Screening for Paroxysmal Nocturnal Hemoglobinuria (PNH) in Patients with Cytopenia and Suspected Myelodysplasia Using the Fluorochrome-Conjugated Derivative of Aerolysin, FLAER
Authors: Kern Wolfgang , Haferlach Claudia , Schnittger Susanne , Haferlach Torsten 
Published: April 2020

Abstract
Evaluation of patients with peripheral blood cytopenias includes bone marrow assessment by cytomorphology and cytogenetics for findings suggestive of a myelodysplastic syndrome (MDS) or proving this diagnosis. While multiparameter flow cytometry (MFC) may increasingly add diagnostic information regarding MDS, MFC is the essential method for the assessment of an important differential diagnosis, paroxysmal nocturnal hemoglobinuria (PNH). Furthermore, it has been suggested by the International PNH Interest Group to screen patients with MDS once yearly for the presence of PNH clones irrespective of the occurrence of hemolysis. While the flow cytometric analysis for PNH has been based for decades on the detection of GPI-anchored surface antigens the methodological spectrum has been broadened recently by the introduction of FLAER (Protox Biotech, Victoria, BC) which is a fluorochrome-conjugated, mutated and inactivated derivative of the bacterial toxin, aerolysin. It directly binds to the GPI moiety of GPI-linked structures and has been shown to reproducibly detect even small amounts of cells from PNH clones at less than 1%. In order to assess the diagnostic value of FLAER in screening patients with cytopenia and suspected MDS for PNH we applied FLAER in parallel to the standard evaluation for PNH as well as for MDS. Of 29 patients analyzed 10 were diagnosed by cytomorpholgy as having MDS, 5 as possible MDS, 2 as chronic myelomonocytic leukemia, 4 as acute myeloid leukemia (AML), 5 as reactive conditions, 1 as aplastic anemia, 1 as toxic cytopenia following chemotherapy, and 1 was not evaluable. The evaluation for PNH included the assessment of CD55 and CD59 on erythrocytes, granulocytes and monocytes as well as of CD16 on granulocytes and of CD14 on monocytes. In addition, FLAER was used to analyze granulocytes and monocytes. In three PNH cases analyzed as controls both conventional markers as well as FLAER clearly demonstrated the affected populations. While the assessment of CD55, CD59, CD16, and CD14 did not reveal a sign suggestive of PNH in any of the 29 patients there were 7 patients in whom a reduced signal for FLAER was found in a portion of the granulocytes. This portion ranged from 10% to 29% of all granulocytes. Similar findings, although less striking, were obtained in monocytes. Interestingly, all of these 7 cases, with the exception of the case not evaluable by cytomorphology, had a diagnosis of either MDS or AML (aberrant karyotype in 4/7) and no aberrant signal for FLAER was found in patients with non-malignant diagnoses. These data suggest that by applying FLAER GPI-deficient cells and thereby PNH-clones may be detected in patients with MDS and AML. It remains to be proven, however, which diagnostic significance is carried by FLAER in the absence of findings by conventional markers suggestive of PNH. Further studies are warranted to assess the clinical utility of adding FLAER to the standard diagnostic panel for PNH, particularly in the setting of patients diagnosed with MDS.