Red Blood Cell Surface Proteins and Cryopreservation for Tracking Altered Erythropoiesis

ABSTRACT The present study aimed to investigate the potential of selected red blood cell surface proteins as biomarkers of altered erythropoiesis. A secondary aim was to determine whether long‐term cryopreservation affects surface protein expression and clinical red blood cell characteristics. Twelve males received subcutaneous injections of recombinant human erythropoietin (rhEpo, 50 IU/kg epoetin β) three times per week for 3 weeks. Venous blood was collected before, during and after treatment. Fresh blood was analyzed for surface protein expression (CD35, CD47, CD55, CD59) by flow cytometry and standard hematology analyzer‐derived red cell characteristics. Samples were cryopreserved and reanalyzed after 2 years. In fresh blood, mean fluorescence intensity for the decay‐accelerating factor CD55 increased ( p < 0.001) with rhEpo treatment, while intensity for the ageing‐dependent CD35, the antiphagocytic CD47 and the membrane attack complex inhibitor CD59 decreased ( p < 0.001), contrary to expectations based on red cell aging. Importantly, the investigated proteins presented significant baseline variation. Compared with fresh blood, cryopreserved cells exhibited a higher mean fluorescence intensity of CD35 and CD47, a lower intensity of CD55 and CD59 and reduced baseline variation but did not reveal clear treatment‐related changes. Cryopreservation increased ( p < 0.05–0.001) variability of MCV, MCH, MCHC, and RDW‐CV. In conclusion, CD35, CD47, CD55, and CD59 were not found to be reliable biomarkers of rhEpo due to inconsistent expression patterns during treatment and substantial baseline variation. Analysis of cryopreserved cells reduced analytical variability but did not improve detection of rhEpo‐induced changes and introduced systematic differences in protein expression and red cell indices compared with fresh blood.