458 THE TECHNIC OF COMPLEMENT-FIXATION REACTIONS 



four tubes are used in the actual test. Add 0.1 c.c. of fresh unheated patient's 

 blood-serum to each of the first 10 tubes. Then add decreasing amounts of 

 normal salt solution to these tubes, beginning with 1 c.c., then 0.9, 0.8, 0.7, 0.6, 

 0.5, 0.4, 0.3, 0.2, 0.1 c.c. to the succeeding nine tubes. Next add increasing 

 amounts of fresh 5 per cent, suspension of sheep's blood, starting with 0.1 c.c. and 

 ending with 1 c.c. Place the rack in the water-bath for one-half hour. The tube 

 which last shows complete hemolysis constitutes our "hemolytic index"; if it is 

 Tube 4, our index is 4, because this tube had received 0.4 c.c. of sheep's corpuscles. 

 The index determines the amount of sheep's corpuscles to be added to the last four 

 tubes. The first three tubes (11, 12, and 13) constitute the tubes for the actual test, 

 while the last tube in the rack (Tube 14) serves as our serum control tube. Tubes 

 11, 12, and 13 receive, therefore, the patient's serum, the proper amount of sheep's 

 corpuscles (dependent on the hemolytic index), rising strengths of antigen, but no 

 complement and no amboceptor. Tube 14 receives only sheep's corpuscles but no 

 antigen. In our technic we use 0.1 c.c. of a diluted antigen, determined by titration 

 in Tube 11, 0.15 c.c. antigen in Tube 12, and 0.2 c.c. in Tube 13. In order to equalize 

 the volume of fluid in all these tubes we add 0.2 c.c. normal saline to Tube 11, 0.15 

 c.c. to Tube 12, and 0.1 c.c. to Tube 13, and 0.3 c.c. to Tube 14. The tubes are then 

 agitated and placed in the water-bath for half an hour. These last four tubes are 

 filled at the time we make the additions to the first 10 and are left with them in the 

 water-bath for half an hour for fixation of complement; the rack is then taken out 

 and the hemolytic index computed. If the index is low, say from 1 to 4, we add 

 0.1 c.c. of sheep's blood to the last four tubes. If the index is between 5 and 7, we 

 use 0.15 c.c. sheep's blood, and if it is between 7 and 10, we use 0.2 c.c. In our ex- 

 perience in this country we have never found an index above 10, although in France 

 it is not uncommon to obtain an index of 15 or 17. 



"If the patient's serum has an index below 3, we regard the reaction as of doubt- 

 ful value. If it is above 3, we regard it as absolute. The reaction is read off exactly 

 as is the Wassermann, that is, inhibition or non-inhibition of hemolysis." 



MODIFICATION OF STERN 



Margaretta Stern devised a modification of the Wassermann reac- 

 tion, using fresh active serum and the patient's complement, and over- 

 coming non-specific reactions by using f to of the usual dose of 

 extract, and three or four times the amboceptor unit. This method is 

 open to defects inherent in the use of variable amounts of complement 

 and excessive amounts of hemolytic amboceptor, which makes it im- 

 possible to test a specimen a few days after it has been collected. 



MODIFICATION OF TCHERNOGUBOU 



Tchernogubou proposed an antihuman hemolytic system with active 

 serum. Blood is collected in sodium citrate, and therefore contains 

 erythrocytes, complement, and syphilis antibody if the patient is luetic. 

 Antigen is added, and after sufficient time has elapsed for fixation of 

 complement to take place, antihuman amboceptor is added to test for 

 free complement. There are many objections to this method, the chief 

 ones being the variable amount of complement present in human serum, 

 the large amount of hemolytic amboceptor required, the absence of a 

 suitable control on the antigen, and the fact that old blood is entirely 

 unsuited for making the test. 



Tchernogubou has also proposed a system in which the natural 

 amboceptor and complement of human serum are utilized against 



