Ham test

Abstract: Because of the insensitivity of the Ham test, paroxysmal nocturnal haemoglobinuria (PNH) has been inaccurately viewed as a late clonal complication of aplastic anaemia (AA). To clarify the relationship between PNH and marrow failure, we tested for the presence of glycosylphosphatidyl-anchored protein-deficient (GPI-AP) granulocytes in large cohorts of patients with AA, myelodysplasia (MDS), and pure haemolytic PNH. A PNH clone was detected in 32% of new AA patients and 18% of MDS patients. In serial studies, this proportion did not change up to 15 years after diagnosis, suggesting that expansion of aberrant cells is an early event (i.e. prior to initial presentation). For all patients with a PNH clone, on average 14% of PNH granulocytes were found on presentation and 37% at 10 years. Patients with PNH but without cytopenia showed higher percentages of GPI-AP-deficient cells than did those with the AA/PNH syndrome. After immunosuppression, there was no change in the contribution of PNH clone to blood production, arguing against the ‘immune-escape’ theory in PNH. Clinically, a high proportion of GPI-AP-deficient cells correlated with marrow hypercellularity. GPI-AP-deficient cells were similarly present in patients with and without karyotypic abnormalities. Our results indicate that the GPI-AP-deficient clones show quantitative and kinetic differences between classic haemolytic PNH and PNH with marrow failure, in which the evolution rate is low later in the course of the disease.

Abstract: Paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome are diseases of excess activation of the alternative pathway of complement that are treated with eculizumab, a humanized monoclonal antibody against the terminal complement component C5. Eculizumab must be administered intravenously, and moreover some patients with paroxysmal nocturnal hemoglobinuria on eculizumab have symptomatic extravascular hemolysis, indicating an unmet need for additional therapeutic approaches. We report the activity of two novel small-molecule inhibitors of the alternative pathway component Factor D using in vitro correlates of both paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome. Both compounds bind human Factor D with high affinity and effectively inhibit its proteolytic activity against purified Factor B in complex with C3b. When tested using the traditional Ham test with cells from paroxysmal nocturnal hemoglobinuria patients, the Factor D inhibitors significantly reduced complement-mediated hemolysis at concentrations as low as 0.01 μM. Additionally the compound ACH-4471 significantly decreased C3 fragment deposition on paroxysmal nocturnal hemoglobinuria erythrocytes, indicating a reduced potential relative to eculizumab for extravascular hemolysis. Using the recently described modified Ham test with serum from patients with atypical hemolytic uremic syndrome, the compounds reduced the alternative pathway-mediated killing of PIGA-null reagent cells, thus establishing their potential utility for this disease of alternative pathway of complement dysregulation and validating the modified Ham test as a system for pre-clinical drug development for atypical hemolytic uremic syndrome. Finally, ACH-4471 blocked alternative pathway activity when administered orally to cynomolgus monkeys. In conclusion, the small-molecule Factor D inhibitors show potential as oral therapeutics for human diseases driven by the alternative pathway of complement, including paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome.