The one hour fixation period in the water bath at 37°C. appears to be the optimum time and temperature for the complement fixation test with our antigens.
Specially tested complement, if only one guinea-pig is killed should be used, but untested pooled complement from six or more guinea-pigs apparently gives satisfactory results.
Natural antisheep hemolysin is markedly thermolabile.
If the serum is kept sterile the natural antisheep hemolysin does not depreciate very rapidly due to ageing.
Heating tubercle bacillus antigen at 100°C. for three hours does not seem to impair its antigenic value in the slightest. The addition of 25 per cent alcohol or ½ per cent carbolic acid to the finished antigen as preservative tends to make the antigen anticomplementary to a greater or less degree.
The best method for preparing the tubercle bacillus antigen seems to be by killing the bacilli with alcohol, and after the finished antigen is bottled and corked in small vials, sterilizing at 100°C. for one hour on two or three successive days to kill all contaminating spores and bacteria.
An increase in the percentage of positive results can probably be obtained by absorption of the natural antisheep amboceptor before making the test or by using the fresh non-inactivated sera according to the technic advocated by Hecht and Gradwohl. However, while both of these modifications give a higher percentage of specific positive reactions than the standard test, they also give a moderate percentage of non-specific reactions. Therefore, they should only be used as control tests along with the standard technic.
The increase in the strength of the fixation and the increase in the percentage of positive findings after preserving the patient's sera for seven days in the ice chest is due, probably, to one or more of the following reasons: the formation of thermostable antilysins in the kept sera, loss of natural antisheep amboceptor due to ageing, loss of natural antisheep amboceptor due to reheating and other unknown non-specific causes. This change in sera after preserving in the ice chest for one week is apparently non-specific.
No cross fixation was apparent between syphilitic antibodies and the tubercle bacillus antigens which were used in this study.
The results of the von Pirquet reaction apparently do not parallel the results obtained with the complement fixation test.
The National Association Classification of tuberculosis patients appears to be too complicated to be used in successfully interpreting this test. If used in its simplified form, the results of the test do not run parallel with the classification.
By using the writer's simplified interchangeable classification a more accurate interpretation of the results of this test is possible. However, even with this simpler classification it was found impracticable accurately to tabulate the results by types in a routine way as the classification of these same patients varied considerably when made by different clinicians. In general, we may say that the largest percentage of negative and weak positive results are in the cases having few or no constitutional symptoms, while the strength and percentage of positive reactions increase progressively with the increased constitutional symptoms, until the patient reaches the far advanced condition, when the reverse is true; i.e., when the patient loses his power of resistance, the complement fixation reaction becomes negative.
The age of the patient apparently has no effect on the complement fixation test for tuberculosis.
About 70 per cent of all types of tuberculosis patients except those clinically healed or inactive gave positive fixation results on repeated tests. Normal non-tuberculous cases gave almost no positive results on repeated tests. Moderately and far advanced cases in good condition showing constitutional symptoms (type III) gave an average of 85.2 per cent positive fixations for all six series.
This complement fixation reaction will not be very valuable, as an aid in diagnosis, to the tuberculosis specialist except as a confirmatory test. However, a positive fixation reaction will be of very great value to the general practitioner not only as a confirmatory test but also as an aid in diagnosis and prognosis.