Rapid and Sensitive Single Tube Screen for Paroxysmal Nocturnal Hemoglobinuria (PNH) Clones in Aplastic Anemia, Myelodysplastic Syndrome and Bone Marrow Failure Syndromes by Multiparameter Flow Cytometry.
Authors: Movalia Mayur K. , Illingworth Andrea 
Published: October 2019

Abstract
Several studies have shown that the presence of glycosylphosphatidylinositol (GPI) anchored protein-deficient PNH-type cells in patients with aplastic anemia and myelodysplastic syndrome may indicate favorable response to therapy and an overall favorable prognosis. Multiparameter flow cytometry is a powerful tool in the detection of even minimal populations of these cells. Evaluation for PNH-type clones has typically required in-depth analysis of red blood cells, granulocytes and monocytes using three tubes looking for the loss of multiple GPI-anchored proteins. Our laboratory has used this method in over 2,100 patients with known aplastic anemia and myelodysplastic syndrome. Accurate PNH-type clone evaluation requires careful backgating, strict gating strategies and a certain level of experience as immature cells, including blasts, may mimic PNH-type clones. The aim of this study was to develop a single tube five-color assay that could be used as a rapid and sensitive screen for the presence of PNH-type cells. We prospectively analyzed the granulocytes in the peripheral blood of 27 patients with known aplastic anemia and myelodysplastic syndrome using a single tube 5-color assay with FLAER-Alexa488 / CD24-PE / CD14-ECD / CD15-PC5 / CD45-PC7. This one-tube approach allows lineage specific gating on granulocytes looking for CD24 and FLAER deficiency. Also, to a more limited extent, the monocytes can be evaluated for CD14 and FLAER deficiency. The results were compared to a more in-depth three tube analysis for PNH-type cells analyzing the red blood cells (GPA-FITC/CD59-PE), granulocytes (CD66b-FITC/ CD24-PE/ CD45-ECD/ CD15-PC5/ CD16) and monocytes (CD64-FITC/ CD55-PE/ CD45-ECD/ CD33-PC5/ CD14). The results are summarized in Table 1. Using a detection sensitivity of 0.05% among the granulocytes showing loss of CD24 and FLAER, the screening assay was able to detect all six patients with PNH-type clones (range 0.4% – 79.1%), which was confirmed by the multiple tube panel. Of the 21 negative samples, all were below the 0.05% limit of detection. Four samples did show a population of CD24-negative, FLAER-negative granulocytes ranging from 0.01%–0.02%. The overall sensitivity and specificity of the single tube screen was 100%. This single tube five-color approach allows for a rapid and simple approach to screening patients for PNH-type clones with a detection sensitivity of 0.05%. Although an extended multiple tube analysis may still be required on positive screening samples to evaluate for the type of red blood cell PNH clone, the single tube screen optimizes the testing approach for PNH-type cells.
Single tube screen versus multiple tube analysis for PNH-type Clones Multiple tube Multiple tube Positive Negative Total Positive = greater than or equal to 0.05% PNH-type cells detected Single tube Positive 6 (22%) 0 (0%) 6 Single tube Negative 0 (0%) 21 (78%) 21 Total 6 21