Multilineage glycosylphosphatidylinositol anchor‐deficient haematopoiesis in untreated aplastic anaemia

Abstract

Summary. 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 channelforming 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.

Detection of PNH cells by flow cytometry, using multiparameter analysis

“…The presence of PNH clones in 25% of patients diagnosed as AA is in line with literature data, which describe the frequency of PNH cells in 22% to 68% of AA patients (5,8,9,12,19,20,25) . This prevalence variability occurs due to methodological differences between the used tests, which define different limits of analytical sensitivity.…”

Clinical significance of a minor population of paroxysmal nocturnal hemoglobinuria–type cells in bone marrow failure syndrome

“…[1][2][3][4][5][6] We previously demonstrated that a minor (less than 1%) population of CD55 Ϫ CD59 Ϫ granulocytes could be detected in the peripheral blood of patients with AA at a much higher frequency than previously reported. 7 Given that the proportion of PNH-type granulocytes decreased in most patients in association with hematologic recovery after successful immunosuppressive therapy, such PNH-type cells appeared to be derived from PNH-type progenitor cells that evaded immune system attack against hematopoietic progenitor cells.…”

The small population of PIG-A mutant cells in myelodysplastic syndromes do not arise from multipotent hematopoietic stem cells

“…11 Interestingly, small populations of PNH-like granulocytes can be found in up to 50% of patients with MDS (usually 0.01-5% PNH granulocytes) 12 and AA (usually 0.1-10% PNH granulocytes). 13,14 PIG-A mutant blood cells (~0.002%) can also been found in healthy control subjects. 15,16 In contrast to PNH, most PIG-A mutations in healthy controls are transient and arise from colony forming cells that have no self-renewal capacity and no ability to differentiate into T lymphocytes.…”