Orianne Wagner-Ballon

The clinical significance of PNH Type II white blood cells (WBCs) remains unclear. We assessed the relative percentage (rel%) of Type II neutrophils in 355 patients with a PNH clone ≥ 1% on neutrophils enrolled by 33 flow cytometry laboratories in the 5‐year French nation‐wide multicenter prospective observational study. We first analyzed 127 of 133 patients with a major PNH clone (≥ 50%) and evaluable Type II neutrophil rel%. Using hemolysis data available for 107 patients, a threshold of 3% Type II neutrophils rel% distinguished two groups: 22 “high Type II” and 105 “low Type II” patients. Hemolysis was less frequent in the “high Type II” group. Thrombosis was more frequent at diagnosis and during cumulative long‐term follow‐up in this group. High neutrophil rel% ≥ 3% was independently associated with thrombotic events. Among 222 patients with smaller PNH clones (1%–50%), 207 displayed evaluable Type II neutrophil rel%, of which 87 were classified as “high Type II” and 120 as “low Type II”. Although no thrombosis was present at diagnosis, two thrombotic events occurred during follow‐up in the “high Type II” group, whereas none were observed in the “low Type II” group. Long‐term follow‐up showed no major changes in Type II neutrophil rel%, irrespective of variations in total clone size. These findings suggest that, regardless of the total PNH clone size, PNH Type II neutrophil rel% is a stable parameter and may represent a reliable predictor of thrombosis, potentially supporting earlier consideration of anticomplement therapy initiation.

Circulating monocyte partitioning refers to the relative quantification of the three main monocyte subsets in the peripheral blood, namely classical (cMo), intermediate (iMo), and non‐classical (ncMo) monocytes, as assessed by flow cytometry, a new nomenclature described 15 years ago. This distribution is influenced by physiological variation as well as by certain therapeutic interventions. In addition, pathological alterations in monocyte partitioning are now well characterized in chronic hematological neoplasms. Most notably, a relative accumulation of cMo exceeding 94% of total circulating monocytes was identified more than a decade ago as a phenotypic hallmark of chronic myelomonocytic leukemia (CMML) and has since been incorporated into the most recent revision of the WHO classification. Altered monocyte partitioning has also been reported in patients with myelodysplastic syndromes (MDS) and myeloproliferative neoplasms (MPNs), highlighting its broader relevance across myeloid disorders.

Background: Cytomorphological examination and classification of bone marrow (BM) cells using a microscope is essential for diagnosing various diseases affecting the haematopoietic system. However, this requires expertise, is effort‐intensive and is inherently subjective. Methods: We developed a method for automatically capturing BM cell images at the single‐cell level using an existing cell image analyser designed for peripheral blood. The captured BM cell images were pre‐classified by the same analyser and subsequently viewed on a large screen by cytomorphologists for BM cell classification. The data consistency in BM blast percentages between the digital and conventional optical microscopy (CM) methods was examined. Results: During the 3.5‐year study period, 657 (96.2%) of the 683 aspirated samples underwent BM cell classification using the digital method. Digital cell images were captured at 2000× magnification and stored permanently, which allowed high‐quality cell image analysis at any time by anyone. Cell images could be compared side‐by‐side across different cell classes, which improves the cell‐classification accuracy. The detailed cell morphology in some cases differed slightly between the digital images and CM. BM blast percentage was comparable between the developed method and CM when examining patients with myelodysplastic syndromes (MDS) and other conditions that need to be differentiated from MDS. In a multicentre study, cytomorphologists who used this method for the first time could perform cell classification. Conclusion: This machine‐assisted and human‐centred approach enhances objectivity in BM cell classification, aligns with the needs of the digital era and facilitates data sharing. Trial Registration : The authors have confirmed clinical trial registration is not needed for this submission.

Introduction: The PNH working group was created in 2010 to initiate a workshop on testing white blood cells to share international recommendations. Methods: Thirty‐five French and Belgian laboratories equipped with 2‐ or 3‐laser cytometers applied three different panels with or without FLAER reagent in 305 samples. This multicenter study demonstrated the interplatform applicability of setting harmonization and the benefits of using a harmonized gating method. A 10 ⁻⁴ limit of detection was achieved with harmonized multiparametric approaches in both six‐color combinations, as well as a robust quantification of minor PNH clones. Results: Following this workshop, the group has developed a quality control scheme since 2013 to improve PNH diagnosis practices. This annual program includes two surveys, a virtual one and one based on fresh whole‐blood sample analysis. Over the last 10 years, the regular participation of the French‐speaking registered centers has demonstrated their strong commitment to our program, which offers various PNH clinical situations. Alongside, the conformity of the final reports provided by the participants has increased to 96%. Conclusion: In 2016, our PNH working group initiated a nationwide multicenter prospective observational study, collecting all PNH cases or GPI‐deficient cells above 0.01% to monitor the long‐term evolution of minor PNH clones < 1% and to further investigate the relevance of classifying type II and type III PNH cells in WBCs in clones > 1%. The strong network of cytometrists built led us to create in 2018 the French‐speaking flow cytometry association in Hematology, namely CytHem, to harmonize the diagnostic practices in various hematological diseases.

Chronic myelomonocytic leukemia (CMML) is a severe myeloid malignancy with limited therapeutic options. Single-cell analysis of clonal architecture demonstrates early clonal dominance with few residual WT hematopoietic stem cells. Circulating myeloid cells of the leukemic clone and the cytokines they produce generate a deleterious inflammatory climate. Our hypothesis is that therapeutic control of the inflammatory component in CMML could contribute to stepping down disease progression. The present study explored the contribution of immature granulocytes (iGRANs) to CMML progression. iGRANs were detected and quantified in the peripheral blood of patients by spectral and conventional flow cytometry. Their accumulation was a potent and independent poor prognostic factor. These cells belong to the leukemic clone and behaved as myeloid-derived suppressor cells. Bulk and single-cell RNA-Seq revealed a proinflammatory status of iGRAN that secreted multiple cytokines of which CXCL8 was at the highest level. This cytokine inhibited the proliferation of WT but not CMML hematopoietic stem and progenitor cells (HSPCs) in which CXCL8 receptors were downregulated. CXCL8 receptor inhibitors and CXCL8 blockade restored WT HSPC proliferation, suggesting that relieving CXCL8 selective pressure on WT HSPCs is a potential strategy to slow CMML progression and restore some healthy hematopoiesis.

The boundary between myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemia (CMML) has been revised in the latest World Health Organization classification of myeloid malignancies. These changes were motivated by the description of a subgroup of MDS patients identified as oligomonocytic chronic myelomonocytic leukemia (OM-CMML) at risk of evolving into overt CMML. Various studies will be reviewed describing the clinical and biological features of MDS patients evolving to CMML. The efforts to discover biomarkers enabling the identification of these patients at the time of MDS diagnosis will be discussed. Finally, the molecular landscape of these patients will be presented with a specific focus on the biallelic inactivation of TET2 in light of its functional impact on hematopoietic stem cells, granule-monocytic differentiation, and its tight interplay with inflammation.

The monocyte subset partitioning by flow cytometry, known as “monocyte assay,” is now integrated into the new classifications as a supporting criterion for CMML diagnosis, if a relative accumulation of classical monocytes above 94% of total circulating monocytes is observed. Here we provide clinical flow cytometry laboratories with technical support adapted for the most commonly used cytometers. Step‐by‐step explanations of the gating strategy developed on whole peripheral blood are presented while underlining the most common difficulties. In a second part, interpretation recommendations of circulating monocyte partitioning from the dedicated French working group “CytHem‐LMMC” are shared as well as the main pitfalls, including false positive and false negative cases. The particular flow‐defined inflammatory profile is described and the usefulness of the nonclassical monocyte specific marker, namely slan, highlighted. Examples of reporting to the physician with frequent situations encountered when using the monocyte assay are also presented.

Chronic myelomonocytic leukemia (CMML) is a severe myeloid malignancy with limited therapeutic options. Single-cell analysis of clonal architecture demonstrated early clonal dominance with few residual wildtype hematopoietic stem cells. Circulating myeloid cells of the leukemic clone and the cytokines they produce generate a deleterious inflammatory climate. Our hypothesis is that therapeutic control of the inflammatory component in CMML could contribute to stepping down disease progression. The present study explores the contribution of immature granulocytes (iGRANs) to CMML progression. iGRANs can be detected and quantified in the peripheral blood of patients by spectral and conventional flow cytometry. Their accumulation is a potent and independent poor prognostic factor. These cells belong to the leukemic clone and behave as myeloid-derived suppressor cells. Bulk and single cell RNA sequencing revealed a pro-inflammatory status of iGRAN that secrete multiple cytokines of which CXCL8 at the highest level. This cytokine inhibits the proliferation of wildtype but not CMML hematopoietic stem and progenitor cells (HSPCs) in which CXCL8 receptors are epigenetically downregulated. CXCL8 receptor inhibitors and CXCL8 blockade restore wildtype HSPC proliferation, suggesting that relieving CXCL8 selective pressure on wildtype HSPCs is a potential strategy to slow CMML progression and restore some healthy hematopoiesis.