CD71-based immature RBC (iRBC) analysis in peripheral blood has a better correlation with leukocyte PNH clone size and improves the delineation of different PNH clone types.

Abstract

Flow cytometric analysis of leukocytes and red blood cells (RBCs) for loss of GPI-linked proteins is the gold standard for the diagnosis of PNH. Assessment of mature RBCs is often confounded by recent transfusions and the variable survival of normal versus PNH erythrocytes. Analysis of immature RBCs (iRBCs) provides a more accurate reflection of the underlying marrow clone size compared with mature RBCs (mRBCs). CD71 (transferrin receptor) serves as a robust marker for identifying iRBCs, thereby enabling the evaluation of PNH clones. This approach is still under investigation and has not yet been incorporated into the guidelines. We evaluated it in a large cohort of patients to validate its utility. FCM-based screening of leukocytes was carried out using a single-tube five-color panel (CD45-V500, FLAER-AF488, CD157-PE, CD15-PC 5.5, CD64-APC). The three-color RBC tube (CD235a-FITC, CD59-PE, CD71-APCH7) was used only in cases in which the leukocyte PNH clone size was >1%. Clone sizes across different cell populations, CD59 staining intensity, and the ability to distinguish among normal RBCs and type II and type III clones were compared between mRBCs (CD71-) and iRBCs (CD71+). A total of 782 samples were screened over a period of 24 months for various indications, including AA, non-immune hemolytic anemia, and unexplained thrombosis. PNH clones with a leukocyte clone size >1% were detected in 120 samples. The iRBC-PNH clone size was significantly higher than the mRBC-PNH clone size (median [range]: 49.9% [0.8-97.3] vs. 12.0% [0-86.0]; p = 0.002). The iRBC-PNH clone size corresponded closely with those of neutrophils and monocytes (median [range]: 49.9% [1-97.3] vs. 54.5% [1-99.5] vs. 58.7% [6.3-98.5], respectively). The MFI of CD59 in iRBCs was higher than that in mRBCs (8996 vs. 5687; p < 0.001). Normal RBCs and type II and III clones were better delineated in iRBCs than in mRBCs. Among cases with clinical evidence of hemolysis and/or thrombosis, the iRBC-PNH clone size reflected the disease burden better than the mRBC-PNH clone size (median [range]: 80.4% [20-95.6] vs. 26.6% [1.1-85.6]). This study validates the role of CD71-based analysis of iRBC-PNH clones, which was found to improve the detection, delineation, and sizing of PNH RBC clones and to correlate better with leukocyte clone sizes and clinical features. Therefore, incorporation of CD71 into high-sensitivity FCM-based PNH screening is suggested.