Multilineage glycosylphosphatidylinositol anchor-deficient haematopoiesis in untreated aplastic anaemia

Aplastic anaemia and paroxysmal nocturnal haemoglobinuria (PNH) are closely related disorders. In PNH, haematopoietic stem cells that harbour PIGA mut ations give rise to blood elements that are unable to synthesize glycosylph osphatidylinositol (GPI) anchors. Because the GPI anchor is the receptor fo r the channel-forming protein aerolysin. PNH cells do not bind the toxin an d 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 0.0 04% of total red cells could be detected when cells were pretreated with ae rolysin to enrich the PNH population. All PNH patients displayed CD59-defic ient 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 a erolysin. However, 14 (61%) had detectable PNH cells after enrichment in ae rolysin. The inactive fluorescent proaerolysin variant (FLAER) that binds t he 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 showe d that 12 out of 18 (67%) aplastic anaemia patients had FLAER-negative gran ulocytes, but none of the MDS patients or normal control subjects had GPI a nchor-deficient cells. These studies demonstrate that aerolysin-based assay s can reveal previously undetectable multilineage PNH cells in patients wit h untreated aplastic anaemia. Thus. clonality appears to be an early featur e of aplastic anaemia.