A method for the separation of erythrocytes on the basis of size using counterflow centrifugation

Abstract

A method has been developed for separation of erythrocytes on the basis of size using counterflow centrifugation. Human red blood cells with an original mean corpuscular volume (MCV) of 89.2 ± 4.1 fl were isolated, free of plasma proteins and other cell contaminants, into seven fractions ranging in size from 77.0 ± 2.7 fl to 98.5 ± 4.8 fl. The ratio of the age‐related enzyme, erythrocyte glutamic oxaloacetic transferase (EGOT), to hemoglobin (Hb) increased progressively through the fractions, suggesting a correlation between erythrocyte volume and age. Reticulocytes, though present in all fractions, were selectively enriched in the larger subpopulations. To verify the biochemical evidence that erythrocytes decrease in volume with aging, in vivo cohort labeling of red blood cells with ⁵⁹Fe was performed in baboons. A similar relationship of EGOT to Hb was observed to that in the human subpopulations. The peak activity of ⁵⁹Fe/RBC appeared initially in the red blood cells with the highest MCV and progressed from the erythrocytes with the largest MCV to the erythrocytes with the smallest MCV over the next 10–12 weeks, confirming the hypothesis that red blood cells decrease in volume as they age. The technique of counterflow centrifugation appears to provide a simple, rapid, and reproducible method for the separation of erythrocytes on the basis of size.