MECHANISM OF HAEMOLYSIS 231 



After centrifuging for the third time dilute the red cells with sufficient 

 sterile normal saline solution to bring the total volume up to the volume of 

 blood originally used. 



It is perhaps unnecessary to say that these operations should be carried out 

 under aseptic conditions. 



2. Inoculate the animal (a guinea-pig if using rabbit cells, a rabbit for 

 sheep cells, etc.) sub-cutaneously, or better into the peritoneal cavity, on 

 five occasions at intervals of 1 week with 5-8 c.c. of a suspension of red cells 

 prepared as described above. Experience has shown that this amount does 

 not produce any toxic symptoms. The serum of the animal is best collected 

 about 1 week after the last immunizing inoculation. 



Mechanism of haemolysis. 



The phenomena of haemolysis are a counterpart of those of bacteriolysis. 



The haemolytic serum is specific and only haemolyzes red cells of the animal 

 species used for inoculating the animal from which the serum has been 

 drawn. 



It contains two substances : 



1. A specific thermostable immune body. 



2. A complement, non-specific, present in all normal serums and only 

 becoming attached to the red cells through the immune body. The pro- 

 perties of haemolytic serums can be demonstrated by means of the following 

 experiments. 



Experimental illustrations. Use the serum of a guinea-pig inoculated with 

 rabbit red cells as an haemolytic serum and prepare an emulsion of red cells 

 by mixing O'l c.c. of washed rabbit red cells with 2 c.c. of normal saline 

 solution. 



(i) Mix the emulsion of red cells with O'l c.c. of the haemolytic serum and 

 incubate at 37 C. for 1 hour. On taking the tube out of the incubator it 

 will be seen by simply looking at the tube that haemolysis is complete : the 

 haemoglobin has been discharged from the red cells and imparts an uniform 

 colour to the solution. 



A control tube in which a little normal guinea-pig serum has been added 

 to an emulsion of red cells shows no haemolysis ; the fluid contents are clear. 



(ii) To an emulsion of red cells, add Ol c.c. of haemolytic serum previously 

 heated to 55 C. for half an hour and incubate at 37 C. 



The red cells are not haemolyzed but simply agglutinated at the bottom 

 of the tube. In this case haemolysis has failed because the complement was 

 destroyed by heating the serum to 55 C. 



(iii) To the mixture used in the preceding experiment and which is quite 

 clear add O'l c.c. of normal guinea-pig serum (complement) and incubate 

 again. The red cells will now undergo haemolysis. 



(iv) Kepeat experiment (ii) and after showing that under the conditions of 

 the experiment no haemolysis occurs, centrifuge the mixture and pipette off 

 the serum from the red cells. 



(a) The centrifuged serum has been deprived of its immune body (which 

 has combined with the red cells) and any attempt to re-activate it by the 

 addition of complement (normal guinea-pig serum) fails ; it is no longer 

 able to haemolyze fresh rabbit cells if these be added to it. 



(6) The red cells separated from the serum by centrifuging have combined 

 with the immune body ; so that even after being repeatedly washed with 

 normal saline solution and centrifuged, they are rapidly haemolyzed on the 

 addition of O'l c.c. of normal guinea-pig serum (complement) if a mixture of 

 the two be put in the incubator at 37 C. 



