Underestimation of the expression of cellular prion protein on human red blood cells

BACKGROUND: Recent transmissions of variant Creutzfeldt-Jakob disease by blood transfusion emphasize the need for the development of prion screening tests. The detection of prions in blood is complicated by the presence of poorly characterized cellular prion protein (PrPC) in both plasma and blood cells. According to published studies, most of PrPC in blood cells resides in platelets (PLTs) and white blood cells. STUDY DESIGN AND METHODS: To clarify conflicting reports about the quantity of PrPC associated with human red blood cells (RBCs), quantitative flow cytometry, Western blot (WB), and enzyme-linked immunosorbent assay (ELISA) were used to measure protein levels in healthy donors. RESULTS: RBCs expressed 290 +/- 140 molecules of PrPC per cell, assuming equimolar binding of monoclonal antibody (MoAb) 6H4 to PrPC. Binding of alternate PrPC MoAbs, FH11 and 3F4, was substantially lower. WB estimated the level of PrPC per cell on RBCs to be just four times lower than in PLTs. A similar level of PrPC was detected using ELISA. The weak binding of commonly used MoAb 3F4 was not caused by PrPC conformation, truncation, or glycosylation, suggesting a covalent modification, likely glycation, of the 3F4 epitope. CONCLUSIONS: Taken together, human RBCs express low but significant amounts of PrPC/cell, which makes them, due to high RBC numbers, major contributors to the pool of cell-associated PrPC in blood. Previous reports utilizing MoAb 3F4 may have underestimated the amount of PrPC in RBCs. Likewise, screening tests for the presence of the abnormal prion protein in blood may be difficult if the abnormal protein is modified similar to RBC PrPC.