Flow cytometric analysis of CD34+ CD38- cells; cell frequency and immunophenotype based on CD45RA expression pattern.

INTRODUCTION: The CD34+ CD38- population in bone marrow includes hematopoietic stem/progenitor cells. Recently, in acute myeloid leukemia, the focus has shifted to flow cytometry analysis targeting CD34+ CD38- leukemic cells due to their effectiveness in minimal/measurable residual disease detection and prognosis prediction. Nevertheless, the immunophenotype and cell frequency of these cells in the bone marrow, in the absence of leukemic cells, remains unknown. We aimed to evaluate detailed characteristics of CD34+ CD38- cells in both normal and leukemic cells by flow cytometry. METHODS: We compared the cell frequency and immunophenotype of the CD34+ CD38- fraction in the following groups: patients with idiopathic thrombocytopenic purpura and malignant lymphoma as controls (n = 17), post-treatment patients without abnormal blasts (n = 35), and patients with myeloid malignancies (n = 86). The comparison was based on the presence or absence of CD45RA expression, a marker commonly used to prospectively isolate lymphoid-primed cell populations within the CD34+ CD38- fraction. RESULTS: The CD34+ CD38- CD45RA+ cell population exhibited a significant expansion in bone marrow without leukemic cells 1 month after cord blood transplantation and in various type of myeloid malignancies, compared to the control group (p < 0.01). Continuous CD45RA expression and notable expansion of the CD34+ CD38- CD45RA- population were exclusively observed in myelodysplastic syndrome-related diseases. The CD34+ CD38- CD45RA+ population displayed frequent expression of various markers in both leukemic and non-leukemic cells, in contrast to the CD34+ CD38- CD45RA- population. CONCLUSIONS: The CD34+ CD38- fraction should be carefully evaluated considering the nature of normal hematopoietic precursor cells, their cell frequency and immunophenotype, including CD45RA expression pattern, for improving the accuracy of myeloid malignancy diagnosis.

VS38 staining contributes to a novel gating strategy in flow cytometry for small B cell lymphoma, especially in lymphoplasmacytic lymphoma/Waldenström macroglobulinemia.

BACKGROUND: Multi-parametric flow cytometry (MFC) is a helpful tool for detecting neoplastic cells in malignant lymphoma; however, lymphoma cells can be difficult to detect when characteristic immunophenotypic abnormalities are not evident. We evaluated the stainability of VS38, which is used for multiple myeloma, in normal and abnormal B cells using MFC to develop a new strategy for detecting lymphoma cells. METHODS: We compared the median fluorescence intensity of VS38 staining in lymphocytes from patients without hematopoietic neoplasms and in B cells from 26 patients with B cell lymphoma (BCL). To evaluate the performance of VS38 gating, we compared VS38-positive B cells with the percentages of BCL cells, and with the mutation ratios of MYD88 L265P measured by droplet digital PCR in patients with lymphoplasmacytic lymphoma (LPL)/Waldenström macroglobulinemia (WM). RESULTS: CD27-positive memory B cells were stained with VS38, whereas normal lymphocytes were faintly stained. Lymphoma cells were stained with VS38 in 11 of 12 patients with LPL/WM, 3 of 3 with chronic lymphocytic leukemia, 3 of 5 with mantle cell lymphoma, 2 of 4 with follicular lymphoma, and 1 of 1 with splenic marginal zone lymphoma. The percentages of VS38-positive B cells in VS38-positive BCL were equivalent to those of lymphoma cells and the mutation ratios of MYD88 L265P in LPL/WM. CONCLUSIONS: VS38 identified neoplastic cells in plasma cell disorders and BCL. This might improve the accuracy of BCL diagnosis, especially in patients with LPL/WM.

Sensitive detection of GATA1 mutations using complementary DNA-based analysis for transient abnormal myelopoiesis associated with the Down syndrome.

INTRODUCTION: GATA1 mutation plays an important role in initiating transient abnormal myelopoiesis (TAM) and in the clonal evolution towards acute megakaryoblastic leukaemia (AMKL) associated with Down syndrome (DS). This study aimed to develop and validate the clinical utility of a complementary DNA (cDNA) analysis in parallel with the conventional genomic DNA (gDNA) Sanger sequencing (Ss), as an initial screening test for GATA1 mutations. METHODS: GATA1 mutations were evaluated using both gDNA and cDNA in 14 DS patients using Ss and fragment analysis (FA), respectively. RESULTS: The detection sensitivity of conventional gDNA sequencing was limited in low blast percentage TAM (LBP-TAM); however, cDNA-based Ss readily detected all the pathognomonic GATA1 mutations. The cDNA-based FA readily detected GATA1 frameshift mutation with a reliable sensitivity ranging from 0.005% to 0.01% of clonal cells. CONCLUSIONS: GATA1 mutations are heterogeneous; therefore, we would like to propose a dual cDNA and gDNA analysis as a standard diagnostic approach, especially for LBP-TAM. cDNA-based FA promises an excellent sensitivity for detecting frameshift GATA1 mutations in the longitudinal clonal evolution towards AMKL without using a patient specific primer.

Efficacy of bezafibrate for preventing myopathic attacks in patients with very long-chain acyl-CoA dehydrogenase deficiency.

BACKGROUND: Very long-chain acyl-CoA dehydrogenase deficiency (VLCADD) is a mitochondrial fatty acid oxidation disorder that causes episodic attacks, such as general fatigue, hypotonia, myalgia, and rhabdomyolysis accompanied by lack of energy. As yet, there are no preventative drugs for these VLCADD-associated metabolic attacks. PATIENTS AND METHODS: We conducted an open-label, non-randomized, multi-center study into the effects of bezafibrate on five patients with VLCADD. Bezafibrate was administered for 4 years, and we analyzed the number of myopathic attacks requiring hospitalization and treatment infusions. RESULTS: The number of myopathic attacks requiring infusions of 24 h or longer significantly decreased during the study period. The patients' ability to conduct everyday activities was also improved by the treatment. CONCLUSION: Our findings show the potential long-term efficacy of bezafibrate in preventing myopathic attacks for patients with VLCADD.

Cosmetic color improvement of the nipple-areola complex by optimal use of tretinoin and hydroquinone.

BACKGROUND: A successful treatment to improve the color of nipple-areola complex (NAC) has never been reported, although the number of women seeking the more attractively colored NAC is not small. OBJECTIVE: To determine the effectiveness of our bleaching protocol for cosmetic improvement of the NAC. METHODS: The protocol was composed of two phases: bleaching phase (4-8 weeks) and healing phase (4-6 weeks). 0.2-0.4% tretinoin aqueous gel was applied concomitantly with 5% hydroquinone, 7% lactic acid ointment for bleaching twice a day. Tretinoin was applied to the NAC with a small cotton applicator, while hydroquinone was widely applied beyond the NAC area. After obtaining sufficient improvement in NAC color, the application of tretinoin was discontinued and hydroquinone alone was continually applied in the healing phase until the reactive erythema was eliminated. Fifteen female patients were involved in this study. RESULTS: The average treatment period was 16.6 weeks. Improvement of NAC color was obtained in 12 patients (80%) by the physician's estimation, and 11 patients (73%) satisfied with their final results. The treatment was repeated after a 1-month interval of tretinoin application in 4 patients: 2 desired further improvement in color, and 2 had the second course conducted to treat the postinflammatory hyperpigmentation on the surrounding mound induced by the first course. CONCLUSION: This approach appeared to be most effective for cosmetic improvement of NAC color among treatments available so far.