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An optimized method for Paroxysmal Nocturnal Hemoglobinuria screening flow cytometric diagnosis

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare clonal item cell disorder results from an absence of the glycosylphosphatidylinositol (GPI) anchor. Flow cytometry (FC) is considered the gold standard for detecting, traditionally CD55 and CD59. However, there has been no consensus on monoclonal antibodies or immunophenotypic criteria. We describe a technique for PNH diagnosis by FC. A hospital‐based, case–control immunophenotyped study was done of 84 patients with positive >1 PNH symptoms. GPI‐linked antigens and antibodies CD14 (FITC), CD4 (PE) and CD45 (APC) were used to assess the PNH+ monocytes and CD16 (PE), CD24 (FITC) and CD45 (APC) to establish the proportion of GPI‐deficient granulocytes and CD33 (PE), CD55 (APC) and CD59 (FHC). Screening was positive in 11% cases. Patients associated 38% thrombosis, 11–22 % autoimmune haemolytic anemia, aplastic anemia (AA) or leukopenia, 6–7% haemoglobinuria or myelodysplastic syndromes (MDS). Among the markers CD45, CD16, CD24, CD55, and CD59 all were effective in the detection of PNH+ granulocytes, although the combination of CD16 and 24 was most sensitive in monocytes. Conversely, CD55 and CD59 showed limited usefulness. Patients with clones >30% have evidenced of thrombosis and classic PNH and <10% AA and MDS. This simple FC inmunophenotyping screening panel might constitute a rapid and cost‐effective indicator for further confirmatory testing of PNH.