Human erythrocyte separation according to age on a discontinuous "Percoll" density gradient.

Abstract

It is well known that during ageing, erythrocyte density increases [l-3]. Density gradient centrifugation is the technique most generally used for fractionating erythrocytes of different mean age. Materials employed to construct density gradients, such as bovine serum albumin (BSA) [4], Stractan II [5], Ficoll [6] and Dextran [7], have many disadvantages. The development of Percoll (colloidal silica particles coated with polyvinylpyrrolidone) has overcome these problems. Owing to its low viscosity, low osmotic pressure and non-toxicity this material can be easily adjusted to physiological conditions. Percoll methods allow separation of erythrocytes on discontinuous gradients [&lo], linear gradients [ 111, or self-generated gradients [ 121. In this work, red cell separation in a discontinuous gradient similar to that reported by Alderman et al. [9] is described. To obtain Percoll with the closest approximation to physiological conditions, a suitable modification of Rennie’s buffer system [ 1 l] has been used. Separated red cell populations have been tested for cell recoveries, reticulocyte count and some age-dependent erythrocyte enzyme activities (pyruvate kinase, hexokinase, glucose-6-phosphate dehydrogenase, aldolase) and indices (mean cell volume and red cell volume distribution width index, mean cell haemoglobin concentration). Two non age-dependent measurements were also made (phosphoglycerate kinase and mean cell haemoglobin). The effectiveness and reliability of the method have been evaluated.