Ham Test For Therapeutic Monitoring Of Eculizumab In Paroxysmal Nocturnal Hemoglobinuria

Introduction Paroxysmal nocturnal hemoglobinuria (PNH) is a rare acquired clonal disease, caused by an inactivating mutation in the PIG-A gene in a hematopoietic stem cell. The PIG-A gene encodes an enzyme required for glycosylphosphatidylinositol (GPI) anchor synthesis. Its inactivation results in a deficiency of many plasma membrane GPI-anchored proteins -including CD55 and CD59, natural inhibitors of the complement cascade- in the involved stem cell and all its progeny (the PNH clone). Intravascular hemolysis, anemia, thrombosis, acute and chronic renal damage, pulmonary hypertension, abdominal pain, esophagic spasm, erectile dysfunction -among others manifestations- are consequences of complement mediated damage of the sensitive PNH blood cells. In 2007 both the FDA and the EMA approved eculizumab, a monoclonal chimeric antibody targeted against C5 fraction of complement, as the first specific treatment of complement mediated PNH manifestations. Flow cytometry (FC) is the gold standard method for diagnosis. The former diagnostic test -the Ham test- is based on the susceptibility of PHN red blood cells (RBC), when they are incubated with both normal and patient sera to lysis mediated by the alternative pathway of complement (APC). APC is activated, in the Ham test, through sera acidification. Despite its physiopathological value, Ham test has been replaced with flow cytometry to diagnose PNH due to a much higher sensitivity and reproducibility. Aims To evaluate the Ham test in PNH treated patients, to monitor the eculizumab-mediated blockade of APC. Patients and methods Ham test was used to monitor APC blockade in the patient serum, testing the ability of the acidified patient serum to lyse his or her own PNH-RBC. Eight patients were diagnosed as PNH by FC and were treated with eculizumab. Six had a good therapeutic response, with decreased levels of both, LDH and the serum total complement hemolytic capacity (CH50). Ham test, in these six patients, showed hemolysis when PNH-RBC were mixed with normal acidified serum but absence of hemolysis when the acidified serum of eculizumab treated patient was added to the PNH-RBC. This result was called “blockade profile” and shows the “ex vivo” APC blockade, confirming thus the eculizumab success. The remaining two patients showed a persistent positivity of the Ham test at day 14 of eculizumab administration (as PNH-RBC lysis continued taking place with both normal and patient acidified sera). One patient demonstrated break through hemolysis occurring near the end of eculizumab dosing period as indicated by increase in LDH. As LDH may increase due to other possible factors (ie hepatic lesions) the positive Ham test confirmed that intravascular hemolysis was taking place, possibly due to a shorter eculizumab half life. An increase of the eculizumab dose to 1,200 mg/14 days reinstated lower LDH levels and the blockade profile in the Ham test (Table). There has been a single patient treated with eculizumab where LDH did not reduce. There was a persistently positive Ham test, elevated LDH and free hemoglobin levels and normal CH50 values despite a dose of 1,200 mg of eculizumab every 14 days (Table). A genetic study found in this case a C5 mutation, which seems responsible of the lack of response to eculizumab. Conclusions In our experience, the Ham test has proved to be a useful and economic method to monitor the effectiveness of eculizumab treatment in cases with high LDH levels due to either a) other causes than intravascular hemolysis, or b) no responsive patients due to pharmacokinetic (inadequate eculizumab concentration) or pharmacodynamic causes. Disclosures: Brodsky: Alexion Pharmaceuticals: Consultancy, Speakers Bureau. Colin:Alexion Pharmaceuticals: Consultancy.