Human blood PIG‐A mutation and micronucleated reticulocyte flow cytometric assays: Method optimization and evaluation of intra‐ and inter‐subject variation

Abstract

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 intersubject 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 intersubject 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 automationcompatible 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.

Assessment of systemic genetic damage in pediatric inflammatory bowel disease

“…The methods for preparing blood samples for MUT RET scoring via flow cytometric analysis have been described in detail by Torous et al . (2020). A brief description follows.…”

“…We sought to extend these genotoxicity‐centric investigations to a pediatric IBD population using two cross‐species compatible flow cytometry‐based biomarkers that are applicable to human studies: the frequency of mutant phenotype (CD59‐/CD55‐) reticulocytes (MUT RET) as a reporter of PIG‐A gene mutation, and the frequency of micronucleated reticulocytes (MN‐RET) as an indicator of chromosomal damage (Torous et al ., 2020). We compared MUT RET and MN‐RET data from IBD patients (18 new‐onset and 46 established) to age‐matched controls consisting of 30 constipation or irritable bowel syndrome patients from the same clinic, as well as 25 young healthy adults.…”

“…Micronucleus assay: Sample processing and flow cytometric analysisThe methods for preparing blood samples for MN-RET scoring via flow cytometric analysis have been described in detail(Torous et al, 2020). A brief description follows.…”

Evaluation ofPIG‐A‐mutated granulocytes and ex‐vivo binucleated micronucleated lymphocytes frequencies after breast cancer radiotherapy in humans

“…However, in the last months, PIG‐A gene mutation has been used as a genotoxicity biomarker in various exposure scenarios in humans, in combination with the formation of micronuclei (Cao et al ., 2020a, 2020b). Moreover, optimized protocols related to human blood PIG‐A mutation and micronucleated reticulocyte flow cytometric assays have been published (Torous et al ., 2020). This highlights the importance of developing tests that can quantify DNA damage in humans.…”

“…optimized protocols related to human blood PIG-A mutation and micronucleated reticulocyte flow cytometric assays have been published (Torous et al, 2020). This highlights the importance of developing tests that can quantify DNA damage in humans.…”