Art. LIII.—The Anticomplementary Properties observed in certain Serum Reactions.
[Read before the Philosophical Institute of Canterbury, 1st December, 1920; received by Editor, 5th December, 1920; issued separately, 12th August, 1921.]
The notes put on record in this paper have been made in connection with the determination of nearly ten thousand Wassermann reactions carried out for the New Zealand Expeditionary Force during the author's overseas service.
In general the Wassermann reaction was determined in conformity with the recommendations of the Medical Research Committee, using the method elaborated by Colonel L. W. Harrison, K.H.P., D.S.O.,* the measurement of the reagents being carried out by the method adopted by Donald.†
When care is exercised in standardizing the pipettes used, Donald's dropping method was found by comparison to give complete concordance with, methods using hand-pipettes. The principle involved in Donald's method is that “at constant temperature and pressure, and at a constant delivery-rate which does not exceed one drop per second, the size of a drop of any given liquid which is delivered by a vertically held nozzle is constant, and depends on the circumference of the delivery-nozzle at its outlet.”
When large numbers of tests have to be carried out the monotony and eye-strain involved in using the hand-pipettes are considerable; with the dropping-pipettes, after a standardization is made, the determination is almost automatic, and accuracy is independent of fatigue. This principle is also applicable to many determinations involved in ordinary chemical analyses, as well as those carried out in connection with bio-chemical reactions.
An important consideration in the Wassermann reaction, as well as in other serum tests, is the nature of the antigen used. While, doubtless, individual workers obtain concordant results with various antigens, it has been the writer's experience that the human-heart extract, with cholesterin, as recommended by the Medical Research Committee, gives the most satisfactory and concordant results, if prepared in strict conformity with the instructions laid down by Fildes and McIntosh‡ and from fresh heart-muscle, the extract-heart-cholesterin being diluted 1 in 15 with normal physiological saline (0.85 per cent. NaCl).
[Footnote] * Medical Research Committee's Report, Path. Methods, No. 1, pp. 13–27, 1918.
[Footnote] † R. Donald, Proc. Royal Soc., vol. 86, pp. 198–202, 1913.
[Footnote] ‡ P. Fildes and J. McIntosh, Brain, vol. 36, p. 193, 1913.
This antigen, if accurately and carefully prepared, shows little inhibitory action upon the complement used: it is immaterial whether the heart is diseased or not; it should not, however, be decomposed, otherwise certain substances soluble in alcohol may be extracted which are in action inhibitory to complement.
One such antigen was prepared from a human heart removed at autopsy thirty-six hours after death and while not obviously decomposed. The resulting preparation deviated 0.75 minimum haemolytic doses (M.H.D.) of complement. The use of antigen prepared from guinea-pig heart was not found to be satisfactory, owing to a similar marked anti-complementary action.
Anticomplementary Reaction of Human Sera.
Normal human-blood sera show, in the majority of cases, certain anti-complementary properties. In a series of tests carried out to determine the inhibitory power towards complement it was found that with a number of normal sera the average deviation of complement was 0.5 M.H.D. In conjunction with these tests the sera were also quantitatively examined to ascertain what, if any, complement-deviation occurred in the presence of the antigen prepared as noted above, such antigens having been found to be non-inhibitory; it was found that the average deviation of complement towards such negative sera was 0.75 M.H.D.
It is thus evident that the normal subject contains in the blood small amounts of antibodies, similar to, and having complementary deviation-properties identical with, the antibody upon which the Wassermann reaction depends for its specificity.
Of course, in the actual determination of the Wassermann reaction the controls adequately secure the true interpretation of the reaction, and allow for the small amounts of inhibitory antibody, as well as the anticomplementary properties of the patient's serum. The specificity of the Wassermann reaction depends upon its quantitative and not its qualitative determination.
Browning's* observation that the blood-serum of the normal rabbit gives a positive Wassermann reaction is confirmed by the writer's experiments; and, whatever interpretation may be placed upon this, it is nevertheless established that there are present in rabbit-sera sufficient antibodies similar to the Wassermann substance in complement-deviation power to produce a positive quantitative reaction. Controls demonstrated that the inhibitory properties were not of themselves merely anti-complementary, but depended upon the presence of the specific antigen in addition to bring about the reaction, which amounted to the deviation of as much as 3 M.H.D. of complement in one case.
In the course of the work one serum was encountered which showed very strongly marked anticomplementary power, deviating by itself nearly 7.00 M.H.D. of complement; later the serum from this patient showed but 4.00 M.H.D. of complement-deviation; and some months later the serum was normal in complement-deviation power. This serum was so abnormal that its properties were examined at the Bland Sutton Institute of Pathology, Middlesex Hospital, and the results of the investigation published in a separate paper.†
[Footnote] * C. H. Browning, Applied Bacteriology.
[Footnote] † E. L. Kennaway and A. M. Wright, Jour. Hygiene, vol. 18, pp. 255–59, 1919.
An interesting point in connection with this serum showed that the first specimens were frozen and examined two months later, when it was found that the anticomplementary properties had disappeared.
Specificity of the Wassermann Reaction.*
In connection with the routine determination of the Wassermann reaction, the blood-sera from fifty-nine patients suffering from, malaria were examined for the evidence of specific reaction to the Wassermann test. It has been recorded by various observers that malarial subjects have given a positive reaction.
While a number of the fifty-nine patients from whom the blood-sera was taken had either just had a rigor or were in the midst of one, and so should, if malarial antibodies influenced the Wassermann reaction, have been expected to show a positive test, yet the whole fifty-nine patients were found to give a negative reaction.
The influence of chloroform anaesthesia was also determined in a number of cases, the blood being taken before, and at twelve and also at twenty-four hours after, anaesthesia: in all cases the reactions were negative.
[Footnote] * J. W. Marohildon, “Wassermann Reaction.”