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

Abstract

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 treatmentinduced 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 analyzer‐generated quantitative bone marrow differential counts in cynomolgus monkeys, Beagle dogs, and CD‐1 mice

“…Although the Sysmex XT‐2000iV has been previously validated for the assessment of rat BM, this report shows, for the first time, a similar validation in cynomolgus monkeys, Beagle dogs, and CD‐1 mice. The ability to perform automated BM differential counts in multiple species greatly increases the efficiency and consistency of data collection across preclinical studies.…”

“…Although the Sysmex XT‐2000iV hematology analyzer is not capable of lineage‐specific fluorescence labeling, it has the unique option of customizable gating, thereby allowing the operator to select and analyze discrete populations. By incorporating magnetic bead separation of lymphocytes and a highly structured BM processing protocol, we were able to produce an automated differential that mirrored microscopic results in untreated rats and those with abnormal hematologic changes . This study expands on the initial study and demonstrates that the Sysmex XT‐2000iV is a highly versatile platform that can also produce accurate 5‐part BM differential counts in cynomolgus monkeys, Beagle dogs, and CD‐1 mice; important species used frequently in preclinical safety studies.…”

“…Previously, to verify the true position of BM lymphoid, myeloid, and erythroid lineages within the Sysmex cytogram, antibody‐specific magnetic capture and separation of lymphoid and myeloid lineages was performed in rats . For this validation, five mouse, three monkey, and four dog BM samples were analyzed with the rat BM gating for proliferating and maturing myeloid and erythroid cell gating.…”

“…The fact that late‐stage nucleated erythroid and lymphoid cells cannot be adequately distinguished by forward and side scatter analysis alone is not surprising since these cell types can be similar in size and morphologic detail. We have previously shown that rat BM lymphocytes and late‐stage erythroblasts occupied the same cytogram location when evaluated by flow cytometry or by the Sysmex‐2000iV technologies . This coincident location of multiple cell types contributes to inaccuracies in automated BM differential counts.…”

“…We have previously shown that the Sysmex XT‐2000iV can produce an automated rat bone marrow (BM) 5‐part differential that is comparable to microscopic differential counts in untreated rats . Additionally, the Sysmex XT‐2000iV analyzer was capable of producing accurate BM differential results and could be used to monitor hematopoietic changes in rats treated with cyclophosphamide, erythropoietin, or serial phlebotomy . These studies demonstrated that automated quantitative BM differential counts could improve the precision and timeliness of hematopoietic safety evaluations in preclinical rat toxicity studies.…”