Erythrocyte Age-fractionation Using a Percollκ-Renografin® Density Gradient: Application to Autologous Red Cell Antigen Determinations in Recently Transfused Patients

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Abstract
A rapid technic for the age-fractionation of human erythrocytes into reticulocyte-enriched (young) red blood cells and reticulocyte-poor (old) red blood cells using an isopycnic density gradient centrifugation through Percoll™-Renografin® was evaluated for use in autologous red blood cell antigen determinations in multiply-transfused patients. The fractionation was demonstrated by statistically significant density-related changes in pyruvate kinase and acetylcholinesterase activities (P = 0.002 and 0.042, respectively) and by the distribution of reticulocytes on the gradient (P < 0.005). With initial reticulocyte counts of ≤1.5%, reticulocyte counts up to 78% were achieved (x = 25%; n = 31). When starting with reticulocyte counts > 5%, samples containing up to 98% reticulocytes were obtained (X = 64%; n = 7). The technic requires less than two hours, uses isotonic media, and is nontoxic to red blood cells. Volumes of red blood cells up to 10 mL can be fractionated at one time and the gradient medium is stable when refrigerated at 4°C. Red blood cell typing was performed in six patients who had received from 4–29 units of blood within a 12-hour period. Within 72 hours posttransfusion, typing of the reticulocyte enriched fraction correctly identified the patient’s red blood cell antigens with all 16 antisera tested. This technic for typing reticulocyte-enriched samples is of importance for confirmation of antibody specificity in determining whether an antibody is an alloantibody or autoantibody, and in the selection of donor blood for transfusion to patients having autoimmune hemolytic anemia.