Automated human blood micronucleated reticulocyte measurements for rapid assessment of chromosomal damage

Abstract

This study evaluated the utility of human blood micronucleated reticulocyte (MN CD71+ ) frequency measurement as a cytogenetic damage biomarker. The analytical methodology was flow cytometry in conjunction with a previously described three color fluorescence labeling technique that includes anti-CD71 to focus analyses on the most immature fraction of reticulocytes [Dertinger et al., Environ. Molec. Mutagen., 44:427-435 (2004)]. Blood specimens from fifty self-reported healthy adult volunteers were studied. In addition to MN CD71+ measurements, blood plasma folate and B 12 levels were assessed, since these variables tend to influence other indices of cytogenetic damage. Timecourse data are also provided for ten cancer patients undergoing treatment. For these subjects, frequency of MN CD71+ was measured immediately before therapy, and daily during the first week of chemotherapy and/or fractionated radiotherapy. For the group of healthy volunteers, the variables of age, and folate and B 12 levels demonstrated no significant effect on MN CD71+ frequency. In addition, no difference was observed between pre-treatment MN CD71+ values for cancer patients compared with healthy volunteers. Regarding chemotherapy and/or partial body radiotherapy, elevated frequencies were observed upon initiation of treatment for 9 of the 10 patients studied. Maximal effects were observed three to five days following initiation of therapy. The largest increases in frequency of MN CD71+ (up to 25.9-fold) were observed in those patients exposed to anti-neoplastic drugs, presumably due to the systemic red marrow exposure provided by these agents. Taken together, these data support the hypothesis that the MN CD71+ endpoint represents a valuable biomarker of cytogenetic damage that does not require cell culture or microscopy-based scoring.

Genotoxicity associated with hydroxyurea exposure in infants with sickle cell anemia: Results from the BABY‐HUG phase III clinical trial

“…Rodent-based micronucleus tests have become an important component of regulatory safety assessment [23, 24]. More recently, studies involving human blood samples have indicated its effectiveness at detecting chromosomal damage caused by a variety of exposure situations, including a variety of anti-neoplastic chemo- and radiotherapies [25, 26], tobacco use [27], and Zidovudine for the prevention of HIV transmission from infected mothers to their infants [28]. To enumerate micronucleated reticulocytes, whole blood was collected and fixed with −80°C methanol.…”

Biomarkers of splenic function in infants with sickle cell anemia: baseline data from the BABY HUG Trial

“…4 Quantitative HJB HJB were quantitated in a central laboratory by flow cytometric evaluation of at least 10 6 mature (CD71 ؊ ) erythrocytes (Litron Laboratories). 6 Higher proportions of HJB in peripheral blood suggest a decline in spleen function. Values Ͼ 300/10 6 erythrocytes have been associated with loss of splenic filtrative function.…”

Molecular markers of radiation-related normal tissue toxicity

“…Chromosomal aberrations, at present specifically the dicentric assay, remain the gold standard for radiation biodosimetry [69, 70] (Figure 2). New approaches to biodosimetry are in development that also quantify chromosomal damage and include assays that examine for micronuclei in hematopoietic cells [71], reticulocytes [72], and cutaneous and pulmonary fibroblasts [73]. Flow cytometric assays are also in development for biodosimetry [71] as are techniques for measuring DNA repair complexes such as histone H2AX [17-19].…”