 工具对 PID 诊断工作中 FC 标准化的贡献。
使用 Infinicyt™软件（Cytognos）平行分析了 44 例患者（13 例 CVID、12 例 PID、19 例非 PID）和 26 例健康供者（HD）血液样本的 PIDOT 染色 FC 数据文件，采用 MG 和 AG&I。为了比较，计算了每个淋巴细胞亚群的绝对细胞计数/µL 的百分比差异。根据年龄匹配的百分位数（p5-p95）参考范围，对数据文件中显示差异>20%的内容进行了潜在临床相关性检查。同时，在 12 个样本的子集上评估了 MG 与 AG&I 的内和间观察者重现性。
AG&I 方法能够识别绝大多数淋巴样事件（>99%），与 MG 相比，其具有更高的内和间观察者重现性。对于大多数 HD（83%）和患者（68%）样本，MG 和 AG&I 模块之间获得了高度一致（绝对细胞计数/µL 的数值差异<20%）。这转化为对最终临床解释的最小影响（<5%的观察值）。除了三个样本外，对 AG&I 方法的扩展专家审查没有发现错误。在其余三个样本中，观察到异常成熟和/或异常标记表达谱，在所有三个病例中，AG&I 都会产生数值警报。
总的来说，我们的结果表明，用 AG&I 模块替代 MG 将与 PID 疑似患者诊断工作中结果的更高重现性和稳健性相关。然而，在数值改变和异常 B 细胞和 T 细胞成熟和/或标记表达谱的样本中，仍然需要对 AG&I 的 PIDOT 数据结果进行专家审查。
