Accurate and High Sensitivity Identification of PNH Clones by Flow Cytometry

Flow cytometry performs a key role in the diagnosis of paroxysmal nocturnal hemoglo- binuria (PNH). Careful selection and validation of antibody conjugates have allowed the development of reagent cocktails suitable for the high sensitivity detection of PNH red blood cells (RBCs) and white blood cells (WBCs) in PNH and related diseases such as aplastic anemia (AA) and some subsets of myelodysplastic syndromes (MDS). A CD235a- FITC/CD59-PE assay was developed capable of detecting Type III PNH RBCs at a limit of quantification (LOQ) of 0.01% or better. While separate 4-color Fluorescent Aerolysin (FLAER), CD24, CD15 and CD45-based neutrophil and FLAER, CD14, CD64 and CD45-based monocyte assays were developed to detect PNH WBC phenotypes, 5-, 6- and 7-color assays have subsequently been developed for more modern cytometers equipped with five or more fluorescence detectors. For instrumentation with five detectors, a single tube 5-color FLAER, CD157, CD15, CD64 and CD45-based assay to simultaneously detect PNH neutrophils and monocytes has been developed. For instruments with six or more detectors and multiple lasers, a variety of 5-, 6- and 7-color assays have been developed using combinations of FLAER, CD24, CD14 and CD157. All WBC assays have a limit of quantification (LOQ) of 0.1% or better. Using these standardized approaches, results have demonstrated good intra- and inter-laboratory performance characteristics even in laboratories with little prior experience performing PNH testing.