The Influence of Salt- Concentration on Hemolysis. 405 



the red cells and antibody combine at a temperature of 0° C, while the 

 further combination of complement with the red-cell-antibody complex only 

 occurs when the temperature is raised. 



An attempt was made to determine whether the combination of red cells 

 and complement in a salt-free medium conformed to this rule, as we should 

 expect if the union were really a direct or unaided one. This observation is 

 of considerable importance, since it would obviously be theoretically possible 

 for an antibody to be involved, which was present in the guinea-pig serum, 

 but which only acted in salt-free media, and which therefore was not removed 

 when the undiluted serum was absorbed on ice with sheep corpuscles. 



The following experiment yields a definite indication as regards this 

 possibility :— 



Experiment. — A number of tubes were prepared, each containing 1 c.c. of 

 a 7"8-per-cent. saccharose solution. To one, red cells and complement were 

 added and incubation was carried out for one hour at 37° C. This tube was 

 controlled by another containing the red cells and complement in 1 c.c. of the 

 normal saline-saccharose solution. To further tubes of saccharose solution 

 were added in some cases red cells and complement, and in others complement 

 alone, and these tubes were kept for one hour at 0° C. Some of these tubes 

 then rapidly centrifugalised, and the supernatant fluid and deposit treated as 

 indicated. 



The red cells were added as O'OS c.c. of a 50-per-cent. suspension in 

 saccharose solution ; 0-075 c.c. of complementary serum was added in every 

 case. 





Haemolysis. 



Incubation for 1 hour at 37° C. — 



(2) „ „ „ +0 -8 per cent. NaCl 



1 hour at 0° C. followed by incubation for 1 hour at 37" C, after the 

 following treatment — 



(4) Deposit from one (red cells + complement) tube + supernatant 



fluid from another 



(5) Supernatant fluid from one (red cells + complement) tube + fresh 



red cells 



(6) Deposit from one (red cells + complement) tube + 1 c.c. of 



saccharose solution 



(7) Supernatant fluid from a tube containing complement alone + 



fresh red cells 



(8) A tube containing complement alone shaken + red cells 



Very marked. 

 None. 



Very marked. 

 >> )> 



3) J J 



None. 



Very marked. 



)J 3) 



Prom these results it follows that : — 



(1) The red cells do not absorb the whole complement at 0° C, since when 

 they are separated by centrifugalisation and, after being suspended in fresh 



