Aplastic anaemia and paroxysmal nocturnal haemoglobinuria (PNH) are closely related disorders. In PNH, haematopoietic stem cells that harbour PIGA mutations give rise to blood elements that are unable to synthesize
glycosylphosphatidylinositol (GPI) anchors. Because the GPI anchor is the receptor for the channel-forming
protein aerolysin, PNH cells do not bind the toxin and are unaffected by concentrations that lyse normal cells. Exploiting these biological differences, we have developed two novel
aerolysin-based assays to detect small populations of PNH cells. CD59 populations as small as 0.004% of total red cells could be detected when cells were pretreated with
aerolysin to enrich the PNH population. All PNH patients displayed CD59-deficient erythrocytes, but no
myelodysplastic syndrome (MDS) patient or control had detectable PNH cells before or after enrichment in
aerolysin. Only one
aplastic anaemia patient had detectable PNH red cells before exposure to
aerolysin. However, 14 (61%) had detectable PNH cells after enrichment in
aerolysin. The inactive fluorescent
proaerolysin variant (FLAER) that binds the GPI anchors of a number of
proteins on normal cells was used to detect a global GPI anchor deficit on granulocytes. Flow cytometry with FLAER showed that 12 out of 18 (67%)
aplastic anaemia patients had FLAER-negative granulocytes, but none of the MDS patients or normal control subjects had GPI anchor-deficient cells. These studies demonstrate that
aerolysin-based assays can reveal previously undetectable multilineage PNH cells in patients with untreated
aplastic anaemia. Thus, clonality appears to be an early feature of
aplastic anaemia.