Three‐color labeling method for flow cytometric measurement of cytogenetic damage in rodent and human blood

Abstract

Experiments described herein were designed to evaluate the performance characteristics of a flow cytometry‐based system that scores the incidence of peripheral blood micronucleated reticulocytes (MN‐RETs). These procedures represent the continued refinement of a previously reported anti‐CD71‐based method (Dertinger et al. [1996]: Mutat Res 371:283–292), with the following modifications: incorporation of a third fluorescent label to exclude platelets from the MN‐RET region, and use of a CD71‐associated fluorescence thresholding technique to increase data acquisition rates. Mouse, rat, and human blood samples were analyzed using both the previously described two‐color procedure (anti‐CD71‐FITC and propidium iodide) and a newly developed three‐color technique (which adds an antiplatelet‐PE antibody). The rodent specimens were also evaluated by standard microscopy procedures (acridine orange staining). Mouse blood was collected via heart puncture of vehicle‐ and 5‐fluorouracil‐treated CD‐1 mice; blood samples from saline‐treated Sprague‐Dawley rats were collected from the tail vein and via heart puncture. Rodent blood samples were analyzed by both the two‐ and three‐color methods. Human blood specimens, obtained via arm venipuncture from cancer patients undergoing radiation therapy, were analyzed for MN‐RETs using the two‐color method. Subsequently, blood samples from a single chemotherapy patient were analyzed by both the two‐ and three‐color methods. Finally, the chemotherapy patient blood samples and blood samples from 15 healthy volunteers were evaluated at very high densities in conjunction with a CD71‐associated fluorescence thresholding technique. Results of these investigations showed that data from mouse blood analyzed by the two‐ and three‐color procedures correlated well with microscopy data (r values = 0.917 and 0.937 for the two‐ and three‐color methods, respectively); all three methods confirmed the genotoxicity of 5‐FU. Data from rat tail vein samples showed improved reproducibility with the three‐color technique, but no significant difference between the two techniques was seen with the heart puncture specimens. Human blood analyzed according to the two‐color procedure produced unreliable results, as platelets and platelet aggregates impacted the rare MN‐RET scoring region. The three‐color technique effectively overcame this problem and produced reproducible measurements that fell within expected ranges. For human blood analyses, the high cell density/CD71‐thresholding technique provided significant improvements over the low‐density technique, as it allowed data acquisition to occur approximately six times faster with no loss of sensitivity. Environ. Mol. Mutagen. 2004.