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.

Cytobrush cytology patterns of early postpartum dairy cows.

Endometrial cytobrush cytology has been recommended as a reliable method for determining the percentage of polymorphonuclear leukocytes (PMN%) in cattle smears to diagnose cytological endometritis (CE). In this study, the clarity of cytobrush　cytological smears and the influence of different sample evaluation methods (number　and types of cells counted) on CE diagnosis were evaluated. Samples from 28 lactating　Holstein cows were collected weekly between 3 and 7 weeks postpartum. Smear clarity,　based on cell density, quality of cell morphology, and red blood cell contamination,　was significantly poorer at 3 weeks than between 5 and 7 weeks postpartum. Five　different cell counting methods (C100, C200, C300, C400, and C500) were used,　where 100-500 nucleated cells (endometrial epithelial cells, PMN consisting of neutrophils, eosinophils and basophils, lymphocytes, and macrophages) were counted. Agreement of diagnostic results for CE between C300 and C500 and between C400 and C500 was excellent at all observation times. In calculations of the PMN% based on whether the number of lymphocytes and macrophages were or were not excluded in the denominator, exclusion of these cells in the calculations did not affect the diagnosis of CE. While reduced clarity in earlier stage samples might interfere with the accuracy of cytobrush cytology, C300 can be recommended to determine the endometrial PMN%.

A simple and practical short-term timed artificial insemination protocol using estradiol benzoate with prostaglandin F2α in lactating dairy cows.

To minimize costs and labor for short-term ovulation synchronization protocol, we developed one wherein each treatment-drug administration and timed artificial insemination (TAI)-was performed 24 h apart. The objective of the present study was to evaluate this short-term ovulation synchronization protocol in lactating dairy cows. Data were derived from 133 inseminations performed in 120 cows (32 primiparous and 88 multiparous), and the ovaries of these cows were scanned using ultrasound. The cows detected to have a functional corpus luteum (CL) received prostaglandin F2α (PGF) as a luteolytic agent. The cows were randomly assigned to two treatment groups: (1) treatment with estradiol benzoate (EB) 24 h after PGF treatment, and TAI 24-28 h after EB treatment (EB group); and (2) treatment with gonadotropin-releasing hormone agonist (GnRH) 56 h after PGF treatment, and TAI 16-20 h after GnRH treatment (GnRH group). As a luteolytic agent, either dinoprost (DP; 25 mg) or D-cloprostenol (DCLP; 0.15 mg) was administered intramuscularly in each treatment group. Pregnancy per AI (P/AI) was significantly higher in the DP- or DCLP-treated cows in the EB group when compared with their counterparts in the GnRH group (64.5% vs. 33.3%, P = 0.03 in the DP-treated cows and 51.1% vs. 27.3%, P = 0.04 in the DCLP-treated cows, respectively). Regarding parity, multiparous cows had greater P/AI in the EB group than in the GnRH group (52.8% vs. 26.7%, P = 0.01), whereas primiparous cows showed no significant intergroup difference (65.2% vs. 41.7%, P = 0.28). To conclude, the use of a convenient synchronization protocol comprising the administration of PGF and EB 24 h apart, rather than PGF and GnRH 56 h apart, has greater potential to improve pregnancy rates after TAI in lactating dairy cows given that a functional CL was accurately detected. This beneficial effect of the protocol using EB was clearly demonstrated in multiparous cows.