Previously, flow cytometric determination of peroxidase activity, cell size, and reactivity to lymphocyte antibodies were used to produce bone marrow differentials in untreated rats. In the present study, abnormal hematologic profiles were induced with erythropoietin (EPO), recombinant murine stem cell factor (rm‐SCF), granulocyte–macrophage stimulating factor (GM‐CSF), and cyclophosphamide (CP). Manual and flow cytometric data showed comparable levels of erythroid and myeloid hyperplasia in EPO‐ and rm‐SCF/GM‐CSF‐treated animals, respectively. In CP‐treated animals, flow cytometric data revealed significant decreases in cellularity at concentrations of CP ≥ 5 mg/kg. In contrast, 20 mg/kg CP were necessary to induce microscopically apparent hypoplasia in histologic bone sections, showing that the automated methodology was a more sensitive indicator of bone marrow hypocellularity than was the more conventional manual method. Megakaryocyte counts were consistently higher by flow cytometer than by manual counts performed on cytocentrifuge preparations made from the same cell suspensions but were similar to megakaryocyte counts performed on histologic sections of femur, indicating that the automated methodology produced a more accurate reflection of true megakaryocyte numbers. Induction of hematologic abnormalities in the present study showed that manual bone marrow differentials can be replaced with the more efficient and reliable flow cytometric method in most preclinical toxicology studies. Cytometry 32:18–27, 1998. © 1998 Wiley‐Liss, Inc.
Preclinical drug trials frequently require the evaluation of animal bone marrow, a time‐consuming process requiring the skills of a highly trained hematologist. In the present study, a flow cytometric technique was developed that could effectively replace the need for manual bone marrow differentials in rats. Peroxidase activity, measured indirectly with 2′7′‐dichlorofluorescin, was coupled with the use of species‐specific T‐ and B‐lymphocyte antibodies and cell size to produce a flow cytometric analysis of rat bone marrow. Accurate identification of lymphocyte, proliferating and maturing erythroid and myeloid, and megakaryocyte populations was confirmed by cell sorting. Flow cytometry yielded differentials that were indistinguishable from manual differentials and published reference ranges. Enumeration of lymphocyte numbers with monoclonal markers is a key advantage of flow cytometric differentials because misidentification of lymphocytes in poorly prepared or stained bone marrow smears is a common problem. The most apparent advantage is increased throughput and reproducibility. Operator training for analysis using flow cytometry can be readily accomplished within a few days as opposed to the extensive training required for individuals performing manual bone marrow differentials. This methodology provides a high‐volume, rapid, and relatively low‐cost tool for the reliable evaluation of rat bone marrow differentials that has been heretofore unavailable. Cytometry 32:9–17, 1998. © 1998 Wiley‐Liss, Inc.
A simple procedure was developed for rapid analysis of animal bone marrow by flow cytometry using the lipophilic cationic dye 3,3′‐dihexyloxacarbocyanine iodide (DiOC6(3)). The batch process allows differentiation of bone marrow cells into lymphoid, erythroid, and myeloid populations and enables classification of erythroid and myeloid cells into proliferating and maturing subpopulations. From these data, myeloid:erythroid (M:E) ratios and maturation indices for erythroid and myeloid cells (EMI and MMI, respectively) can be derived. This procedure provides the opportunity to analyze bone marrow quantitatively and offers distinct advantages to current manual methods in terms of simplicity, throughput, and reproducibility. The method has been tested successfully using marrow from Wistar rats, B6C3F1 mice, beagle dogs, and cynomolgus monkeys. This technique facilitates the evaluation of bone marrow samples taken from preclinical safety studies or from animal colonies of large size. © 1992 Wiley‐Liss, Inc.
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