Separation of rabbit red cells by density methods and characteristics of separated layers

Abstract

Abstract Red cells from rabbits (reticulocyte count 1–2%) injected 4–6 days previously with Fe 59 were centrifuged in isotonic hypodense media (plasma, buffer, saline) for 30 minutes at 1,600 G and layers were removed to achieve density fractionation. An important criterion of separation was the fractionation ratio (specific activity of hemoglobin in any layer/specific activity of hemoglobin in unfractionated blood) which indicated whether a layer contained mainly “new” or “old” red cells. The most important factor in enhancing the fractionation ratio was the centrifugal force, but time also had an effect. All isotonic media were essentially equivalent, but hypertonic media (sucrose) were quite poor. Dextran and PVP were also not good in promoting density separation. The exact degree of separation achieved by various combinations of conditions is documented, but no system achieves a true equilibrium, as shown by the fact that refractionation can split any fraction into subfractions of higher and lower hemoglobin specific activity. Upper layers were characterized by greater cell size, more reticulocytes, more free cholesterol, phospholipid, and ATP, and a faster rate of glycolysis. The distribution of Fe 59 in the fractionated blood was followed serially for up to ten weeks, the most homogeneous distribution being seen only in the first week or two. After that the Fe 59 was not sharply restricted to any density fraction. This suggested that the most significant density change takes place as the reticulocyte matures.