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.