364 IMMUNOGENETICS 



have all been used for the quantitative identification of cellular mixtures in chimeras 

 deriving from either natural or experimental transplants of erythropoietic tissues. In 

 some mixtures, however, only minute quantities of positive cells may be present in an 

 overwhelming predominance of negative cells, or, in the reverse situation, very small 

 numbers of nonreactive cells may be present among very large numbers of positive ones. 

 Several techniques have been developed to evaluate these minute populations. No 

 detailed description of techniques will be attempted here, but reference should be made 

 at least to an isotope-dilution method together with use of plant lectins for the selective 

 removal of positive cells; 36 to the "mixed agglutination" found with detector cells 

 added to a suspension ; 666 and to the potential utility of antibody marked with a radio- 

 active tracer. For a general discussion of this subject, see Cotterman. 228 



Preserving red cells. — Contamination of blood samples can be controlled by adding 

 certain antibiotics. In a procedure described by Cahan, 154 200,000 units of penicillin 

 G (crystalline-potassium) is dissolved in 100 ml. of a citrate solution (6.0 g. sodium 

 citrate, 7.0 g. sodium chloride, 1 liter distilled water). One gram of Streptomycin 

 sulfate (Squibb) is dissolved in 100 ml. of a phosphate buffer (16.4 g. Na 2 P0 4 -7H 2 0, 

 5.36 g. NaH 2 P0 4 H 2 0, 1 liter distilled water). Stone and Beckstrom 1284 report 

 that, by using 0.25 ml. of the penicillin solution and 0.1 ml. of the streptomycin solution 

 in the anticoagulant provided for 10 ml. of whole cattle blood, the blood can be shipped 

 for long distances without refrigeration and can be stored under refrigeration for 

 extended periods. 



Blood cells can also be stored frozen for years in proper media. A technique 

 effective for cattle erythrocytes has been described by Stone et a/. 1285 Whole blood 

 warmed to 37° C. is centrifuged, and the plasma is removed and replaced with 40 per cent 

 ethylene glycol at 37° C. made up in a 6 per cent sodium citrate solution. After 

 mixing, the blood is put into plastic tubes, stoppered and stored at —20° C. When 

 needed, the blood is thawed at room temperature or 37° C. A minimum of 4 ml. is 

 centrifuged and the supernatant discarded. The cells are resuspended in an excess of 

 20 per cent ethylene glycol, centrifuged, and the procedure is repeated with solutions 

 of 10, 5, and 2 per cent ethylene glycol in sequence. The cells are then suspended in 

 0.9 per cent saline. Although many of the cells may lyse during this procedure, 

 enough remain for typing, and they type normally after periods of storage in excess of 

 two years. 



For some species, glycerine or glycerine and plasma provide excellent media for 

 freezing and preserving red cells and other tissues. 195 For others, such as cattle, the 

 ethylene-glycol method described above seems to work much better. Several papers 

 on red-cell preservation were included at a meeting of the American Association of 

 Blood Banks in San Francisco in August, 1960, and abstracts appear in the published 

 proceedings of that meeting. 



Tests on contaminated blood samples should be regarded with caution, because 

 particular types of bacterial contamination may change the test reactions with particular 

 reagents. 1287 



