Sheep’s Corpuscles. A 5% suspension of sheep’s corpuscles in 0.85% salt solution is prepared from defibrinated sheep’s blood. The corpuscles are washed three times and for each washing four to five times as much 0.85% salt solution is used as the original volume of the defibrinated blood.
Amboceptor. The amboceptor is prepared by injecting sheep’s corpuscles into a rabbit. The serum of this rabbit which contains amboceptor is diluted with 0.85% salt solution so that 0.25 cc. will hemolyze 0.5 cc. of a 5% suspension of sheep’s corpuscles. In the test twice the quantity or 0.5 cc. of amboceptor is used.
Sensitized Cells. The sensitized cells consist of equal parts of washed sheep’s corpuscles and diluted amboceptor. This mixture is incubated in a water bath at 37° C. for a half hour to effect the sensitization of the cells.
Technique of the Wassermann Test. One-tenth cubic centimeter of each inactivated specimen of serum and 0.5 cc. of each uninactivated specimen of spinal fluid is pipetted into a separate tube. A mixture is freshly prepared in salt solution, each cubic centimeter of which contains the proper amount of antigen A (the most sensitive antigen), and two units of a 10% solution of guinea pig serum (complement). One cubic centimeter of this mixture is pipetted into each test tube. These tubes are then incubated for forty minutes in a water bath at 37° C. At the end of this period, sensitized cells are added, and the tubes are again incubated in a water bath at 37° C. for one hour. Each specimen which shows any degree of inhibition of hemolysis is retested in the afternoon. For this second test antigen A is again used and in addition antigens B and C. A control is also made for each specimen retested to eliminate any possibility of the inhibition of hemolysis being due to anti-complementary substances in the serum or spinal fluid tested. The technique of the second test differs in no wise from that of the first, except for the use of a control in each retested specimen and the employment of three antigens instead of one. The degree of positiveness is noted for each retested specimen and compared with the degree of positiveness obtained for the corresponding specimen with the same antigen-complement-salt solution mixture in the morning’s test. The specimen is retested on the next day when discrepancies occur between the morning reading for antigen A and the afternoon reading for antigen A. From the above description it will be noted that the negative specimens have but a single test with one antigen only, while the positive specimens are retested, thus permitting a confirmation of any positive reaction. In this way attention is focalized on the positive specimens.
Interpretation of Results. Antigen C (the weakest of the three antigens) is used entirely for diagnostic purposes and any specimen showing the slightest degree of inhibition with this antigen and stronger degrees of inhibition with the other antigens is reported as positive. The specimens which are strongly or moderately positive with antigens A and B and negative with antigen C are reported as doubtful. In testing spinal fluids by the titration method, antigen C is used and the readings are based upon the degree of inhibition of hemolysis noted. The intensity of this inhibition is indicated by Arabic numerals: “5” indicates complete inhibition, while “1” means a faint cloudiness, hence a weak reaction. Intermediate numbers show relative intensity varying between complete inhibition “5” (strong positive) and slight inhibition “1” (weak positive); “—” equals no inhibition (negative).
Although it is commonly believed that the recent administration of antisyphilitic treatment will affect the reaction by making it negative, this is not our experience, and it is, therefore, not necessary that treatment be withdrawn for a short period before the specimen is submitted for examination.
The reaction as carried out in this laboratory has the following diagnostic significance: Positive indicates syphilis, except very rarely in acute febrile conditions such as malaria and pneumonia. Negative does not exclude syphilis. In obscure conditions a series of less than three negatives has little diagnostic significance. Doubtful suggests syphilis. It is therefore advisable to submit three or more specimens in such a case, and interpret a persistently or predominatingly doubtful reaction as indicative of syphilitic infection.
Bruck Test. A new serum test for syphilis has recently been described by C. Bruck.[[152]] Following are recent results in our laboratory with this test.[[153]]
This new test for the diagnosis of syphilis by C. Bruck has aroused much interest. The scientific standing of Bruck and the simplicity of the technique led us to overcome our prejudice, that has been the offspring of the numerous tests that have been offered of late. Bruck states that since the discovery of the complement fixation test for syphilis by Wassermann, Neisser and himself in 1906, he has been trying to find a simple chemical reaction that would take the place of the complicated technique of the Wassermann reaction. This method, as he has published it, was worked out and is being used at the front, in the present war, where complete laboratory equipment is not available.
Commencing our experiments with a great deal of scepticism, we were much surprised at the results obtained, which are given below. Whatever may be the final status of the test in the determination of syphilis, we feel that there is a great deal of interest in the fact that this simple chemical reaction does pick out certain differences in the composition of blood sera and that apparently a large number of syphilitic sera differ in their chemical composition percentage from the majority of non-syphilitic sera.