Abstract : ABSTRACT Paroxysmal nocturnal hemoglobinuria (PNH) is a rare hematopoietic stem cell disorder characterized by a somatic mutation in the PIGA gene, leading to a deficiency of proteins linked to the cell membrane via glycophosphatidylinositol (GPI) anchors .These include complement defence structures like CD55 and CD59 , absence of which on RBC is responsible for intravascular hemolysis.CD16,CD 24 and CD66 are GPI linked antigens on granulocytes and CD14 is a GPI –linked marker expressed on monocytes.Flow cytometry is the method of choice for identifying cells deficient in GPI-linked proteins and is, therefore, necessary for the diagnosis of PNH . Fluorescent aerolysin (FLAER) is another reagent that binds to GPI linked antigens on leucocytes and is preferred reagent for PNH testing . CD 157 is a member of CD38 supergene family is another GPI linked structure expressed at relatively high antigen density on neutrophils and monocytes. The aim of our study was to assess the ability of CD157 to replace CD24 and CD14 as a GPI anchor in our predicate 5 color granulocyte and monocyte high resolution flow cytometry .We also assessed CD64 as a lineage gating agent for monocytes with respect to CD45 being used alone for lineage gating of monocytes. PNH testing was done prospectively in patients diagnosed with Aplastic anemia / cytopenias. The study period was two years from 2016-2018.Total number of patients were 27. Acquisition and analysis were performed on a BD FACS Canto II cytometer equipped with lasers and BD FACS Diva software.Two tubes were tested using 5 color flow cytometry; Tube 1 used the predicate method with the following 5 antibodies , FLAER FITC , CD24PE, CD14APCH7,CD15APC and CD45PerCP. Tube 2 used the following 5 antibodies, FLAER FITC ,CD157PE ,CD64APCH7,CD15APC and CD45PerCP.All individual antibodies and FLAER were verified for appropriate reactivity with target cells prior to being cocktailed for use in the high sensitivity granulocyte and monocytes assays. The frequency of cells with PNH phenotype among granulocytes and monocytes were determined in 10 normal samples using FLAER-CD24-CD14-CD15-CD45.The normal samples were similarly screened using 5 color CD157 based granulocyte and monocytes assays. Data was acquired in both the tubes with 1,00,000 total number of gated granulocyte events and 20,000 gated monocytes or a maximum of 10 minutes .Results were determined by the number of FLAER negative /CD24 negative events , FLAER negative/CD14 negative events or FLAER negative/CD157 negative events. Statistical analysis was done by the Wilcoxon –signed rank test for paired samples at level 0.05 using Epi info and Microsoft excel sheet 2017 Of the total 27 subjects 7 were pediatric patients and 20 adult patients. Mean age of pediatric patients was 11 years(range 4-15 years).Mean age of Adults was 37 years ( range 18-63 years).There were 15 males and 12 female patients .25 patients were diagnosed with Aplastic anemia , 1 for refractory anemia with unilineage dysplasia and 1 for unexplained cytopenia. We applied the Wilcoxon –signed rank test for CD24/CD157 for granulocyte and and CD14/CD157 and were significant but rejected the null hypothesis .3 patients were PNH positive and 4 with severe Aplastic anemia underwent allogenic bone marrow transplantation . CD157 was superior to CD24 and CD14 for assessing GPI deficiency and diagnosing PNH.CD 64 was superior for gating monocytes than using CD45 alone for assessing monocytes.CD157 is a rapid and cost effective reagent that can be coupled with FLAER diagnosing PN Keywords: Paroxysmal nocturnal hemoglobinuria, CD157

Keyword : Paroxysmal nocturnal hemoglobinuria, CD157