Optimization of monocyte gating to quantify monocyte subsets for the diagnosis of chronic myelomonocytic leukemia

Abstract

BackgroundThe presence of >94% classical monocytes (MO1, CD14++/CD16‐) in peripheral blood (PB) has an excellent performance for the diagnosis of chronic myelomonocytic leukemia (CMML). However, the monocyte gating strategy is not well defined. The objective of the study was to compare monocyte gating strategies and propose an optimal one.MethodsThis is a prospective, single center study assessing monocyte subsets in PB. First, we compared monocyte subsets using 13 monocyte gating strategies in 10 samples. Then we developed our own 10 color tube and tested it on 124 patients (normal white blood cell counts, reactive monocytosis, CMML and a spectrum of other myeloid malignancies). Both conventional and computational (FlowSOM) analyses were used.ResultsComparing different monocyte gating strategies, small but significant differences in %MO1 and percentually large differences in %MO3 (nonclassical monocytes) were found, suggesting that the monocyte gating strategy can impact monocyte subset quantification. Then, we designed a 10‐color tube for this purpose (CD45/CD33/CD14/CD16/CD64/CD86/CD300/CD2/CD66c/CD56) and applied it to 124 patients. This tube allowed proper monocyte gating even in highly abnormal PB. Computational analysis found a higher %MO1 and lower %MO3 compared to conventional analysis. However, differences between conventional and computational analysis in both MO1 and MO3 were globally consistent and only minimal differences were observed when comparing the ranking of patients according to %MO1 or %MO3 obtained with the conventional versus the computational approach.ConclusionsThe choice of monocyte gating strategy appears relevant for the monocyte subset distribution test. Our 10‐color proposal allowed satisfactory monocyte gating even in highly abnormal PB. Computational analysis seems promising to increase reproducibility in monocyte subset quantification.

Technical, gating and interpretation recommendations for the partitioning of circulating monocyte subsets assessed by flow cytometry

“…These results were first confirmed by a single center study from Eric Padron's team, which reported a 86% specificity and 100% sensitivity based on 29 CMML patients (Talati et al, 2017). Three other studies highlighted the high specificity of the 94% cMo cut‐off, from 94% to 95% (Hudson et al, 2018; Jurado et al, 2023; Pophali et al, 2019), yet with lower sensitivity, 88% based on 16 CMML cases (Hudson et al, 2018) and 75% based on 43 CMML cases (Pophali et al, 2019). A French oligocenter study using data from real life conditions also confirmed the good performances obtained with the 94% threshold, with a specificity of 89.7% and a sensitivity of 93.6% with 47 CMML patients included (Tarfi et al, 2018).…”

“…Abnormal partitioning of peripheral blood monocyte subset is now recognized as an essential tool for the screening of monocytosis in order to distinguish CMML from reactive monocytosis (Khoury et al, 2022) (Talati et al, 2017). Three other studies highlighted the high specificity of the 94% cMo cut-off, from 94% to 95% (Hudson et al, 2018;Jurado et al, 2023;Pophali et al, 2019), yet with lower sensitivity, 88% based on 16 CMML cases (Hudson et al, 2018) and 75% based on 43 CMML cases (Pophali et al, 2019).…”

“…Hence, computational analysis may be helpful. A first attempt for unsupervised analysis using FlowSOM was recently performed and revealed a greater cMo percentage compared to conventional analysis (Jurado et al, 2023).…”

“…An independent study validated this approach in real life conditions yet experienced poor interobserver concordance for ncMo percentages, hence limiting its useability (Barge et al, 2023). Recently, an elegant comparison of 13 gating strategies highlighted the variation of the ncMo quantification (Jurado et al, 2023).…”

Issue highlights—July 2023

“…The last article in this issue, Jurado et al focused on optimizing the monocyte gating strategy for diagnosing chronic myelomonocytic leukemia (CMML). They proposed a 10‐color tube and computational analysis to improve the reproducibility of monocyte subset quantification (Jurado et al, 2022). Further studies highlight the robustness of the “monocyte assay” for CMML diagnosis, the complexities in distinguishing CMML from blastic plasmacytoid dendritic cell neoplasm (BPDCN), and the potential of immunophenotypic analysis in differentiating CMML from reactive monocytosis (Espasa et al, 2021; Feng et al, 2018; Wagner‐Ballon et al, 2023).…”