We previously described flow cytometry‐based methods for scoring the incidence of micronucleated reticulocytes (MN‐RET) and PIG‐A mutant phenotype reticulocytes (MUT RET) in rodent and human blood samples. The current report describes important methodological improvements for human blood analyses, including immunomagnetic enrichment of CD71‐positive reticulocytes prior to MN‐RET scoring, and procedures for storing frozen blood for later PIG‐A analysis. Technical replicate variability in MN‐RET and MUT RET frequencies based on blood specimens from 14 subjects, intra‐subject variability based on serial blood draws from 6 subjects, and inter‐subject variation based on up to 344 subjects age 0 to 73 years were quantified. Inter‐subject variation explained most of the variability observed for both endpoints (≥77%), with much lower intra‐subject and technical replicate variability. The relatively large degree of inter‐subject variation is apparent from mean and standard deviation values for MN‐RET (0.15 ± 0.10%) and MUT RET (4.7 ± 5.0 per million, after omission of two extreme outliers). The influences of age and sex on inter‐subject variation were investigated, and neither factor affected MN‐RET whereas both influenced MUT RET frequency. The lowest MUT RET values were observed for subjects <11 years old, and males had moderately higher frequencies than females. These results indicate that MN‐RET and MUT RET are automation‐compatible biomarkers of genotoxicity that bridge species of toxicological interest to include human populations. These data will be useful for appropriately designing future human studies that include these biomarkers of genotoxicity, and highlight the need for additional work aimed at identifying the sources of inter‐individual variability reported herein.

中文翻译：

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人血 PIG-A 突变和微核网织红细胞流式细胞术分析：方法优化和受试者内和受试者间变异的评估。

我们之前描述了基于流式细胞术的方法，用于对啮齿动物和人类血液样本中的微核网织红细胞 (MN-RET) 和PIG-A突变表型网织红细胞 (MUT RET)的发生率进行评分。目前的报告描述了人类血液分析的重要方法学改进，包括在 MN-RET 评分之前对 CD71 阳性网织红细胞进行免疫磁性富集，以及为以后的PIG-A储存冷冻血液的程序分析。基于 14 名受试者的血液样本的 MN-RET 和 MUT RET 频率的技术重复变异性、基于 6 名受试者的连续抽血的受试者内变异性以及基于多达 344 名 0 至 73 岁受试者的受试者间变异被量化. 受试者间变异解释了两个终点观察到的大部分变异性（≥77%），受试者内和技术重复变异性要低得多。从 MN-RET (0.15 ± 0.10%) 和 MUT RET (百万分之 4.7 ± 5.0，省略两个极端异常值后) 的平均值和标准偏差值可以看出，受试者间的差异程度相对较大。研究了年龄和性别对受试者间变异的影响，两个因素都不影响 MN-RET，而两者都影响 MUT RET 频率。在<11 岁的受试者中观察到最低的 MUT RET 值，男性的频率略高于女性。这些结果表明，MN-RET 和 MUT RET 是自动化兼容的遗传毒性生物标志物，可将具有毒理学意义的物种与人类群体联系起来。这些数据将有助于适当设计包括这些遗传毒性生物标志物的未来人类研究，并强调需要开展旨在确定本文报告的个体间变异来源的额外工作。