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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]

Background: Cryopreservation of ovarian tissue is a fertility-preservation option for women before gonadotoxic treatments. However, cryopreserved ovarian tissue transplantation must be performed with caution in women with malignancies that may metastasize to the ovaries. For this purpose, detecting minimal residual disease (MRD) in the ovarian cortex using sensitive methods is a crucial step. We developed an automated ovarian tissue dissociation method to obtain ovarian cell suspensions. Results: We assessed MRD by multicolor flow cytometry (MFC) in cryopreserved ovarian cortex of 15 leukemia patients: 6 with B-cell acute lymphoblastic leukemia (B-ALL), 2 with T-cell acute lymphoblastic leukemia (T-ALL) and 7 with acute myeloid leukemia (AML). Ovarian MRD was positive in 5 of the 15 leukemia patients (one T-ALL and 4 AML). No B-ALL patient was positive by MFC. Quantitative reverse-transcribed polymerase chain reaction was performed when a molecular marker was available, and confirmed the MFC results for 3 patients tested. Xenografts into immunodeficient mice were also performed with ovarian cortical tissue from 10 leukemia patients, with no evidence of leukemic cells after the 6-month grafting period. Conclusions: In conclusion, this is the first study using MFC to detect MRD in ovarian cortical tissue from acute leukemia patients. MFC has been accepted in clinical practice for its ease of use, the large number of parameters available simultaneously, and high throughput analysis. We demonstrate here that MFC is a reliable method to detect MRD in cryopreserved ovarian tissue, with a view to controlling the oncological risk before ovarian tissue transplantation in leukemia patients. [ABSTRACT FROM AUTHOR]

Purpose: This study aimed to develop a method to detect ovarian residual disease by multicolor flow cytometry in acute leukemia patients. Methods: We designed an experimental model consisting in adding acute leukemia cells to a cell suspension obtained from healthy ovarian cortex. Leukemic cell detection within the ovarian cell suspension required the development of a specific myeloid antibody panel different from that commonly used for minimal residual disease (MRD) monitoring in bone marrow. The method was then used to detect ovarian residual disease in 11 acute leukemia patients. Results: Multicolor flow cytometry is able to evaluate the presence of viable leukemic cells in the ovarian cortex with good specificity and robust sensitivity of 10−4. We observed a good correlation between multicolor flow cytometry and quantitative polymerase chain reaction results. Ovarian residual disease detection by multicolor flow cytometry was positive in 3 out of 11 acute leukemia patients. Conclusion: Multicolor flow cytometry can potentially be applied to ovarian tissue from all acute leukemia patients and is essential to evaluate the risk of cancer re-seeding before autograft of ovarian tissue in case of acute leukemia. [ABSTRACT FROM AUTHOR]

Background: Cryopreservation of ovarian tissue is a fertility-preservation option for women before gonadotoxic treatments. However, cryopreserved ovarian tissue transplantation must be performed with caution in women with malignancies that may metastasize to the ovaries. For this purpose, detecting minimal residual disease (MRD) in the ovarian cortex using sensitive methods is a crucial step. We developed an automated ovarian tissue dissociation method to obtain ovarian cell suspensions. Results: We assessed MRD by multicolor flow cytometry (MFC) in cryopreserved ovarian cortex of 15 leukemia patients: 6 with B-cell acute lymphoblastic leukemia (B-ALL), 2 with T-cell acute lymphoblastic leukemia (T-ALL) and 7 with acute myeloid leukemia (AML). Ovarian MRD was positive in 5 of the 15 leukemia patients (one T-ALL and 4 AML). No B-ALL patient was positive by MFC. Quantitative reverse-transcribed polymerase chain reaction was performed when a molecular marker was available, and confirmed the MFC results for 3 patients tested. Xenografts into immunodeficient mice were also performed with ovarian cortical tissue from 10 leukemia patients, with no evidence of leukemic cells after the 6-month grafting period. Conclusions: In conclusion, this is the first study using MFC to detect MRD in ovarian cortical tissue from acute leukemia patients. MFC has been accepted in clinical practice for its ease of use, the large number of parameters available simultaneously, and high throughput analysis. We demonstrate here that MFC is a reliable method to detect MRD in cryopreserved ovarian tissue, with a view to controlling the oncological risk before ovarian tissue transplantation in leukemia patients. [ABSTRACT FROM AUTHOR]

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]

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]

Submicron-sized extra-cellular vesicles generated by budding from the external cell membranes, microparticles (MPs) are important actors in transfusion as well as in other medical specialties. After briefly positioning their role in the characterization of labile blood products, this technically oriented chapter aims to review practical points that need to be considered when trying to use flow cytometry for the analysis, characterization and absolute counting of MP subsets. Subjects of active discussions relative to instrumentation will include the choice of the trigger parameter, possible standardization approaches requiring instrument quality-control, origin and control of non-specific background and of coincidence artifacts, choice of the type of electronic signals, optimal sheath fluid and sample speed. Questions related to reagents will cover target antigens and receptors, multi-color reagents, negative controls, enumeration of MPs and limiting artifacts due to unexpected (micro-) coagulation of plasma samples. Newly detected problems are generating innovative solutions and flow cytometry will continue to remain the technology of choice for the analysis of MPs, in the domain of transfusion as well as in many diverse specialties. [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]

STUDY QUESTION How can leukemic cells be detected in cryopreserved ovarian tissue? SUMMARY ANSWER Multicolor flow cytometry (FCM) is useful to evaluate the presence of viable leukemic cells in the ovarian cortex with a high specificity and a robust sensitivity. WHAT IS KNOWN ALREADY Storing ovarian tissue is an option to preserve fertility before gonadotoxic radiotherapy or chemotherapy treatments. However, transplantation of cryopreserved ovarian cortex to women cured of leukemia is currently not possible due to the risk of cancer re-seeding. STUDY DESIGN, SIZE, DURATION We developed an automated ovarian cortex dissociation technique and we used eight-color FCM to identify leukemic cells with a series of dilutions added to ovarian single cell suspensions obtained from healthy cortex. PARTICIPANTS/MATERIALS, SETTINGS, METHODS Healthy ovarian cortex originated from women between 23 and 39 years of age undergoing laparoscopic ovarian drilling for polycystic ovary syndrome. Blood or bone marrow cells were collected in acute lymphoblastic leukemia (ALL) patients at diagnosis. MAIN RESULTS AND THE ROLE OF CHANCE The tissue dissociation technique yield was 1.83 ± 1.49 × 106 viable nucleated cells per 100 mg of ovarian cortex. No cell exhibiting a leukemic phenotype was present in the normal ovarian cortex. Added leukemic cells were detected using their leukemia-associated phenotype up to a dilution of 10–4. When specific gene rearrangements were present, they were detected by real-time quantitative PCR at the same dilution. The ovarian cortex from two leukemia patients was then used, respectively, as positive and negative controls. LIMITATIONS, REASONS FOR CAUTION Making available minimal residual disease (MRD) detection techniques (multicolor FCM, PCR and xenograft), that can be used either alone or together, is essential to add a fail-safe oncological dimension to pre-autograft monitoring. WIDER IMPLICATIONS OF THE FINDINGS This approach can be performed on fresh ovarian tissue during cryopreservation or on frozen/thawed tissue before reimplantation and it is currently the only available technique in cases of ALL where no molecular markers are identified. This new perspective should lead to studies on ovarian tissue from leukemia patients, for whom the presence of MRD should be established before autograft. STUDY FUNDINGS/COMPETING INTEREST(S) The study was supported by the BioMedicine Agency, the Committee of the League against Cancer, the Besançon University Hospital, DGOS/INSERM/INCa and the regional Council of Franche-Comté. There were no conflicts of interest to declare. [ABSTRACT FROM AUTHOR]