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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: 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]

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]