A-257 Monocyte Subset Repartitioning by Flow Cytometry: A Three-Year Retrospective Study Differentiating Chronic Myelomonocytic Leukemia from Other Causes of Monocytosis

Abstract

Chronic myelomonocytic leukemia (CMML) is a rare, non-classical chronic myeloproliferative neoplasm whose diagnostic criteria were updated by the World Health Organization (WHO) in 2022 incorporating peripheral blood (PB) monocyte subset analysis by flow cytometry (FCM) into the diagnostic framework. An increased proportion of classical monocytes (MO1; CD14++/ CD16-) =94% facilitates the differential diagnosis among causes of monocytosis, particularly in settings with limited access to molecular testing. The aim of this study was to describe the experience of a Brazilian diagnostic laboratory applying monocyte subset analysis by FCM in patients presenting with monocytosis. This retrospective study included 25 patients with suspected CMML evaluated between January 2022 and March 2025. Complete blood counts, karyotyping, and BCR-ABL testing (when available) were reviewed, along with FCM performed on PB (n=14) or bone marrow (n=11) samples. FCM acquisition was performed using a 13-color DxFLEX Flow Cytometer (Beckman Coulter). Daily instrument performance and fluorescence stability were monitored according to manufacturer recommendations and internal quality control procedures. The antibody panel included CD3, CD4, CD8, CD11b, CD13,CD14, CD16, CD19, CD33, CD34, CD36, CD45, CD56, CD64, IREM, and HLA-DR, and was internally validated according to the 2022 WHO classification. Up to 100,000 leukocyte events were acquired, depending on sample quality. Doublets were excluded using forward scatter height versus area parameters, and debris was removed based on FSC/SSC properties. Leukocytes were identified by CD45 expression and SSC characteristics. Monocyte subsets were defined by CD14 and CD16 expression as MO1 (CD14++/CD16-), MO2 (CD14+/CD16+), and MO3 (CD14low/-/CD16+). Patients were classified as “suspected CMML” when MO1 =94% was observed in association with absolute monocytosis =1,000/µL and =10% monocytes in PB. Among the 25 patients (age range: 15–87 years), four (16%) demonstrated a PB immunophenotypic profile suggestive of CMML, with MO1 =94% and monocytosis =1,000/µL and =10%. Among these patients, one had negative BCR-ABL result and another presented a normal karyotype (46,XY); the remaining two lacked molecular and cytogenetic data. Four patients (16%) exhibited a pattern compatible with reactive monocytosis, characterized by proportional increases in MO2 and MO3 subsets. The remaining 17 patients (68%) were classified as indeterminate based solely on monocyte subset distribution. Monocyte subset repartitioning by FCM, particularly a classical monocyte proportion =94% in PB, is highly suggestive of CMML and effectively distinguishes clonal from reactive monocytosis, with high reported sensitivity and specificity. In our cohort, the identifying of 16% of patients as suggestive of CMML and 16% as probable reactive monocytosis supports the applicability of this method in a real-world Brazilian laboratory setting. Study limitations include the use of bone marrow samples in 44% of cases, as monocyte subset distribution in marrow differs from PB and is not included in WHO diagnostic criteria; notably, none of the CMML suggestive cases were marrow-based. The lack of comprehensive molecular and cytogenetic data also limited definitive classification. Nevertheless, FCM proved to be a robust, reproducible, and accessible diagnostic tool, playing a central role in the structured evaluation of monocytosis and in guiding further molecular investigation, particularly in resource-limited settings.