Diagnosis of paroxysmal nocturnal haemoglobinuria by phenotypic analysis of erythrocytes using two-colour flow cytometry with monoclonal antibodies to DAF and CD59/MACIF.

We investigated the relationship between the complement lysis sensitivity test and two-colour flow cytometric analysis using monoclonal antibodies to decay accelerating factor (DAF) and CD59/membrane attack complex inhibitory factor (MACIF) in patients with paroxysmal nocturnal haemoglobinuria (PNH) and other haematological diseases. Flow cytometry showed that all 59 PNH patients had two or three erythrocyte populations, while all 74 patients with other haematological diseases and all 31 healthy volunteers had a single erythrocyte population. We compared the percentage of PNH III erythrocytes in the lysis test with the percentage of negative cells shown by flow cytometry in 52 PNH patients, and found a significant correlation (r = 0.960, P < 0.001). However, in 13 patients the erythrocyte phenotypes did not correspond in both tests. This was generally related to difficulty of detecting PNH II erythrocytes in the lysis test. In the PNH patients the ranges of mean fluorescence intensity for the negative, intermediate and positive erythrocyte populations were respectively 1.1-2.5, 2.2-29, and 61-600 for CD59/MACIF positivity and 1.9-7.2, 3.6-22. and 31-350 for DAF positivity. In contrast, the mean intensities in healthy volunteers ranged from 190 to 720 for CD59/MACIF and from 150 to 350 for DAF. These findings suggest that PNH can be diagnosed and phenotypic analysis of PNH erythrocytes can be performed by respectively assessing the fluorescence profiles and mean fluorescence intensities of both proteins using flow cytometry. Flow cytometry may provide a superior diagnostic method to the traditional tests for PNH.