Evaluation of the haemopoietic reservoir in de novo haemolytic paroxysmal nocturnal haemoglobinuria

Paroxysmal nocturnal haemoglobinuria (PNH) is an acquired clonal disorder of the haemopoietic stem cell (HSC). The pathogenetic link with bone marrow failure is well recognized; however, the process of clonal expansion of the glycosylphosphatidylinositol (GPI)-deficient cells over normal haemopoiesis remains unclear. We have carried out detailed analysis of the stem cell population in 10 patients with de novo haemolytic PNH using the long-term culture-initiating cells (LTC-IC) assay in parallel with measurements of CD34(+) cells and mature haemopoietic progenitors, granulocyte-macrophage colony-forming unit (CFU-GM) and CFU-erythroid [burst-forming units erythroid (BFU-E) + CFU granulocyte/erythroid/macrophage/megakaryocyte (GEMM)]. All patients had hypercellular bone marrows with erythroid hyperplasia, normal blood counts or mild peripheral blood cytopenias, increased reticulocyte counts and evidence of deficient GPI-anchored proteins. We found a significant reduction in the LTC-IC frequency in the CD34(+) compartment of PNH patients (mean 2, range 1.3-3.0; n = 6) compared with normal donors (mean 13, range 5.2-45.5; n = 21) (P < 0.0001). Furthermore, there was a significant reduction in the erythroid compartment [CFU-E/10(5) bone marrow mononuclear cells (BMMC) and CFU-E/10(5) CD34(+) cells] of PNH patients, but no significant difference in the granulocyte-monocyte precursors (CFU-GM/10(5) BMMC or CFU-GM/10(5) CD34(+) cells) compared with normal donors, suggesting that there is a defect in the early stem cell pool in PNH patients without clinical or haematological evidence of bone marrow failure.