Figure 2. Background levels of phosphatidylinositol glycan class A (PIG-A ) mutant erythrocytes in healthy populations from published research. Blue bars represent publications that have used antibodies targeting CD55 only and red bars represent publications that have used antibodies targeting CD55 and CD59 in combination. The green bar shows the weighted mean. The bars represent mean values and the error bars represent standard deviation.
We have assessed repeated measurements of individuals over time to provide further evidence relating to the reproducibility of the assay and the stability of the erythrocyte PIG-A mutant phenotype. Figure 3A shows repeat measurements from the same 4 individuals (2 females and 2 males) over a period of 17 weeks (see (Lawrence, 2020 #4) for methods). Although intra-individual variation exists, erythrocytePIG-A mutant frequency remains relatively stable. For example, the mutant frequency of Participant 2 remains relatively high between 7 and 14 x 10-6 mutants (average 10.53 \(\pm\) 2.16 x 10-6 mutants) whilst the mutant frequency of Participant 4 is consistently below 5 x 10-6 mutants (average 3.56 \(\pm\) 1.22 x 10-6 mutants). This is similar to what has been previously published where Cao and colleagues repeatedly measured the RBC PIG-A mutant frequency of 3 individuals and discovered that whilst 2 subjects had MF’s consistently below 5 x 10-6 mutants, one subject’s MF always remained around 15 x 10-6 mutants (Cao et al., 2016). The average coefficient of variation (CV) (indicator of intra-individual variation) of the 4 individuals presented here was 35.2%. This is lower than the intra-individual variation observed in granulocytes by Rondelliet al where 32 healthy subjects were tested for PIG-Astatus on 3 separate occasions which produced a CV of 44.3% (Rondelli et al., 2013). Figure 3B further shows the erythrocyte PIG-Amutant frequency for 9 healthy volunteers at 2 different time points. The duration between each time point ranged from 92 to 516 days with an average duration of 270 days. Participant HV112 had consistently high mutation frequency of 9.61 – 10.75 x 10-6 mutant erythrocytes and HV038 mutant frequency remained low between 2.14 and 2.43 x 10-6 mutants over this period. This data further demonstrates the general stability of PIG-A mutant phenotype over a longer period in self-reported healthy volunteers.