An Affinity Chromatography
Method for the Preparation of Human Reticulocytes An affinity chromatography method utilizing transferrin-liganded agarose for the separation of reticulocytes from erythrocytes and leucocytes in samples obtained from anemic (reticulocyte-rich) and normal human donors is described.
Studies on reticulocytes from human sources have been limited by the difficulty of separating reticulocytes from whole blood. While populations of red cells containing high proportions of reticulocytes can be obtained by induced anemia in animals (l), thus circumventing this problem, the proportion of reticulocytes in blood from anemic humans rarely exceeds 40% and is more often 15 to 20%, even in response to vitamin Blz and folate treatment (2). Here we describe a simple affinity method utilizing transferrin-liganded agarose gels for the separation of reticulocytes from erythrocytes and nonerythroid cells in whole blood. The method is based on that of Edelman et al. ( 3) and exploits the fact that, unlike erythrocytes, reticulocytes transport iron and hence are able to bind transferrin (4).
Transferrin-liganded Sepharose 4B was prepared by the method of Adair and Kornfeld (5). Any uncoupled sites were blocked by incubating the agarose in 1.0 M ethanolamine for 2 hr at room temperature. The transferrin-coupled Sepharose 4B was then washed sequentially in 0.1 M sodium acetate, pH 4.0,O. I5 M NaCl, and phosphate-buffered saline (PBS, 8 g of NaCl, 0.2 g of KCl, 1. I5 g of Na2HP04, 0.2 g of KH2POI, 0.1 g of CaCl,, and 0.1 g of MgClz .6Hz0 per liter of solution). After dilution (1: 1, v/v) with unactivated Sepharose 4B, the gel was packed into a column over siliconized glass beads.
Blood was obtained (generally l-2 ml) from human patients responding to vitamin B,,/folate treatment for severe anemia. The cells were washed four times in PBS, but the buffy coat was not removed after each wash because the reticulocyte fraction sediments preferentially in the uppermost half of the red cell pellet (6). The cells were suspended in PBS to give a final concentration of 4% (3). The cell suspension was then slowly applied (typically, 1.5-20 ml/hr) at room temperature to a SO-ml gel column (15 x 2 cm). The column was then washed through with PBS for 2 to 4 hr before elution of the bound cells with a 10 mg/ml solution of transferrin in PBS. It was found that some agitation of the column increased the rate of elution. The results for two samples of blood obtained from patients treated for megaloblastic anemia and pernicious anemia (containing 18 and 20%