DIAGNOSIS OF PAROXYSMAL-NOCTURNAL HEMOGLOBINURIA BY PHENOTYPIC ANALYSIS OF ERYTHROCYTES USING 2-COLOR FLOW-CYTOMETRY WITH MONOCLONAL-ANTIBODIES TO DAF AND CD59 MACIF/

We investigated the relationship between the complement lysis sensitiv ity test and two-colour flow cytometric analysis using monoclonal anti bodies to decay accelerating factor (DAF) and CD59/membrane attack com plex inhibitory factor (MACIF) in patients with paroxysmal nocturnal h aemoglobinuria (PNH) and other haematological diseases. Flow cytometry showed that all 59 PNH patients had two or three erythrocyte populati ons, 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 per centage of negative cells shown by flow cytometry in 52 PNH patients, and found a significant correlation (r = 0.960, P < 0.001). However, i n 13 patients the erythrocyte phenotypes did not correspond in both te sts. This was generally related to difficulty of detecting PNH II eryt hrocytes in the lysis test. In the PNH patients the ranges of mean flu orescence intensity for the negative, intermediate and positive erythr ocyte 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 DA F positivity. In contrast, the mean intensities in healthy volunteers ranged from 190 to 720 for CD59/MACIF and from 150 to 350 for DAF. The se findings suggest that PNH can be diagnosed and phenotypic analysis of PNH erythrocytes can be performed by respectively assessing the flu orescence 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.