Evaluation of HEPES and phosphate buffers for rheological studies of sickle cells

Abstract

Determination of the optimal buffer for rheological studies of sickle cells is of particular importance for in vitro screening of potential anti-sickling compounds and for ex vivo studies of erythrocyte deformability in patients with sickle cell disease. We have shown that sickle cells can be incubated in HEPES buffered saline (20 mmol/l HEPES with 5 mmol/l glucose) for 6 hours at 37°C without significant change in morphology, haemolytic rate, mean cell volume, mean cell haemoglobin concentration, osmolality, pH, ATP content, filterability through 5 µm diameter pores, and oxygen affinity. Continuous mixing of sickle cells during the 6 hour incubation was essential to maintain stable values for oxygen tension and erythrocyte rheology.