Figure 1. Flow cytometric analysis of red blood cell phosphatidylinositol glycan class A (PIG-A ) gene status. Anti-CD235A antibodies can be used to isolate erythrocytes in a whole blood sample and anti-CD55/CD59 antibodies are used to detect the presence of the glycosylphosphatidylinositol (GPI)-linked proteins CD55 and CD59. PIG-A wild-type cells present functional (GPI) anchors and corresponding GPI-linked proteins at the extracellular surface (top panel) whilst PIG-A mutant cells do not fluoresce as they lack GPI-anchors and corresponding anchored proteins (made using biorender.com).
Blood cell PIG-A mutations were first described in patients with paroxysmal nocturnal haemoglobinuria (PNH). A de novoPIG-A mutation in haematopoietic stem cells results in clonal expansion of the mutant and subsequent GPI (and GPI-AP) deficiency (measured using antibodies targeting CD55/CD59). Clinical symptoms of PNH include intravascular haemolysis and thrombosis (Endo, Ware, Vreeke, Howard, & Parker, 1996). Whilst there are around 150 GPI-APs, many GPI-APs are immune effectors. This includes CD55 and CD59 which regulate the complement immune system. In PNH, therefore, their widespread deficiency results in complement mediated erythrolysis (Boccuni, Del Vecchio, Di Noto, & Rotoli, 2000). Clinical presentation of symptoms similar to that of PNH have also been observed in patients with germline mutations resulting in systemic loss of the GPI-AP CD59 (Yamashina et al., 1990).
This mutation test has been widely researched in rodents to test novel products for mutagenic effects as part of regulatory safety assessment (Dobrovolsky, Cao, Bhalli, & Heflich, 2020; Olsen et al., 2017). It is also highly suitable for human application as the same biochemical pathway is conserved across mammals (Kawagoe, Takeda, Endo, & Kinoshita, 1994). Typical analysis time is 3-4 hours, not requiring further culturing or sub-cloning of mutant cells and costing as little as £50 per sample. The suitability of the PIG-A mutation assay as a tool for human biomonitoring and cancer detection has been examined and reported in the literature and is discussed in detail in this review.