846 THE COMPLEMENT FIXATION TEST FOR SYPHILIS 



employing cholesterinized antigen with cold fixation and a relatively small amount of 

 complement in proportion to the total volume of the test, the least sensitive results 

 with a method employing water-bath fixation at 37° C. and relatively more comple- 

 ment. The desirability of using a sensitive cholesterinized antigen was again demon- 

 strated, and there was no indication that such an antigen, if carefully standardized, 

 will give definite reactions with specimens from normal individuals. Two laboratories 

 using identical procedures secured results that agreed remarkably well. If a satis- 

 factory standardization can be secured, therefore, results obtained throughout the 

 various parts of the world should be uniform. 



The great variation in procedures that are being employed in different laboratories 

 and the diificulties encountered in efforts made to secure adequate standardization 

 of the test emphasize the importance of determining the fundamental basis for 

 complement fixation. Study directed toward the determination of the nature of the 

 substances in patient's serum, antigen, and complement that take part in the reac- 

 tion, the nature of the reactions that occur, as well as the effect of physical conditions 

 on these combinations, will be of the greatest value. The solution of these problems 

 will no doubt be most readily obtained through investigation of the reactions that 

 occur between bacterial antigens, immune sera, and complement. 



Almost regardless of the method used, there are certain factors essential to the 

 satisfactory performance of complement fixation tests. It is of prime importance that 

 the persons responsible for the work have the proper educational background and 

 adequate training and experience. The apparatus used should be adequate to permit 

 accurate measurement of the reagents, and all glassware should be scrupulously 

 clean. Proper precautions are necessary to insure uniformity of procedure from day 

 to day, and special care should be observed in the performance of the details of the 

 test in order to detect possible technical errors. The patient's serum should be free 

 from evidence of contamination or hemolysis. Complement, if obtained from guinea 

 pigs, should consist of a pool of serum from three or more healthy, non-pregnant 

 animals that have had a comparatively uniform diet consisting in part of leafy 

 vegetables. The refrigeration of the serum used as complement after it has been re- 

 moved from the clot, as well as its protection from unnecessary agitation, is of im- 

 portance. The individual sera should be tested for hemolytic activity and fixability 

 with the antigens to be used, and unsatisfactory ones discarded prior to making the 

 pool. The antigen should be prepared and diluted in a uniform manner and tested for 

 anticomplementary and hemolytic activities and for fixability with serum from a large 

 series of cases of syphilis as well as healthy individuals and if possible from those 

 suffering from diseases other than syphilis, to insure that a satisfactory extract and a 

 proper dilution of it has been chosen. The blood cells employed should be tested for 

 fragility and proper control of the density of the suspensions made. Color standards 

 should be prepared for use in reading the results of the test, and there should be uni- 

 formity in the volume of the test and the various titrations. The reagents should al- 

 ways be mixed in the same order, preferably — I believe — patient's serum, then anti- 

 gen, and finally complement, the mixture being shaken after each addition. For con- 

 trol purposes, each serum should be tested without antigen and each series should in- 

 clude tests with antigen and complement without patient's serum and a routine test 



