Low Level Residual Extravascular Haemolysis Is Commom Following Eculizumab Treatment In Paroxysmal Nocturnal Haemoglobinuria(PNH), but Does Not Affect Transfusion Requirement

PNH is an acquired haemolytic anaemia which is characterised by intravascular haemolysis. The disease is due to a somatic mutation of the PIG-A gene in haematopoietic stem cells resulting in loss (complete or partial) of glycosylphosphatidylinositol-(GPI) anchored proteins on the cell surface. The lack of CD59, one of the GPI-anchored proteins, on the red cell surface leads to increased susceptibility to complement-mediated intravascular haemolysis. Eculizumab is a monoclonal antibody that inhibits the complement protein C5 by preventing its cleavage and leads to transfusion independence in 50% of the patients.

Objective: To determine the role of C3 –mediated opsonisation of PNH cells, as we found a significant number of patients treated with Eculizumab still needing transfusion.

Twenty six PNH patients treated with Eculizumab and 22 patients from an untreated non transfusion dependant PNH cohort were analysed. Transfusion dependency was present in 14 of 26 patients (54%) compared with transfusion independence in 12/26 (46%) patients. Direct anti-globulin test (DAT) was positive in 21 of 26(81%) patients during eculizumab treatment, while DAT was positive in 2 of 24 untreated patients (8%).All except one patient (20/21) with positive DAT had C3d positivity at varying levels. There was no correlation between the strength of C3d positivity and transfusion dependency.

Of the 21 patients who had a positive DAT during eculizumab treatment, samples were available for 16 patients prior to treatment, and 7 of 16 were DAT positive. All the 7 patients were weakly positive for IgG and only one patient had C3d positivity at a low level. The two untreated patients were also only weakly positive for IgG.

Among the eculizumab treated patients, 14 of the 21(67%) with a positive DAT test received at least two transfusions compared with no transfusions in the 5 DAT negative patients (p<0.007). There was a reduction in transfusion requirements compared to pre-eculizumab requirement in both DAT positive and DAT negative groups. Interestingly, 7 out of 21(33%) who were DAT positive did not require transfusion post-eculizumab. The median age of the eculizumab treated patient cohort was 51years (range 22–73 years). The median duration of Eculizumab treatment in the 14 DAT positive patients who needed transfusion post eculizumab was 17 months (3.8-98 months) while the median duration of Eculizumab treatment in the 5 DAT negative patients was 7.9 months (1.8-98.5 months). There was no difference in the neutrophil PNH clone size and mean haemoglobin levels (p=0.3), in the DAT positive and negative patients. The median LDH levels did not differ between eculizumab treated DAT positive and negative cases.

Eculizumab treatment in PNH patients has resulted in significant reductions in transfusions in a majority of patients, though some patients still require transfusion at a low intensity. The observation of C3 fragments on PNH red cells reveals an extravascular haemolytic pathway which is unmasked by Eculizumab treatment as evidenced by finding new DAT positive tests. The finding of positive DAT test in a 9 of 50(18%) untreated haemolytic PNH patients demonstrates the utility of flow cytometry to analyse PNH clones when clinical suspicion is high, as conventionally finding DAT positivity indicates autoimmune haemolytic process and excludes diagnosis of PNH. The true detection of C3d on red cells causing a positive DAT test post treatment, rather than due to allo-antibodies is important. There is a proportion of patients (33%) who became DAT positive but never needed transfusion indicating the contribution of other factors like bone marrow failure, intercurrent infections in the transfusion dependent DAT positive group.

Eculizumab is effective in resolution of intravascular haemolysis, but unmasking of low level C3d mediated haemolysis is an important phenomenon which is not entirely responsible for the persistent transfusion requirement in a subset of patients.

No relevant conflicts of interest to declare.