Comparison of the Sysmex XT-2000iV and microscopic bone marrow differential counts in Wistar rats treated with cyclophosphamide, erythropoietin, or serial phlebotomy.

BACKGROUND: In a previous study, it was demonstrated that bone marrow analysis using the Sysmex XT-2000iV hematology analyzer produced differential counts in untreated rats that were comparable to microscopic differential counts. OBJECTIVE: The aim of this study was to modulate hematopoiesis in rats in vivo either through pharmacologic treatment or serial phlebotomy, and to determine whether the Sysmex XT-2000iV could accurately analyze bone marrow quantitative changes when compared with results obtained by microscopy. METHODS: Rats were treated once with 0, 5, 20, and 40 mg/kg cyclophosphamide (CP), 0, 50, 100 IU/kg erythropoietin (EPO) on 4 consecutive days, or serial phlebotomy of 1-2 mL of blood for 4 days. Modulation of hematopoietic populations in bone marrow was evaluated using the Sysmex XT-2000iV hematology analyzer, and compared with microscopic differential counts. RESULTS: Correlation coefficients between M:E ratios determined by Sysmex and the microscopic method were 0.94, 0.96, and 0.98 for CP, EPO, or serial phlebotomy treatments, respectively. Mean concordance correlation coefficients for M:E demonstrated method agreement of 0.63, 0.92, and 0.85 for the 3 treatments. Quantitative automated and microscopic bone marrow differential counts were within the expected 95% confidence intervals for CP, EPO or serial phlebotomy. CONCLUSIONS: The Sysmex XT-2000iV provides quantitative bone marrow differential counts of bone marrow cell series in rats with treatment-induced changes which are comparable to microscopic differential counts. Reliable automatic bone marrow differential counting allows increased throughput, sensitivity, reproducibility, and enhanced interpretation of bone marrow evaluation in rodent preclinical studies.

Validation of Sysmex XT-2000iV generated quantitative bone marrow differential counts in untreated Wistar rats.

BACKGROUND: Preclinical drug trials frequently require assessment of bone marrow toxicity in animals to evaluate hematopoietic safety. Since the gold standard, cytologic evaluation, is time consuming and requires highly trained individuals, automated methods remain intriguing. OBJECTIVE: The Sysmex XT-2000iV hematology analyzer allows user-developed customizable gating. This study was conducted to validate the gating of bone marrow cell populations in Sysmex cytograms from untreated rats. METHODS: B- and T-lymphocytes and myeloid cells were experimentally depleted from Charles River Wistar Han IGS (CRL: WI [Han]) rat whole bone marrow suspension using a magnetic cell sorting (MACS) method. The positively and negatively selected populations were used to verify select gates within the Sysmex cytogram. Intra- and inter-animal precision, comparability between right and left femur, as well as agreement with microscopic myelograms based on 500 counted cells, were determined. RESULTS: Intra-sample precision and right-to-left femur comparability confirmed that gating was reproducible and stable. In 50 tested rats, myeloid to erythroid ratios (M:E) were 1.32 ± 0.33 in males and 1.38 ± 0.29 in females by Sysmex compared to 1.36 ± 0.32 in males and 1.42 ± 0.32 in females by microscopic evaluations. Bland-Altman differences between methods was ≤ ± 0.35 units for M:E, ≤ 5.4% for maturing myeloid cells, ≤ 3.4% for proliferating myeloid cells, ≤ 6.0% for maturing myeloid cells, ≤ 3.4% for proliferating myeloid cells, and ≤ 4.1% for lymphocytes. CONCLUSIONS: In untreated control Charles River Wistar Han IGS (CRL: WI [Han]) rats, the Sysmex XT-2000iV produced an automated M:E and 5-part differential count equivalent to microscopic differential counts.

Pregabalin induces hepatic hypoxia and increases endothelial cell proliferation in mice, a process inhibited by dietary vitamin E supplementation.

The preceding article identified key components of pregabalin's mode of action on nongenotoxic hemangiosarcoma formation in mice, including increased serum bicarbonate leading to decreased respiratory rate, increased blood pH, increased venous oxygen saturation, increased vascular endothelial growth factor and basic fibroblast growth factor expression, increased hepatic vascular endothelial growth factor receptor 2 expression, and increased iron-laden macrophages. Increased platelet count and platelet activation were early, species-specific biomarkers in mice. Dysregulated erythropoiesis, macrophage activation, and elevations of tissue growth factors were consistent with the unified mode of action for nongenotoxic hemangiosarcoma recently proposed at an international hemangiosarcoma workshop (Cohen, S. M., Storer, R. D., Criswell, K. A., Doerrer, N. G., Dellarco, V. L., Pegg, D. G., Wojcinski, Z. W., Malarkey, D. E., Jacobs, A. C., Klaunig, J. E., et al. (2009). Hemangiosarcoma in rodents: Mode-of-action evaluation and human relevance. Toxicol. Sci. 111, 4-18). In this article, we present evidence that pregabalin induces hypoxia and increases endothelial cell (EC) proliferation in a species-specific manner. Dietary administration of pregabalin produced a significant 35% increase in an immunohistochemical stain for hypoxia (Hypoxyprobe) in livers from pregabalin-treated mice. Increased Hypoxyprobe staining was not observed in the liver, bone marrow, or spleen of rats, supporting the hypothesis that pregabalin produces local tissue hypoxia in a species-specific manner. Transcriptional analysis supports that rats, unlike mice, adapt to pregabalin-induced hypoxia. Using a dual-label method, increased EC proliferation was observed as early as 2 weeks in mouse liver and 12 weeks in bone marrow following pregabalin administration. These same assays showed decreased EC proliferation in hepatic ECs of rats, further supporting species specificity. Dietary supplementation with vitamin E, which is known to have antioxidant and antiangiogenic activity, inhibited pregabalin-induced increases in mouse hepatic EC proliferation, providing confirmatory evidence for the proposed mode of action and its species-specific response.