METHODS IN MAMMALIAN IMMUNOGENETICS 365 



OTHER CELLS IN SUSPENSION 



Although suspensions of red blood cells have provided the most convenient 

 material for mammalian immunogenetics in the past, increasing attention is being 

 given to the antigenic characterization of other types of cells as well. Some of the red- 

 cell specificities seem to be limited to the erythrocytes; others are found in or on 

 diverse types of cells and tissues. Particularly in man, a variety of white-cell antigens 

 has become available for immunogenetic analysis. In some instances, these are of 

 significance in systems of maternal-fetal incompatibility, comparable to those involving 

 the Rh and ABO red-cell antigens. 152 - 586 - 744 - 1071 



Terasaki 1317 has described a procedure for obtaining rather pure lymphocyte 

 suspensions from chicken blood, and for conducting agglutination tests with lymphocyte 

 suspensions. Agglutinating test systems have also been applied to mammalian 

 leucocyte suspensions; in the past, however, such tests have often given inconsistent 

 results. 242 Absorption tests with A and B human white cells have given straight- 

 forward results, as have such tests with spermatozoa, though sperm are not agglutinated 

 directly by anti-^4 or anit-i?. 750 Race and Sanger 1036 report that the ability of 

 spermatozoa to absorb anti-^4 and anti-5 antibodies is not removed by as many as nine 

 successive washings of the sperm, indicating that this property of the spermatozoa is 

 probably not simply adsorbed from the seminal fluid. Kiddy and his colleagues 706 

 have reported rather extensive tests on the antigenic qualities of rabbit sperm. 



The sensitive and versatile technique of mixed agglutination was reported by 

 Coombs and Bedford in 1955. 223 Essentially, it involves sensitizing test cells with anti- 

 body, then adding red cells of known type. If the test cells adsorb the antibody to 

 their surface, red cells of the corresponding type cluster on the surfaces of the sensitized 

 cells, whereas failure of the test cells to combine with the antibody results in their 

 failure to accumulate a shell of the corresponding red cells. This technique, with 

 relatively minor modifications, has been used to type human epidermal cells, 224 cells in 

 tissue culture, and other cells. A basically similar procedure was reported by Gull- 

 bring 511 to fractionate sperm-cell suspensions from AB men; anti-^4 sensitized A red 

 cells combined with part but not all of the sperm, leaving an unreactive fraction, and 

 anti-5-sensitized red cells had a similar effect. The question of whether a sperm cell 

 may express its own antigenic genotype, however, remains open at present. 



Another useful and versatile technique depends upon the cytotoxic effects of anti- 

 body on cells, especially in the presence of complement. 456 Several procedures are 

 applied ; the one we use, based entirely on the experiences of others and most directly 

 from Vos et a/. 1351 is as follows: Murine spleen-cell suspensions are prepared by pressing 

 the spleen through a fine stainless steel screen, and suspending in Tyrode's solution. 

 Small clumps are suspended by flushing the suspension in and out of a syringe with a 

 22-gauge needle. One drop of the cellular suspension is mixed with one drop of 

 murine antiserum or normal serum. One drop of rabbit complement, which has been 

 absorbed with red cells of the mouse in the cold in order to remove normal rabbit-anti- 



