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Introduction: Monocytosis is a frequent trigger for blood smear review in a routine hematology laboratory whereas chronic myelomonocytic leukemia (CMML) is infrequent and arises mostly in elderly patients. In order to define the best workflow for monocytosis, we studied three diagnostic approaches: the classical morphology approach (blood smear review), the flow cytometry assay (quantification of monocyte subsets as described by Selimoglu‐Buet et al in 2015), and the "mono‐dysplasia‐score" also referred to as "Monoscore (as described by our team in 2018 using the structural parameters of the Sysmex XN™ analyzers). Methods: Studying a multicentric cohort of 196 nonclonal monocytoses and CMML patients aged over 50 years, we compared the diagnostic performance of the three approaches alone and in combination to propose a diagnostic decision tree. Results: In patients presenting with additional criteria for slide review to monocytosis (37% of our cohort), we propose to sequentially combine morphology, Monoscore, and flow cytometry. On the contrary, for patients with isolated monocytosis (63%), slide review is not mandatory and we suggest performing flow cytometry depending on the Monoscore value. Using the proposed algorithm, 98% of CMML patients would have been correctly identified, slide review rate drastically reduced, and flow cytometry would have been carried out in 44% of patients. Conclusion: We have shown that implementation of Monoscore is a useful input filter to significantly reduce slide reviews without losing sensitivity and that flow cytometry is a performant technique in the second step of the diagnostic workup of CMML. [ABSTRACT FROM AUTHOR]

Flow cytometry is broadly used for the identification, characterization, and monitoring of hematological malignancies. However, the use of clinical flow cytometry is restricted by its lack of reproducibility across multiple centers. Since 2006, the EuroFlow consortium has been developing a standardized procedure detailing the whole process from instrument settings to data analysis. The FranceFlow group was created in 2010 with the intention to educate participating centers in France about the standardized instrument setting protocol (SOP) developed by the EuroFlow consortium and to organise several rounds of quality controls (QCs) in order to evaluate the feasibility of its application and its results. Here, we report the 5 year experience of the FranceFlow group and the results of the seven QCs of 23 instruments, involving up to 19 centers, in France and in Belgium. The FranceFlow group demonstrates that both the distribution and applicability of the SOP have been successful. Intercenter reproducibility was evaluated using both normal and pathological blood samples. Coefficients of variation (CVs) across the centers were <7% for the percentages of cell subsets and <30% for the median fluorescence intensities (MFIs) of the markers tested. Intracenter reproducibility provided similar results with CVs of <3% for the percentages of the majority of cell subsets, and CVs of <20% for the MFI values for the majority of markers. Altogether, the FranceFlow group show that the 19 participating labs might be considered as one unique laboratory with 23 identical flow cytometers able to reproduce identical results. Therefore, SOP significantly improves reproducibility of clinical flow in hematology and opens new avenues by providing a robust companion diagnostic tool for clinical trials in hematology. © 2019 International Society for Advancement of Cytometry [ABSTRACT FROM AUTHOR]

Background: The procoagulant and proinflammatory microparticles (MPs) released during storage of packed red blood cells (pRBCs) can potentially modify transfusion benefits. A robust method to quantify MPs in pRBCs is needed to evaluate their impact in clinical trials.Study Design and Methods: The objective was to validate the preanalytic conditions required to prepare pRBC supernatant as well as a method to quantify and evaluate MP variations over 42 days of pRBC storage.A flow cytometry method with size-calibrated beads was developed and fully validated. Quantification of MPs in pRBCs (n = 109) was assessed during short-term (7 days) and long-term (42 days) storage at 4°C, during short-term storage (8 hours) at room temperature, and after 2 years frozen.Results: Repeatability, reproducibility, and linearity of the quantification method were validated, and variations during conservation are presented. There was high variability in RBC (erythrocyte) MP (ERMP) and platelet MP (PMP) levels between RBC units, depending on the filter used for leukocyte reduction. During the 42 days of storage at 4°C, significant increases in ERMPs and PMPs occurred (from 58 to 138 ERMPs/µL from Day 2 to Day 42; p = 0.0002; and from 326 to 771 PMPs/µL from Day 2 to Day 42; p = 0.00026).Conclusion: We use a robust method to confirm that ERMPs and PMPs are present to various degrees in pRBCs and that storage for 42 days significantly increases their generation. This method is robust enough to allow MP quantification in pRBCs and is adapted to evaluate the clinical impact of transfused MPs in prospective clinical trials. [ABSTRACT FROM AUTHOR]

We investigated the plasma levels of PMPs in patients with 45 stage III and 45 stage IV melanoma. PMPs were characterised by flow cytometry and their thrombogenic activity. We also investigated the link between PMPs circulating levels and tumor burden. The circulating levels of PMPs were significantly higher in stage IV (8500 μL−1) than in patients with stage III (2041 μL−1) melanoma ( P=.0001). We calculated a highly specific (93.3%) and predictive (91.7%) cut-off value (5311 μL−1) allowing the distinction between high-risk stage III and metastatic stage IV melanoma. The thrombogenic activity of PMPs was significantly higher in patients with stage IV melanoma (clotting time: 40.7 second vs 65 second, P=.0001). There was no significant association between the radiological tumoral syndrome and the plasma level of PMPs. Our data suggest the role of PMPs in metastatic progression of melanoma. [ABSTRACT FROM AUTHOR]

Although purine analogues have significantly improved the outcome of hairy cell leukaemia ( HCL) patients, 30-40% relapse, illustrating the need for minimal residual disease ( MRD) markers that can aid personalized therapeutic management. Diagnostic samples from 34 HCL patients were used to design an 8-colour flow cytometry (8- FC) tube for blood MRD (B/ RD) analysis (188 samples) which was compared to quantitative IGH polymerase chain reaction (Q- PCR) on 83 samples and to qualitative consensus IGH PCR clonality analysis on 165 samples. Despite heterogeneous HCL phenotypes at diagnosis, discrimination from normal B lymphocytes was possible in all cases using a single 8- FC tube, with a robust sensitivity of detection of 10−4, comparable to Q- PCR at this level, but preferable in terms of informativeness, simplicity and cost. B/ RD assessment of 15 patients achieving haematological complete remission after purine analogues was predictive of a clinically significant relapse risk: with a median follow-up of 95 months; only one of the nine patients with reproducible 8- FC B/ RD levels below 10−4 (B/ RDneg) relapsed, compared to 5/6 in the B/ RDpos group ( P = 0·003). These data demonstrate the clinical interest of a robust 8- FC HCL B/ RD strategy that could become a surrogate biomarker for therapeutic stratification and new drug assessment, which should be evaluated prospectively. [ABSTRACT FROM AUTHOR]

Diagnosis of blastic plasmacytoid dendritic cell neoplasm (BPDCN) or plasmacytoid dendritic cell leukemia (pDCL) is mainly based on immunophenotypical characterization of leukemic cells in blood or bone marrow samples. We tested by flow cytometry intracellular expression of the proto-oncogene T-cell leukemia 1 (TCL1), as well as membrane and intracellular expression of immunoglobulin-like transcript 7 (ILT7) in 21 pDCL samples and 61 non-pDC acute leukemia samples [i.e., 14 B-acute lymphoblastic leukemia (B-ALL), 9 T-ALL and 38 acute myeloid leukemia (AML)]. TCL1 is highly expressed in all pDCL samples while at a statistically lower level in all B-ALL and 34% of AML. Statistical analysis shows that intensity of TCL1 expression is a good marker for differential diagnosis of pDCL versus other acute leukemia (area under the receiver-operating characteristic curve, [AUC]: 0.96). By contrast, ILT7 positivity is limited to few pDCL samples and cannot be useful for diagnosis purpose. In conclusion, high intracellular intensity of TCL1 expression is currently the best marker for pDC lineage assignment by flow cytometry, which is particularly useful to distinguish pDCL from CD4+ CD56+/− undifferentiated or monoblastic acute leukemia. Thus, intracellular TCL1 detection should be included in acute leukemia diagnosis panels used in hematology laboratories. © 2012 International Society for Advancement of Cytometry [ABSTRACT FROM AUTHOR]

The current screening for eligibility of unrelated volunteer marrow donors comprises a complete clinical check-up, a blood CBC and serum protein immunoelectrophoresis. This allows to eliminate acute leukemias, myeloproliferative and myelodysplastic disorders, myelomas and MGUS. To date, the risk of transmission of chronic lymphocytic leukemia (CLL) disease is only evaluated by the clinical evaluation and CBC. We report here the case of a CLL-type MBL disease occurring in a 12-year-old boy after unrelated BMT. Deep biological investigations, as Immunophenotyping, cytogenetic and molecular biology allow us to determine the donor origin of the CLL clone. In 2010, 14.2% donor (105/737) for unrelated hematopoietic stem cell transplantation were over 45y. It is currently estimated (USA) that 1 in 210 men and women will be diagnosed with CLL during their lifetime. Given the long asymptomatic phase of CLL, this raises the case for a detection strategy analog to that used for MGUS and myeloma through serum protein electrophoresis. This case-report, to our knowledge, of a CLL-type MBL unrelated donor-to-recipient transmission through BMT raises ethical and practical questions, such as the proper information about disease transmission risk. The cost-effectiveness of a systematic peripheral blood Immunophenotyping in donors elder than 40y at time of stem cell donation should be evaluated. [ABSTRACT FROM AUTHOR]

Minimal residual disease (MRD) has emerged as a major prognostic factor for monitoring patients with B-lineage acute lymphoblastic leukemia (B-ALL). The quantification of MRD by flow cytometry (FC) is based on the identification of a leukemia-associated phenotype (LAP). Because phenotypic switch is common during treatment, multiple LAPs must be available and used for MRD detection over time. We evaluated the potential usefulness of CD304 as a new marker for monitoring MRD. CD304 was expressed in 48% of B-ALL (24/50) with discriminative fluorescence intensity compared with CD304-negative normal B-cell precursors ( n = 15). The sensitivity of CD304-based MRD detection reached 10−4, as with some of established LAPs. The stability of CD304 expression evaluated during therapy and at relapse confirms the usefulness of this marker for MRD quantification. Finally, CD304 was repeatedly expressed in patients with TEL-AML1 gene rearrangement, which warrants further investigation on its potential relevance as a prognosis marker or therapeutic target. © 2011 International Society for Advancement of Cytometry [ABSTRACT FROM AUTHOR]

The diagnosis of plasmacytoid dendritic cell leukaemia (pDCL) is based on the immunophenotypic profile: CD4+ CD56+ lineageneg CD45RA+/ROneg CD11cneg CD116low CD123+ CD34neg CD36+ HLA-DR+. Several studies have reported pDCL cases that do not express this exact profile or expressing some lineage antigens that could thus be misdiagnosed. This study aimed to validate pDCL-specific markers for diagnosis by flow-cytometry or quantitative reverse transcription polymerase chain reaction on bone marrow samples. Expression of markers previously found in normal pDC was analysed in 16 pDCL, four pDCL presenting an atypical phenotype (apDCL) and 113 non-pDC – lymphoid or myeloid – acute leukaemia. CD123 was expressed at significantly higher levels in pDCL and apDCL. BDCA-2 was expressed on 12/16 pDCL and on 2/4 apDCL, but was never detected in the 113 non-pDC acute leukaemia cases. BDCA-4 expression was found on 13/16 pDCL, but also in 12% of non-pDC acute leukaemia. High levels of LILRA4 and TCL1A transcripts distinguished pDCL and apDCL from all other acute leukaemia (except B-cell acute lymphoblastic leukaemia for TCL1A). We thus propose a diagnosis strategy, scoring first the CD4+ CD56+/− MPOneg cCD3neg cCD79aneg CD11cneg profile and then the CD123high, BDCA-2 and BDCA-4 expression. Atypical pDCL can be also identified this way and non-pDC acute leukaemia excluded: this scoring strategy is useful for diagnosing pDCL and apDCL. [ABSTRACT FROM AUTHOR]

CD4+/CD56+ haematodermic neoplasm or ‘early’ plasmacytoid dendritic cell leukaemia/lymphoma (pDCL) was described as a disease entity in the last World Health Organisation/European Organisation for Research and Treatment of Cancer classification for cutaneous lymphomas. These leukaemia/lymphomas co-express CD4 and CD56 without any other lineage-specific markers and have been identified as arising from plasmacytoid dendritic cells. Despite a fairly homogeneous pattern of markers expressed by most pDCL, numerous distinctive features (e.g. cytological aspects and aberrant marker expression) have been reported. This may be related to the ‘lineage-independent developmental’ programme of dendritic cells, which may be able to develop from either immature or already committed haematopoietic progenitors. This highlights the need for specific validated markers to diagnose such aggressive leukaemia. Here, we propose –among others (e.g. T-cell leukaemia 1) – blood dendritic cell antigen-2 and high levels of CD123 expression as potential markers. In addition, we propose a multidisciplinary approach including several fields of haematology to improve pDCL diagnosis. [ABSTRACT FROM AUTHOR]

Psoriasis involves TNF- α secretion leading to release of microparticles into the bloodstream. We investigated the effect of TNF blockers on microparticles levels before and after treatment in patients (twenty treated by anti- TNF- α agents and 6 by methotrexate) with severe psoriasis. Plasmatic microparticles were labelled using fluorescent monoclonal antibodies and were analysed using cytometry. Three months later, 70% of patients treated with anti- TNF- α agents achieved a reduction in PASI score of at least 75%. The clinical improvement in patients treated with anti- TNF- α agents was associated with a significant reduction of the mean number of platelet microparticles (2837/ μl vs 1849/ μl, P = 0.02) and of endothelial microparticles (64/ μl vs 22/ μl, P = 0.001). Microparticles are significantly decreased in psoriatic patients successfully treated by anti- TNF- α. Microparticles levels as circulating endothelial cells represent signs of endothelial dysfunction and are elevated in psoriasis. Then, TNF blockade may be effective to reduce cardiovascular risk through the reduction of circulating microparticles. [ABSTRACT FROM AUTHOR]