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BACKGROUND: The reference method for hematopoietic stem cell enumeration is flow cytometric CD34+ cell analysis. We evaluated using the hematopoietic progenitor cell (HPC) count on the Sysmex hematology analyzer to safely replace some flow cytometric measurements performed in peripheral blood samples to guide apheresis timing. STUDY DESIGN AND METHODS: We compared HPC and CD34+ cell counts in 133 preharvest peripheral blood samples and 124 apheresis products. RESULTS: Pre-apheresis HPC counts ≥24 × 106/L in healthy donors and ≥36 × 106/L in lymphoma patients predicted adequate mobilization with 100% specificity and positive predictive value, saving 79% and 63% of flow cytometry analyses, respectively. Due to a positive bias (mean bias 50.26; 95% CI 36.24-64.29), a higher threshold was needed in multiple myeloma patients (HPC ≥ $$ \ge $$ 132 × 106/L), saving only 24% of flow cytometry analyses. CONCLUSION: When the HPC count is above the corresponding threshold, apheresis could be safely initiated without waiting for the flow cytometry result, thereby reducing time-to-decision. Lower HPC values, however, require confirmation by flow cytometry.
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STUDY QUESTION: What is the contamination rate by cancer cells and spermatogonia numbers in immature testicular tissue (ITT) harvested before the start of gonadotoxic therapy in boys with a hematological malignancy? SUMMARY ANSWER: Among our cohort of boys diagnosed with acute lymphoblastic leukemia (ALL) and lymphomas, 39% (n = 11/28) had cancer cells identified in their tissues at the time of diagnosis and all patients appeared to have reduced spermatogonia numbers compared to healthy reference cohorts. WHAT IS KNOWN ALREADY: Young boys affected by a hematological cancer are at risk of contamination of their testes by cancer cells but histological examination is unable to detect the presence of only a few cancer cells, which would preclude autotransplantation of cryobanked ITT for fertility restoration, and more sensitive detection techniques are thus required. Reduced numbers of spermatogonia in ITT in hematological cancer patients have been suggested based on results in a limited number of patients. STUDY DESIGN, SIZE, DURATION: This retrospective cohort study included 54 pre- and peri-pubertal boys who were diagnosed with a hematological malignancy and who underwent a testicular biopsy for fertility preservation at the time of diagnosis before any gonadotoxic therapy between 2005 and 2021. PARTICIPANTS/MATERIALS, SETTING, METHODS: Among the 54 patients eligible in our database, formalin-fixed paraffin-embedded (FFPE) testicular tissue was available for 28 boys diagnosed either with ALL (n = 14) or lymphoma (n = 14) and was used to evaluate malignant cell contamination. Hematoxylin and eosin (H&E) staining was performed for each patient to search for cancer cells in the tissue. Markers specific to each patient's disease were identified at the time of diagnosis on the biopsy of the primary tumor or bone marrow aspiration and an immunohistochemistry (IHC) was performed on the FFPE ITT for each patient to evidence his disease markers. PCR analyses on the FFPE tissue were also conducted when a specific gene rearrangement was available. MAIN RESULTS AND THE ROLE OF CHANCE: The mean age at diagnosis and ITT biopsy of the 28 boys was 7.5 years (age range: 19 months-16 years old). Examination of ITT of the 28 boys on H&E stained sections did not detect malignant cells. Using IHC, we found contamination by cancerous cells using markers specific to the patient's disease in 10 of 28 boys, with a higher rate in patients diagnosed with ALL (57%, n = 8/14) compared with lymphoma (14%, n = 2/14) (P-value < 0.05). PCR showed contamination in three of 15 patients who had specific rearrangements identified on their bone marrow at the time of diagnosis; one of these patients had negative results from the IHC. Compared to age-related reference values of the number of spermatogonia per ST (seminiferous tubule) (Spg/ST) throughout prepuberty of healthy patients from a simulated control cohort, mean spermatogonial numbers appeared to be decreased in all age groups (0-4 years: 1.49 ± 0.54, 4-7 years: 1.08 ± 0.43, 7-11 years: 1.56 ± 0.65, 11-14 years: 3.37, 14-16 years: 5.44 ± 3.14). However, using a cohort independent method based on the Z-score, a decrease in spermatogonia numbers was not confirmed. LIMITATIONS, REASONS FOR CAUTION: The results obtained from the biopsy fragments that were evaluated for contamination by cancer cells may not be representative of the entire cryostored ITT and tumor foci may still be present outside of the biopsy range. WIDER IMPLICATIONS OF THE FINDINGS: ITT from boys diagnosed with a hematological malignancy could bear the risk for cancer cell reseeding in case of autotransplantation of the tissue. Such a high level of cancer cell contamination opens the debate of harvesting the tissue after one or two rounds of chemotherapy. However, as the safety of germ cells can be compromised by gonadotoxic treatments, this strategy warrants for the development of adapted fertility restoration protocols. Finally, the impact of the hematological cancer on spermatogonia numbers should be further explored. STUDY FUNDING/COMPETING INTEREST(S): The project was funded by a grant from the FNRS-Télévie (grant n°. 7.4533.20) and Fondation Contre le Cancer/Foundation Against Cancer (2020-121) for the research project on fertility restoration with testicular tissue from hemato-oncological boys. The authors declare that they have no conflict of interest. TRIAL REGISTRATION NUMBER: N/A.