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.

Elevated frequencies of micronucleated erythrocytes in infants exposed to zidovudine in utero and postpartum to prevent mother‐to‐child transmission of HIV

“…Samples were processed using flow cytometric micronucleus analysis kits (Prototype Human MicroFlow® kits, Litron Laboratories, Rochester NY). Details of the collection, processing, and analysis of blood samples using this method have been previously described (Dertinger et al, 2004). Briefly, a 60-120 μL sample of whole blood (venous or cord blood) was diluted with 350μL of sodium heparin solution.…”

“…Prompted by the observations of increased frequencies of micronucleated reticulocytes (MN-RET) in mouse pups exposed to ZDV, we designed a pilot study to determine if similar effects were detectable in human infants exposed to ZDV in utero and postnatally. We used a recently developed single-laser three-color flow cytometric system for enumerating MN-RET in human blood (Dertinger et al, 2004). The advent of this new technology was important for our study design because, although the mouse peripheral blood erythrocyte micronucleus test has been used routinely for decades to evaluate genotoxicity (Heddle et al, 1991;OECD, 1997;MacGregor et al, 1990;Albertini et al, 2000;Witt et al, 2000;Hayashi et al, 1994), there has not been a corresponding human assay because the human spleen, unlike the mouse spleen, rapidly removes damaged erythrocytes from circulation.…”

Reticulocyte and micronucleated reticulocyte responses to gamma irradiation: dose–response and time-course profiles measured by flow cytometry

“…Anti-CD71-FITC, anti-CD61-PE, and propidium iodide fluorescence signals were detected in the FL1, FL2, and FL3 channels, respectively (stock filter sets). The gating logic used to ensure that quantitative analyses of erythrocyte subpopulations where not contaminated by other cellular or noncellular events has been described previously [7].…”

“…This laboratory has developed a three-color flow cytometric method for scoring MN-RETs [7]. In this system, whole blood is fixed and then incubated with anti-CD71-FITC and anti-CD61-PE.…”

Sensitivity of the erythrocyte micronucleus assay: Dependence on number of cells scored and inter-animal variability

“…The inter-animal variability of the percentage of MN-RETs among RETs (#MN-RET/#RETs scored × 100) in the peripheral blood of Sprague-Dawley rats, CD-1 mice, and purpose-bred beagle dogs, and also in the bone marrow of Sprague-Dawley rats after removal of nucleated cells on a cellulose mini-column as described by Romagna [9] and Weiner et al [10], was estimated by scoring 20,000 reticulocytes using the flow cytometric method described by Dertinger et al [11][12][13]. These data are summarized in Table 1.…”