Comparative diagnostic accuracy between simplified and original flow cytometric gating strategies for peripheral blood neutrophil myeloperoxidase expression in ruling out myelodysplastic syndromes

Tatiana Raskovalova, Laura Scheffen, Marie-Christine Jacob, Claire Vettier, Bénédicte Bulabois, Gautier Szymanski, Simon Chevalier, Nicolas Gonnet, Sophie Park, José Labarère, Tatiana Raskovalova, Laura Scheffen, Marie-Christine Jacob, Claire Vettier, Bénédicte Bulabois, Gautier Szymanski, Simon Chevalier, Nicolas Gonnet, Sophie Park, José Labarère

Abstract

Background: Flow cytometric analysis of peripheral blood neutrophil myeloperoxidase expression is accurate in ruling out myelodyplastic syndromes (MDS) but might not be suitable for implementation in busy clinical laboratories. We aimed to simplify the original gating strategy and examine its accuracy.

Methods: Using the individual data from 62 consecutive participants enrolled in a prospective validation study, we assessed the agreement in intra-individual robust coefficient of variation (RCV) of peripheral blood neutrophil myeloperoxidase expression and compared diagnostic accuracy between the simplified and original gating strategies.

Results: Cytomorphological evaluation of bone marrow aspirate confirmed MDS in 23 patients (prevalence, 37%), unconfirmed MDS in 32 patients (52%), and was uninterpretable in 7 patients (11%). Median intra-individual RCV for simplified and original gating strategies were 30.7% (range, 24.7-54.4) and 30.6% (range, 24.7-54.1), with intra-class correlation coefficient quantifying absolute agreement equal to 1.00 (95% confidence interval [CI], 0.99 to 1.00). The areas under the receiver operating characteristic (ROC) curves were 0.93 (95% CI, 0.82-0.98) and 0.92 (95% CI, 0.82-0.98), respectively (P = .32). Using simplified or original gating strategy, intra-individual RCV values lower than a pre-specified threshold of 30.0% ruled out MDS for 35% (19 of 55) patients, with both sensitivity and negative predictive value estimates of 100%.

Conclusions: The simplified gating strategy performs as well as the original one for ruling out MDS and has the potential to save time and reduce resource utilization. Yet, prospective validation of the simplified gating strategy is warranted before its adoption in routine.

Trial registration: ClinicalTrials.gov Identifier: NCT03363399 (First posted on December 6, 2017).

Conflict of interest statement

The authors have declared that no competing interests exist.

Copyright: © 2022 Raskovalova et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Figures

Fig 1. Original flow cytometric gating strategy…
Fig 1. Original flow cytometric gating strategy for quantifying peripheral blood neutrophil myeloperoxidase expression.
CD45+ cells were first individualized by crossing the singlet gate (A), FSC-SSC leukocytes (B), and CD45-positive gate (C). Three populations including granulocytes (CD15+ CD14-), monocytes (CD14+ CD15dim/-), and lymphocytes (CD15- CD14-) were identified (D). Eosinophils were individualized by CD45high CD16 low (E). Mature neutrophils were visualized by [CD15+ CD14-] [CD45low CD16 high] [CD16+ CD11b+] and selected by Boolean intersection: [CD15+ CD14-] [CD16+ CD11b+] with exclusion of [CD45high CD16 low] [CD14+ CD15dim/-] [CD15- CD14-] (F). RCV for MPO was estimated on the MPO gate conditioned on all visualized granulocytes (red dots) without threshold (G). The populations identified were lymphocytes (purple), monocytes (green), eosinophils (orange), MPO mature neutrophils (red). Abbreviations: CD = cluster of differentiation; FSC-A = forward scatter area; FSC-H = forward scatter height; MPO = myeloperoxidase; RCV = robust coefficient of variation; SSC-A = side scatter area; SSC-H = side scatter height.
Fig 2. Simplified flow cytometric gating strategy…
Fig 2. Simplified flow cytometric gating strategy for quantifying peripheral blood neutrophil myeloperoxidase expression.
CD45+ cells were first individualized by crossing the singlet gate (A) and CD45 positive gate (B). Population of granulocytes (CD15+ CD14-) was identified (C). Gated cells were selected based on CD16/CD11b double positivity (D) The MPO SSC dot plot (E) was used only to visualize MPO expression of mature neutrophils. The cell population identified was MPO mature neutrophils (red dots). Abbreviations: CD = cluster of differentiation; FSC-A = forward scatter area; FSC-H = forward scatter height; MPO = myeloperoxidase; RCV = robust coefficient of variation; SSC-A = side scatter area; SSC-H = side scatter height.
Fig 3. Agreement in continuous intra-individual robust…
Fig 3. Agreement in continuous intra-individual robust coefficient of variation between simplified and original flow cytometric gating strategies for quantifying peripheral blood neutrophil myeloperoxidase expression (n = 62).
Abbreviations: RCV = robust coefficient of variation.

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