Background mutant frequency in healthy populations
Somatic PIG-A mutations can occur at low frequency through
spontaneous mutational processes during normal haematopoiesis without
inducing PNH-like clinical symptoms. This is perhaps because these
mutated, GPI-negative blood cells occur at a lower frequency than is
required to activate mass complement mediated lysis (Araten, Nafa,
Pakdeesuwan, & Luzzatto, 1999; Hu et al., 2005). These PIG-Amutated erythrocytes are present in healthy individuals at low numbers
(Cao et al., 2016; Dertinger, Avlasevich, Bemis, Chen, & MacGregor,
2015; Dobrovolsky, Elespuru, Bigger, Robison, & Heflich, 2011;
Lawrence, Haboubi, Williams, Doak, & Jenkins, 2020), allowing for easy
observation of any increase in mutant cell levels and association with
individual mutagenic exposures. At low levels, such mutant mammalian
cells are growth neutral, viable and are easily detectable using flow
cytometry (David et al., 2018) (Figure 1).
PIG-A mutations can be measured in all blood cell types and have
been reported for erythrocytes, reticulocytes, granulocytes, and
lymphocytes, with the majority of the reports using human erythrocytes.
A number of publications have measured PIG-A mutant erythrocyte
levels in healthy populations with values ranging from 2.9 - 5.56 x
10-6 mutants per million erythrocytes (Figure 2).
These 7 studies all measured PIG-A status through flow cytometric
analysis, using antibodies that target the GPI-linked protein CD59 (Cao,
Wang, Xi, et al., 2020; Cao et al., 2021; Cao et al., 2016; Dobrovolsky
et al., 2011), or CD55 and CD59 in combination (Dertinger et al., 2015;
Haboubi et al., 2019; Lawrence et al., 2020). Using both CD55 and CD59
in combination may reduce the number of falsely classified mutant cells
by eliminating any effect of reduced CD59 expression (Peruzzi, Araten,
Notaro, & Luzzatto, 2010). However, as seen in Figure 2, there is no
significant difference in mutant cell levels between studies that
applied either CD55 alone (blue bars) or in combination with CD59 (red
bars) (p=0.057). The green bar on the graph represents the weighted
average from these 7 publications which is 4.58 \(\pm\) 4.2 x
10-6 mutants per million erythrocytes. The consistency
across studies is remarkable, considering that mutant frequency can be
easily inflated by any slight technological variation. The number of
mutant cells is subject to technical variation and can be artificially
altered in a number of ways. This includes antibody batch to batch
variation and supplier differences, sample preparation resulting in
shearing of anchored proteins, flow cytometer choice, flow rate, laser
strength and gating procedure. Test reproducibility is essential for
successful biomarker implementation. It is particularly important if
such a test were to be used in the assessment of human genotoxic
exposures where a subtle change in mutant frequency, may be indicative
of a positive genotoxic response.