Cell types investigated for PIG-A status
There are multiple different reports evaluating which human cell type is most suitable for the PIG-A assay, including the use of erythrocytes, reticulocytes (RETs), granulocytes, and peripheral blood mononuclear cells (PBMCs) (Table II). The most frequently used cell type for measuring PIG-A mutations is erythrocytes as only a finger prick volume of whole blood (3-10\(\mu\)l) is required to measure thePIG-A status of over a million cells. This is crucial to identify such rare events as PIG-A mutant cells, where millions of cells need to be interrogated. Furthermore, with the addition of the anti-CD235a antibody, blood pre-processing is minimised, and the high throughput technology of flow cytometry is exploited. Alongside RBCPIG-A analysis, some groups have also measured the percentage of reticulocytes (%RET) to account for abnormalities in haematopoietic function (Cao et al., 2016; Dertinger et al., 2015). However, no compromise in %RET has been observed to date (Dertinger et al., 2015; Xi et al., 2023).
Some groups favour the use of alternative blood cells to measurePIG-A mutation as mature erythrocytes that lack the complement inhibitors CD55 and CD59 may undergo complement mediated lysis (Ruiz-Arguelles & Llorente, 2007). Although we believe this effect to be minimal, evidence suggests mutant erythrocytes may be subject to modest selective pressure (Dertinger et al., 2015). Despite this disadvantage to using erythrocytes over RETs, the population of reticulocytes found in human peripheral blood is low in comparison, and therefore the use of erythrocytes is much more rapid, and simple to carry out. Furthermore, Dertinger and colleagues have noted that inter-individual variation of RET PIG-A mutant frequency may be as high as 30-fold (Dertinger et al., 2015). However, RETs may be the most desirable cell type for analysis when measuring mutant induction post exposure as such an effect may be observed sooner than with RBCs which need to enucleate and mature to yield a mutant phenotype post exposure. Importantly, in vitro Pig-A analysis in the L5178Y cell line has shown a phenotypic expression period of 8 days post treatment (David et al., 2018).
The utility of granulocytes in measuring PIG-A mutation status has also been explored. As with RETs, mutation induction post exposure may be observed sooner than with RBCs. However, the short life span of granulocytes (1-2 days) provides only a short window in which mutant cell levels can be accurately measured following a mutagenic event. Although granulocyte mutant cell levels are present at approximately the same frequency as erythrocyte mutant cells in healthy volunteers (Rondelli et al., 2013), using granulocytes to measure PIG-Amutation requires blood pre-processing and a more complex flow cytometry gating strategy (Bonetto et al., 2021; Peruzzi et al., 2010; Rondelli et al., 2013). Peripheral blood mononuclear cells (PBMCs) have also been interrogated for PIG-A mutation status. Like granulocytes, this process requires cell isolation from whole blood prior to flow cytometry. Two studies carried out by McDiarmid et al., and Ware et al., detected higher background mutant frequencies in PBMCs (compared to healthy volunteer erythrocytes published elsewhere) of 18.13 x 10-6 (McDiarmid et al., 2011) and 18 x 10-6 mutants (Ware, Pickens, DeCastro, & Howard, 2001) respectively. This technique also required the subculture of PBMCs under Aerolysin selection. Although there are advantages and disadvantages to using each cell type (Table II), the decision of which type to use should be based on the type and requirements of the study.
Table II: Advantages and disadvantages of exploiting different blood cell types for the phosphatidylinositol glycan class A (PIG-A ) mutation assay.