Validation of Sysmex XT‐2000iV analyzer‐generated quantitative bone marrow differential counts in cynomolgus monkeys, Beagle dogs, and CD‐1 mice

Abstract

Background: In a previous study, the validation of rat bone marrow (BM) collection, processing, and analysis using the Sysmex XT-2000iV (Sysmex Corporation, Kobe, Japan) hematology analyzer showed that the Sysmex hematology analyzer produced BM differential counts that were comparable to those obtained with microscopic differential counts.Objective: This study was conducted to expand the validation of the Sysmex TNCC (total nucleated cell count) and 5-part BM differential in cynomolgus monkeys, Beagle dogs, and CD-1 mice, which are alternate species that are also frequently used in preclinical safety studies. Methods:The Sysmex 5-part BM differential counts were generated with a twostep process, whereby proliferating and maturing erythroid and myeloid cells were determined by preset gating and lymphocytes were determined using species-specific B-and T-lymphocyte antibodies and a magnetic cell-sorting method (MACS).Agreement with microscopic myelograms with 500-cell differential counts was determined from BM suspensions of 62 cynomolgus monkeys, 47 Beagle dogs, and 44 CD-1 mice. Results:The correlation coefficients between methods for myeloid to erythroid (M: E) ratios in all three species was > 0.928. The Bland-Altman differences between methods were approximately ± 0.3 units for the M:E ratio in dogs and mice, and +0.6 and −0.4 in monkeys. The upper limits of agreement for all three species were ≤7% for maturing myeloid cells, ≤6% for maturing erythroid cells, and ≤4% for proliferating myeloid cells, proliferating erythroid cells, and lymphocytes. Conclusions:The Sysmex XT-2000iV produces an automated M:E ratio and a 5-part differential count equivalent to microscopic differential counts in cynomolgus monkeys, Beagle dogs, and CD-1 mice. K E Y W O R D Sbone marrow, cytology, hematology, hematotoxicity, laboratory animals, preclinical safety

Methodologies and Emerging Technologies for the Evaluation of the Hematopoietic System

“…This method has initially been qualified with success and published for Wistar rats 3 and then for Monkeys, dogs and mice. 4 We were able to reproduce a similar qualification for Sprague Dawley (SD) rats.…”

Opinion on the Optimal Histologic Evaluation of the Bone Marrow in Nonclinical Toxicity Studies

“…These techniques include cytological evaluation of bone marrow smears, flow cytometry or Sysmex XT 2000iV analysis, and, less frequently, clonogenic assays, liquid cultures, and transmission electron microscopy (TEM). 4,[30][31][32][33] Ideally, bone marrow smears are made proactively at the time of necropsy. These smears, stained with various Romanowsky stains (modified Wright's-Giemsa, Giemsa, or May-Grunwald Giemsa), are often not evaluated unless subsequent data suggest there would be value in doing so.…”

“…If evaluating cellular morphology is not useful or sufficient on its own, then flow cytometry or Sysmex XT 2000iV analysis can be utilized to provide some comparable information to a bone marrow smear cytological evaluation, and data can often be obtained faster and more precisely, as they are automated processes that evaluate a larger number of cells. 4,[30][31][32] Flow cytometry can classify hematopoietic cells into major categories such as neutrophilic granulocytic, erythrocytic, and lymphocytic lineages and can provide some information regarding maturation but not a complete classification of all cell types (eg, megakaryocytic and eosinophilic/basophilic granulocytic cell lineages cannot be characterized with standard flow cytometry). 4 Additionally, if the entire marrow of a long bone is processed from rodents, then a total nucleated cell count can be determined.…”

“…4 Similarly, the Sysmex XT 2000iV hematology analyzer (when coupled with magnetic bead isolation, purification and sequestration of bone marrow lymphoid cells, and applying customized gating) can produce an automated M:E ratio and a 5-part differential count (proliferating myeloid, maturing myeloid, proliferating erythroid, maturing erythroid, and lymphocytes) in rats, mice, dogs, and monkeys equivalent to those derived from cytological evaluation of bone marrow smears. [30][31][32] Samples can also be collected for TEM if there is a morphologic change that needs to be further classified or for clonogenic assays if an in vitro system is needed. 4 If there are issues with platelets, liquid culture may be needed for in vitro studies of megakaryocytes.…”