Flow cytometric analysis of micronuclei in peripheral blood reticulocytes III. An efficient method of monitoring chromosomal damage in the beagle dog.

ABSTRACT

Erythrocyte-based micronucleus tests have traditionally analyzed bone marrow because splenic filtration in most species removes micronucleated cells from peripheral blood. We have evaluated a flow cytometric method for monitoring micronucleated reticulocyte frequencies (%MN-RET) in the peripheral blood of beagle dogs treated with cyclophosphamide (CP) and have found that analysis of micronucleated reticulocytes (MN-RETs) in peripheral blood is a suitable surrogate for bone marrow analysis. The three-color flow cytometric method uses anti-CD71 labeling to identify reticulocytes and Plasmodium berghei-containing erythrocytes as a calibration standard. The spontaneous %MN-RET determined by flow cytometry was 0.31 +/- 0.09% (n = 22) for peripheral blood, compared with 0.38 +/- 0.13% (SD, n = 12) for bone marrow, and 0.27 +/- 0.08% (n = 12) for peripheral blood by microscopic scoring with acridine orange staining. The kinetics of appearance and disappearance of MN-RETs in blood were determined by collecting daily samples after iv treatment with CP. The maximum frequency occurred approximately 48 h after dosing. Frequencies of MN-RETs in peripheral blood at steady state following daily CP treatment were 55-68% of corresponding bone marrow values assessed by microscopy and 55-112% as assessed by flow cytometry. This difference is presumably due to splenic removal, which appears slightly less stringent than that previously reported for CP-treated Sprague-Dawley rats. Responses in bone marrow and peripheral blood were highly correlated and similar to or greater than those reported in mice and rats at equitoxic doses.