RUTH GILBERT 839 



that extracts from organs which did not contain Treponema pallidum were satisfac- 

 tory antigens. Landsteiner and Stankovic' demonstrated that alcoholic extracts from 

 the organs of normal animals were even more satisfactory than the aqueous extracts 

 of syphilitic organs which had been used previously. 



Almost from the first the complement fixation test was found very helpful as an 

 aid in the diagnosis of syphilis, and it rapidly came into general use in large public 

 health and hospital laboratories. As the technique is somewhat complicated, how- 

 ever, many and diverse procedures for the performance of the test were proposed. In 

 about twenty years, the literature on the subject has become voluminous, and since 

 the fundamental basis for the reaction has not been determined, most of the research 

 work on the subject has of necessity consisted primarily in the comparison of the 

 results of various procedures with the clinical manifestations in the patient, an at- 

 tempt being made to obtain a large proportion of positive reactions with specimens 

 from syphilitic patients and as few reactions as possible with specimens from patients 

 free from any history or clinical manifestations of the disease. The pendulum has con- 

 tinued to swing between tests which are so lacking in delicacy that specimens from 

 cases of syphilis not infrequently fail to react, and those that give reactions with 

 specimens from patients who present every evidence of being free from syphilis. 



The diversity of the procedures used ranges from variations in minor details to 

 marked differences in essential factors. An outline of the methods' employed in some 

 of the large laboratories in this country and abroad may serve to illustrate the degree 

 of this variability. 



METHOD OFFICIALLY RECOMMENDED FOR USE IN GERMANY^ 



Each component of the test is so diluted that it will be contained in 0.5 cc. The patient's 

 serum is inactivated for one-half hour at from 55° to 56° C. A i : 5 dilution is used in the test. 



The complement employed is a i : 10 and i : 20 dilution of fresh guinea pig serum. 



Three antigens, or preferably five, are used, one of which is an extract of syphilitic liver. 

 They are prepared by manufacturers and tested by the government during a period of two 

 weeks on at least six days. They are tested against eighty syphilitic and forty control sera. 



One hour in the incubator at 37° C. is a'lowed for fixation. After the addition of sensi- 

 tized cells, there is a secondary incubation period until the controls have hemolyzed. 



The amboceptor is titrated daily with 0.5 cc. of a i: 10 dilution of complement, and a 

 similar titration is made with the addition of antigen. The dilution used must contain at 

 least four times the amount necessary for hemolysis without antigen and at least the smallest 

 amount required for hemolysis with antigen. 



Sheep ceUs, washed three times, are used in a 5 per cent suspension. 



The results are recorded according to the following scale: 



The supernatant fluid colorless = 4-H 



The supernatant fluid delicate pink = 3-!- 



The supernatant fluid somewhat red (one-half cells hemolyzed) = 2 -|- 



The supernatant fluid red (three-fourths or more cells hemolyzed) = + 



The supernatant fluid clear bright red = — 



' Landsteiner, K., and Stankovic, R.: "Ueber die Bindung von Komplement durch suspendierte 

 und kolloid geloste Substanzen," Centralbl. f. Bakteriol., Orig., 42, 353. 1906. 



^ The methods outlined have been officially recommended for use in certain countries or were 

 among those used in recent attempts made to standardize the tests, the technique being chosen in 

 eacli case that was thought to illustrate best the dissimilarity in the procedures in routine use. 



3 "Anleitung fiir die Ausfiihrung der Wassermann'schen Reaktion," Sonderbeilage zu Veroffent- 

 Uchiingen des Reichsgesundheitsamts, No. 46. 1920. 



