METHODS IN MAMMALIAN IMMUNOGENETICS 361 



The buffer favored for such purposes was reported by Pillemer 1006 in his studies of 

 complement and properdin. The recipe is as follows: 



85.0 g. NaCl 

 5.75 g. 5,5-diethylbarbituric acid 

 3.75 g. 5,5-diethylbarbiturate 

 5.0 ml. M MgCl 2 

 1 .5 ml. M CaCl 2 



Dissolve in about 1500 ml. hot distilled water. Cool, add distilled water to final 

 volume of 2 liters. Store as stock at 1 ° C. ; add one part stock buffer to four parts dis- 

 tilled water before use; discard diluted portions after 12 hours (pH 7.4). 



One laboratory reports that time can be saved in setting up extensive red-cell 

 hemolytic tests if measured amounts of diluted complement are mixed with the test 

 serum and dropped into the tubes in a single operation, before the cells are added. 

 Another laboratory, however, reports that this technique in some systems leads to some 

 inactivation of the complement, and that it is better to add the complement to the 

 sensitized cells as a final step. Hemolytic tests are often read at an interval of one-half 

 hour after the test has been set up and shaken, in order to detect the quick hemolysis 

 that is often characteristic of cells homozygous for the test antigen, in contrast to the 

 slower hemolysis detected with the cells of heterozygotes. In any case, the cells are 

 then allowed to settle for a further hour and a half before a definitive reading is taken ; 

 often an additional reading is taken after two more hours. Readings are generally 

 recorded on a scale from to 4, representing no hemolysis, in which the supernatant 

 is free of hemoglobin deriving from the test cells. With increasing degrees of hemolysis 

 deeper color appears in the supernatant, and the size of the pellet of sedimented cells 

 decreases. Complete hemolysis (4) leaves no unlysed cells; the shaken tube remains a 

 sparkling clear red. 



Hemolytic tests are especially well adapted to evaluating the proportions of 

 positive and negative cells in a mixture, such as the cellular population found in a 

 chimera. The supernatant of the hemolytic test can be read for hemoglobin colori- 

 metrically; and after subtraction of an appropriate blank prepared to measure the 

 contribution in color, if any, from the non-red-cell components of the test (that is, 

 the complement and the test serum), the degree of color in the test tube can be ex- 

 pressed as a fraction of the color in the tube containing the same amount of red cells 

 osmotically lysed. This technique has proved useful in following the course of re- 

 population of the erythropoietic tissues of irradiated mice after homologous bone- 

 marrow transplants. 452, 981, 985 In other systems, it is reported that more reliable 

 values may be obtained by washing the residual cells after the specific lysis of positive 

 cells in a mixture, then lysing these residual cells osmotically and reading the hemo- 

 globin colorimetrically. 848 



4. Antiglobulin test systems. A sensitive and versatile technique for the detection 

 of incomplete antibodies was described by Coombs, Mourant, and Race in 1945. 225 



