412 IMMUNOLOGY 



6. Has the temperature of the incubator accidentally become 

 so high as to destroy the complement during the period of primary 

 incubation ? 



7. Were the tubes chemically clean and free from traces of acids, 

 alkalies, dyes, etc., that might fix complement? 



Complete Hemolysis in Tube 5. — On the other hand, if Tube 5, 

 mentioned above, shows complete hemolysis, the following questions 

 arise : 



1. Is it because of a deficiency of antibody content? 



2. Was the patient's serum inactivated? 



3. Was too much complement added? 



4. Will the antigen fix this particular amount and kind of 

 complement in the presence of known antibodies ? 



5. Is the antigen hemolytic? 



6. Are the red cells too fragile due to age ? 



7. Is the saline used hypotonic or isotonic ? 



8. Was it due to unusually slow fixation during the primary 

 stage of incubation? 



9. Is the hemolysis due to traces of acids, alkalies, or other 

 reagents that might be present in the test tubes or pipettes? 



Object of Controls. — These nine control tubes are designed 

 to answer most of the questions raised except those pertaining to 

 the temperature of the incubator or to slow fixation during primary 

 incubation. Acid hemolysis can be readily detected by the greenish 

 discoloration of the liquid in the tubes. 



Results When Fixation Is Due to Antibodies. — If fixation in 

 Tube 5 is due only to the presence of antibodies in the patient's 

 serum, then complete hemolysis should be present in serum control 

 Tube 6, where antigen is omitted; complete fixation in Tube 1, 

 the positive serum-antigen control; complete hemolysis in Tube 2, 

 designed as a check on the anticomplementary nature of the posi- 

 tive serum ; complete hemolysis in I'ube 3 containing normal serum 

 and antigen, and in its anticomplementary control Tube 4. Tube 

 7 should show complete hemolysis if the hemolytic system is work- 

 ing. Tubes 8, 9, and 10 should show no hemolysis if the hemolysin 

 has been properly inactivated, if the antigen is not hemolytic, and 

 if the saline is isotonic. In this protocol Tube 9 is used to de- 

 termine if the antigen is hemolytic. Kolmer prefers to use it to as- 

 certain if the antigen is anticomplementary and therefore includes 

 2 full units of complement. In that case complete hemolysis should 



