Imagini ale paginilor


Second Assistant Physician,


JOHN I. WISEMAN, M. D., Medical Interne, Kings Park State Hospital, Kings Park, N. Y. Since the introduction of the Wassermann reaction it has become possible to detect syphilis in cases in which it exists in a latent form. This reaction, besides being an aid in diagnosis, affords important therapeutic indications and constitutes, furthermore, a means of investigation of the relationship between syphilis and various pathological conditions of obscure etiology.

In the work, the results of which are embodied in this contribution, we have made use of the Wassermann reaction, applied to the blood and to the cerebro-spinal fluid, for the purpose of gaining some acute knowledge of the role played by syphilis in the pathogenesis of insanity.

With the same object in view we have also made use of the butyric acid reaction for syphilis recently suggested by Noguchi' and have subjected our findings to control by means of cytological examinations of the cerebro-spinal Auid.


$1. METHODS OF INVESTIGATION. The original technique of the Wassermann reaction has given in the hands of different investigators widely differing, almost contradictory results. To take a few striking instances, Plaut’ examined the blood serum from 159 cases of general paresis and found a positive reaction in 100 per cent; Edel' obtained similar


* Proceedings of the Soc. for Experimental Biol. and Med., 1909, Vol. VI, Pp. 51-54 * Allg. Zeitschr. f. Psychiatrie, Apr. 17, 1909. Allg. Zeitschr. f. Psychiatrie, Feb. 22, 1909.


results in 50 cases of general paresis; Nonne' obtained positive findings in 90 per cent of his cases; while Marie, Levaditi, and Yamonouchi obtained positive findings only in 59 per cent of their cases.' Similar, though slighter, discrepancies are to be found in results of examinations of the cerebro-spinal fluid. These discrepancies are generally attributed to the numerous possibilities of error due to the complexity of the technique of the reaction; there is, however, another and more vital source of error which is inherent in the reaction, namely, the fact that this reaction is not strictly specific. In the first place, as is well known, infections other than syphilis give a positive reaction: leprosy, yaws, trypanosomiasis, scarlet fever; and in the second place, normal sera frequently cause partial inhibition of hæmolysis such as is observed in mild syphilitic infection. By varying the proportions of complement or of hæmolytic amboceptor it is indeed possible to make the reaction more or less sensitive, but not more reliable; for if the proportions of the reagents were regulated so as to make the reaction very sensitive many specimens of serum from non-syphilitic subjects would be found positive; and if the reaction were made less sensitive then many sera from cases of mild syphilitic infection would be found negative. Even if the reagents were stable and easily and permanently standardizable it would not be possible to find proportions which would exclude all error in the reaction.

Thus in all work in which the Wassermann reaction is employed it becomes necessary to take into account not only the danger of error from slips of technique but also the inherent fallacy of the reaction, or the results may turn out to be misleading.

In this work in order to safeguard the reliability of the results the following measures were adopted :

(1) Instead of using Wassermann's original method we used Noguchi's modification. The reader will recall that this modification differs from the original method in three important particulars: in the first place an anti-human hæmolytic system is used instead of an anti-sheep system and thus the error, due to the frequent presence of natural anti-sheep amboceptor in the sera to

[ocr errors]

Quoted by Plaut.

Compt. rend. de la Soc. de Biol., Vol. LXIV, p. 169. Journ. of Experimental Med., Vol. XI, No. 2, 1909.

be tested, is eliminated; in the second place the acetone-insoluble fraction of tissue lipoids is used as antigen instead of watery extract from luetic liver; and in the third place the antigen and the hæmolytic amboceptor are prepared for use in the form of test papers-a form which is comparatively stable and readily standardizable.

(2) A dozen or more specimens were always examined at a time so that in addition to the customary positive and negative controls there were usually specimens of serum and of cerebrospinal fluid showing negative reactions and positive reactions of various degrees of intensity; thus any error of technique was made more obvious and led to a repetition of the tests.

(3) Whenever the reaction was characterized by a partial inhibition of hæmolysis the result was recorded as doubtful (+); although in this way in addition to the groups of positive and negative cases a third group was created—a heterogeneous group undoubtedly containing both positive and negative cases—yet it seems to us that we gained in accuracy by avoiding an arbitrary grouping of cases showing an incomplete reaction.

As Noguchi's modification of the Wassermann reaction has been introduced but recently and, although it is already widely known, it has not yet been generally adopted, we will give here a brief description of the technique.'

Preparation of Reagents.-Antigen may be prepared by steeping thoroughly mashed liver or heart or kidney tissue in ten times its volume of absolute alcohol at 37° C. for several days, filtering, and evaporating the filtrate to the consistency of a thick sticky mass. A portion of this mass is dissolved in some ether and evaporated slowly to a small volume, the consistency still remaining perfectly fluid. To this concentrated solution five times its volume of acetone is then added; the precipitate which forms is allowed to settle to the bottom and the supernatant Auid is decanted. 0.2 gram of the precipitate is dissolved in about 5 cc. of ether, and 100 cc. of normal salt solution is gradually added, the whole mixture well shaken and the resulting emulsion filtered to remove flocculi


'For a full description of the methods of preparing the reagents and of the technique of the reaction the reader is referred to the recently published book by Dr. Hideyo Noguchi, Serum Diagnosis of Syphilis (Philadelphia, 1910).

or solid particles. The filtrate is then tested for its antigenic power. Should it be found to possess insufficient antigenic power or, perhaps, none at all, or should it be found to possess a strong anti-complementary power, as is sometimes the case, then it cannot be used and another lot must be prepared. When a suitable extract has been obtained it is prepared for use by dissolving it in ether and evenly impregnating with it sheets of filter paper; the impregnated sheets are dried in the air, cut into strips 0.5 cm. in width, and standardized so that the exact length of a strip which contains the proper dose of antigen for a test is determined.

In this form the antigen keeps admirably. One lot of antigen test paper that we used, which was kept in a large test tube, often without a stopper, at room temperature in warm summer weather, and exposed to the pronounced humidity of the atmosphere which is characteristic of the Long Island climate, remained good for three months, at the end of which time it began to show a feeble anti-complementary power; no doubt if any attempt had been made to preserve it more carefully it would have remained good for months longer.

Anti-human hæmolytic amboceptor may be derived conveniently from the blood serum of a rabbit which has been immunized to human blood corpuscles by injecting thoroughly washed human blood corpuscles into the peritoneal cavity in five or six doses, starting with 5 cc. and gradually increasing to about 20 cc.; 8 the injections are given at intervals of four days. Ten or twelve days after the last injection the rabbit is bled and the serum is collected and inactivated by heating to 56° C. for 30 minutes. Sheets of rather heavy filter paper are then thoroughly saturated with the inactivated serum and dried in the air. As in the case of antigen paper, the sheets are then cut up into strips 0.5 cm. wide and standardized for their hæmolytic power.

We have found that in this form hæmolytic amboceptor keeps very well. Under the same unfavorable conditions to which our antigen test paper was exposed, as described above, our lot of amboceptor paper ultimately began to show a somewhat weaker


The corpuscles used for injection into a rabbit for the purpose of producing amboceptor must be thoroughly washed at least three times in large amounts of normal salt solution; the suspension prepared for the injection should contain about the same proportion of corpuscles as blood itself.

hæmolytic power than it possessed originally; on being restandardized, however, it could still be used by taking a somewhat longer strip for each test.

Suspension of human blood corpuscles, which is used in the reaction, is prepared by allowing blood from a puncture in the lobe of the ear or the tip of the finger to run into normal salt solution in the proportion of about one drop to 4 cc. (1 per cent). This is placed in the refrigerator and shaken repeatedly during the first four or five hours to break up the coagulum which is apt to form in the shape of a thin, jelly-like, more or less coherent mass, enclosing many of the corpuscles. The corpuscles are then allowed to settle, the supernatant fluid is poured off, and an equal volume of fresh salt solution is added. The suspension is made uniform by shaking again and is now ready for use.

With the aid of a centrifuge the suspension can be prepared much more quickly-in a few minutes.

It is important to obtain the blood for making the corpuscle suspension from a person who is free from syphilis, or all the tubes, including the negative control, may give a positive reaction. To be certain on this point it is best to take the blood from a person whose serum has been examined for the Wassermann reaction and found to be negative.

Complement is derived from fresh guinea-pig serum, the following being the most convenient way. A full grown guinea-pig is held by an assistant over a large Petri dish in a hyper-extended position by grasping the head with one hand and all the four legs with the other. A long slender sharp knife is introduced into the neck at the side just in front of the vertebral column until it is thrust through on the other side, when the edge of the blade is turned ventrally and all the tissues in the front part of the neck are cut through. The blood is caught in the Petri dish which is then covered and set aside in a corner out of direct sunlight and allowed to remain at room temperature for about two hours, at the end of which time the serum may be poured off and used; or the Petri dish may at the end of two hours be placed in the refrigerator where it may be kept over night and used on the following morning; but standing over night at room temperature renders the serum inactive. If kept on ice the activity of the serum is reduced much more slowly, so that it usually remains good for about forty-eight hours.

« ÎnapoiContinuă »