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Introduction While white blood cell (WBC) parameters have been suggested to depend on ethnicity and gender, reference intervals in healthy Asian populations are limited. The present study established reference intervals of WBC parameters for healthy adults in Japan. Methods A total of 750 healthy adults (447 women and 303 men; 18‐67 years old, median 40 years old) at 7 Japanese centers who participated in regular medical checkups entered this study. The WBC parameters were measured using automated hematocytometers and blood film reviews by a manual microscopic examination. Results The reference intervals of the WBC parameters according to gender in healthy adults were determined. Age‐specific decreases in WBC counts of both gender groups and in neutrophil counts of women were noted. Favorable correlations between the hematocytometer and microscopic methods were found in neutrophils, lymphocytes, and eosinophils but not in monocytes or basophils. Conclusion This study suggests the need to consider gender and age in the clinical use of reference intervals of WBC parameters.

Anti-CD19 chimeric antigen receptor T (CAR-T) cell therapy has facilitated progress in treatment of refractory/relapsed diffuse large B cell lymphoma (DLBCL). A well- known adverse event after CAR-T therapy is cytokine releasing syndrome (CRS). However, the etiology and pathophysiology of CRS-related coagulopathy remain unknown. Therefore, we conducted a prospective cohort study to comprehensively analyze coagulation/fibrinolysis parameters present in peripheral blood of adult DLBCL patients treated with Tisagenlecleucel in a single institution. Samples were collected from 25 patients at three time points: before lymphocyte-depletion chemotherapy, and on Day3 and 13 after CAR-T infusion. After infusion, all patients except one experienced CRS, and 13 required the administration of tocilizumab. A significant elevation in the plasma level of total plasminogen activator inhibitor 1 (PAI-1), which promotes the initial step of coagulopathy (mean: 22.5 ng/mL before lymphocyte-depletion, and 41.0 on Day3, p=0.02), was observed at the onset of CRS. Moreover, this suppressed fibrinolysis induced relatively hypercoagulable state was gradually resolved after CRS remission with normalization of total PAI-1 to pre-infusion levels without any organ damage (mean values of soluble fibrin: 3.16 µg/mL at baseline, 8.04 on Day3, and 9.16 on Day13, p<0.01 and mean PAI-1: 25.1 ng/mL on Day13). In conclusion, a hypofibrinolytic and relatively hypercoagulable state concomitant with significant total PAI-1 elevation was observed at the onset of CRS even in DLBCL patients with mild CRS. Our results will facilitate understanding of CRS-related coagulopathy and they emphasize the importance of monitoring sequential coagulation/fibrinolysis parameters during CAR-T therapy.

X-linked sideroblastic anemia (XLSA) is associated with mutations in the erythroid-specific δ-aminolevulinic acid synthase (ALAS2) gene. Treatment for XLSA is mainly supportive, except in pyridoxine-responsive patients. Female XLSA often represents a late onset of severe anemia, mostly due to the acquired skewing of X-chromosome inactivation. Here, we successfully generated active wild-type and mutant ALAS2 induced pluripotent stem cell (iPSC) lines from the peripheral blood cells of an affected mother and two daughters in a family with pyridoxine-resistant XLSA due to a heterozygous ALAS2 missense mutation (R227C). The erythroid differentiation potential was severely impaired in active mutant iPSC lines compared to that in active wild-type iPSC lines. Most of the active mutant iPSC-derived erythroblasts revealed an immature morphological phenotype, and some showed dysplasia and perinuclear iron deposits. Additionally, globin and HO-1 expression and heme biosynthesis in active mutant erythroblasts were severely impaired compared to that in active wild-type erythroblasts. Furthermore, genes associated with erythroblast maturation and karyopyknosis showed significantly reduced expression in active mutant erythroblasts, recapitulating the maturation defects. Notably, the erythroid differentiation ability and hemoglobin expression of active mutant iPSC-derived hematopoietic progenitor cells (HPCs) were improved by the administration of δ-aminolevulinic acid, verifying the suitability of the cells for drug testing. Administration of a DNA demethylating agent, azacitidine, reactivated the silent wild-type ALAS2 allele in active mutant HPCs and ameliorated erythroid differentiation defects, suggesting that azacitidine is a potential novel therapeutic drug for female XLSA. Our patient-specific iPSC platform provides novel biological and therapeutic insights for XLSA.

Early diagnosis and prompt initiation of appropriate treatment are critical for improving the prognosis of acute leukemia. Currently, acute leukemia is diagnosed by microscopic morphological examination of bone marrow smears and flow cytometric immunophenotyping of bone marrow cells stained with fluorophore-conjugated antibodies. However, these diagnostic processes require trained professionals and are time and resource-intensive. Here, we present a novel diagnostic approach using ghost cytometry, a recently developed high-content flow cytometric approach, which enables machine vision-based, stain-free, high-speed analysis of cells, leveraging their detailed morphological information. We demonstrate that ghost cytometry can detect leukemic cells from the bone marrow cells of patients diagnosed with acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML), without relying on biological staining. The method presented here holds promise as a precise, simple, swift, and cost-effective method for the diagnosis of acute leukemia in clinical practice.