Eculizumab prevents intravascular hemolysis in patients with paroxysmal nocturnal hemoglobinuria and unmasks low-level extravascular hemolysis occurring through C3 opsonization

Abstract

Background: Paroxysmal nocturnal hemoglobinuria is an acquired hemolytic anemia characterized by intravascular hemolysis which has been demonstrated to be effectively controlled with eculizumab. However, lactate dehydrogenase levels remain slightly elevated and haptoglobin levels remain low in some patients suggesting residual low-level hemolysis. This may be due to C3-mediated clearance of paroxysmal nocturnal hemoglobinuria red blood cells through the reticuloendothelial system.

Design and methods: Thirty-nine samples from patients not treated with eculizumab and 31 samples from patients treated with eculizumab were obtained (for 17 of these 31 samples there were also samples taken prior to eculizumab treatment). Membrane bound complement was assessed by flow cytometry. Direct antiglobulin testing was carried out using two methods. Lactate dehydrogenase was assayed to assess the degree of hemolysis.

Results: Three of 39 patients (8%) with paroxysmal nocturnal hemoglobinuria not on eculizumab had a positive direct antiglobulin test, while the test was positive in 21 of 31 (68%) during eculizumab treatment. Of these 21 patients who had a positive direct antiglobulin test during eculizumab treatment, 17 had been tested prior to treatment; only one was positive. Flow cytometry using anti-C3 monoclonal antibodies was performed on the 21 direct antiglobulin test-positive, eculizumab-treated patients; the median proportion of C3-positive total red blood cells was 26%. Among the eculizumab-treated patients, 16 of the 21 (76.2%) with a positive direct antiglobulin test received at least one transfusion compared with one of ten (10.0%) of those with a negative test (P<0.01). Among the eculizumab-treated patients, the mean hemoglobin value for the 21 with a positive direct antiglobulin test was 9.6+/-0.3 g/dL, whereas that in the ten patients with a negative test was 11.0+/-0.4 g/dL (P=0.02).

Conclusions: These data demonstrate a previously masked mechanism of red cell clearance in paroxysmal nocturnal hemoglobinuria and suggests that blockade of complement at C5 allows C3 fragment accumulation on some paroxysmal nocturnal hemoglobinuria red cells, explaining the residual low-level hemolysis occurring in some eculizumab-treated patients.

Figure 1.

Surface levels of C3b fragment determined by flow cytometry. A fluorescent anti-C3b antibody was used against (A) washed whole blood from normal volunteers (negative control) and (B) washed whole blood from normal volunteers incubated with neat serum from a patient with cold hemagglutinin disease and neat C8d serum (positive control) (x axis = fluorescent intensity; log scale).

Figure 2.

Direct antiglobulin test (DAT) in patients with PNH in the presence or absence of the complement inhibitor eculizumab. Proportions of DAT-positive individuals among normal volunteers, PNH patients not receiving eculizumab, and PNH patients receiving eculizumab are shown.

Figure 3.

Flow cytometric analysis of C3b fragment deposition on red cells from PNH patients before or during eculizumab treatment. Levels of C3b on A) red cells from a normal volunteer; B) and C) red cells from a PNH patient not on eculizumab; D), E) and F) red cells from PNH patients on eculizumab.

Figure 4.

Two-color flow cytometric analysis of C3b fragment deposition on red cells from PNH patients during eculizumab treatment. A proportion of CD59-negative red cells are C3b-positive whereas the non-PNH (CD59-positive red cells) remain negative for C3b.

Figure 5.

Transfusion requirement and hemoglobin values in DAT-positive or -negative PNH patients treated with eculizumab. Proportion of patients who received at least one transfusion and the level of hemoglobin in DAT-positive (DAT +; n=21) and DAT-negative (DAT -; n=10) PNH patients on eculizumab therapy.