High sensitivity 8-color flow cytometry assay for paroxysmal nocturnal hemoglobinuria granulocyte and monocyte detections

Abstract

Flow cytometry is the gold standard in diagnosis of paroxysmal nocturnal hemoglobinuria (PNH) by detecting the absence of glycol-phosphatidyl inositol (GPI)-linked protein expression on granulocyte and monocyte surfaces. However, the current assays are not optimized and require improvement, particularly in reducing background fluorescence and optimizing sensitivity and specificity. With more fluorochromes available and with advances in instrument engineering, rare populations may be identified with high sensitivity. The present study assessed an 8-color combination of comprehensive GPI-linked markers, namely fluorescein-labeled proaerolysin (FLAER), cluster of differentiation 157 (CD157), CD24 and CD14, and the lineage markers for granulocyte (CD15) and monocyte (CD64) cells to detect PNH clones. Additionally, to optimize the PNH flow assay, a ‘dump’ channel was used, comprised of CD5 and CD19, to exclude non-specific binding in order to reduce background. This method aimed to improve sensitivity and reduce the background to create an optimized PNH flow cocktail. The results demonstrated that the current 4-color PNH combination identifies a CD55− and FLAER+ population that is not PNH clones. By contrast, the 8-color panel delineated PNH clones from both monocyte and granulocytes by using granulocyte antigen (CD15) and monocyte antigen (CD64) as a gating strategy. The sensitivity was 0.01% for granulocytes and 0.05% for monocytes with an acquisition of 100,000 monocyte and granulocyte events. The background on a normal whole blood sample was 0.00076% on monocytes and 0.00277% on granulocytes. Thus, overall, the 8-color PNH assay exhibited high levels of specificity and sensitivity. The 8-color combination facilitated the improvement and enhancement of sensitivity in PNH clone identification, and may provide a useful tool for pathologists in PNH diagnosis and for monitoring patients at risk of developing classical/hemolytic PNH, to enable treatment to be delivered promptly.

CD157 Can Replace CD24 and CD14 in a Single-Tube Flow-Cytometric Assay to Detect Paroxysmal Nocturnal Hemoglobinuria (PNH) Clones on Both Neutrophils and Monocytes: A Prospective Study From North India

“…These observations support the ability of CD157/FLAER-based single-tube assays to detect a wide range of PNH clones on both neutrophils and monocytes with acceptable correlation to the methods based on CD24, CD14, and FLAER. Of note, our technique was not designed as a high-sensitivity PNH leucocyte assay, and hence we might have missed smaller clones measured by the methods adopted by other observers [ 5 , 7 - 12 ].…”

“…Their observation can help in judiciously prescribing PNH assays to individuals with MDS in resource-constrained settings. In another newer approach, a single-tube assay using nine antibodies (CD45/CD15/CD64/CD24/CD14/CD157/FLAER, with CD5/CD19 in the same fluorochrome for a "dump" channel to exclude non-specific binding) achieved a sensitivity of 0.01% for granulocytes and 0.05% for monocytes; the background on a normal whole blood sample was 0.00277% on granulocytes and 0.00076% on monocytes [ 12 ]. These recently published data offer scope for further clinically meaningful research using CD157/FLAER-based PNH leucocyte assays.…”

“…Like the predicate four-color methods, the CD157-based four and five-color assays could satisfactorily delineate PNH leucocytes from leucocytes with normal GPI expression, with a high correlation between the clone sizes detected [ 5 ]. Thereafter, CD157 has been evaluated successfully in single-tube flow-cytometric techniques in several studies [ 7 - 12 ]. The reduction of one antibody in CD157/FLAER-based assays as compared to CD24/CD14/FLAER-based methods improves the technical efficiency of the laboratory and reduces the cost of the test to the patients [ 5 , 7 - 10 ].…”