A standardized flow cytometric method for screening paroxysmal nocturnal haemoglobinuria (PNH) measuring CD55 and CD59 expression on erythrocytes andgranulocytes

PNH is a disorder of the pluripotent stem cells resulting in a deficient ex pression of membrane-bound GPI-anchored proteins in different cell types. S everal flow cytometric approaches are designed to detect this antigen defic iency. But they all require drawing and testing of normal samples as contro l. Therefore, in the present study two flow cytometric assays for the detec tion of CD55 and CD59 deficiency in erythrocytes (REDQUANT(TM) CD55/CD59) a nd granulocytes (CELLQUANT(TM) CD55/CD59) are proposed. Precalibrated beads are used to define the cut off between normal and deficient cell populatio ns. The specificity of the tests has been evaluated in healthy blood donors (n = 52) resulting in a clear and reproducible cut off (3%) for the normal percentage of GPI-deficient cells. This cut off has been confirmed in leuk aemia and lymphoma patients not suspected for developing PNH. The sensitivi ty has been tested in patients suffering from known PNH (n = 23). Both test s performed in combination allowed a reliable detection of PNH in all patie nts showing antigen deficiencies in both cell types in most patients (20/23 ). In contrast, the PNH clones in the investigated patients with MDS (4/19) or AA (4/22) were present in granulocytes or erythrocytes, only. This unde rlines the necessity of analysing erythrocytes as well as granulocytes. Pre liminary data regarding a possible correlation between disease activity and percentage of antigen-deficient cells lead to the assumption that haemolyt ic crises can only be determined on granulocytes whereas deficient erythroc ytes disappeared due to complement-mediated lysis of the PNH clone. In conc lusion, the combination of the test kits enables the differential diagnosis of PNH clones in a standardized, simple and rapid approach which may have therapeutic consequences.