Ficoll Flotation for the Separation of Blood Leukocyte Types
- Huiny CurrS, J. H. Cui-rs
method has been developed in this laboratory using a gum acacia solution of proper specific gravity, hydrogen ion concentration, and osmotic pressure. Supravital studies of leukocytes separated by this method indicate that cells are viable and apparently undamaged . Preparation of solution: To io ml. ofdistilled water in a 100 ml. graduate (which has a stopper), 30 grams of gum acacia, and 30 ml. water are added. The materials are mixed with a glass rod until solution is complete. Then 0.2.5 Gm. of sodium bicarbonate are added. After mixing well, the glass rod is washed down with distilled water and enough water added to make o ml. The graduate is stoppered and heated in a water bath at 50 C for a few days. The solution is agitated at intervals and unstoppered occasionally to permit the escape of carbon dioxide. After a few days, the volume is brought to ioo ml. with distilled H2O. The pH is tested with a Beckman pH meter. If the pH is below 6.9, heating is continued. When the solution has reached a pH of 6.9-7.2. through the combined effects of the original amount of sodium bicarbonate and continued heating, pilot tests are run to determine the combination of gum acacia and citrate-saline which will produce the best separation. Eight tenths ml. of this gum acacia solution is mixed with 0.30, 0.35, 0.40, 0.45 ml. of a i per cent sodium citrate in o. per cent sodium chloride. After the solutions are thoroughly mixed, heparinized blood is added in the proportion of 1.5 ml. blood to 1.0 ml. of gum acacia saline-citrate mixture. With the exception of a few red cells which sink immediately, the blood floats. Tubes are spun at 500 r.p.m. for ten minutes, and 3,000 r.p.m. for thirty minutes. At the end of this time, in correct solutions, the red cells are at the bottom of the tube, and the white cells float at the interface of the layers of gum acacia and plasma where they may be easily collected with a capillary pipet. The collected cells are placed in a small test tube with about twice their volume of 2. per cent citrate and shaken briskly to counteract the agglutination of platelets which was promoted by the colloidal gum acacia solution. To estimate the number of white cells present, the tube may be spun at 2.000 r.p.m. for five minutes, the supcrnatant fluid removed, and the cells resuspended in a measured quantity of 2. per cent sodium citrate. A WBC pipet is used for obtaining the number of cells per ml. The diluent used is a solution of brilliant cresyl blue o.i Gm., sodium citrate 3.8 Gm., and water zoo ml. The solution does not hemolyze the red cells which may be present and stains the white cells a pale blue. The total number of WBC present is estimated by multiplying the count per cubic mm. X 1000 X ml. of citrate added to cells in the last suspension.