A SIMPLE MODIFICATION OF WASSERMANN'S REACTION.

Transcription

1 415 A SIMPLE MODIFICATION OF WASSERMANN'S REACTION. By LIEUTENANT-COLONEL C. BIRT. Royal Army Medical Corp8. A MIXTURE of the serum of a syphilitic subject with a dilution of an alcoholic extract of syphilitic liver or guinea-pig's heart differs from a similar mixture of the serum of a healthy person with these extracts. The reagents which are used to detect this difference consist of complement, red blood corpuscles and a serum hffimolytic to these cells. Wassermann employs as complement the fresh serum of a guinea-pig, as blood corpuscles those of the sheep, and as hffimolytic serum that of a rabbit which has received several inoculations with washed sheep erythrocytes. When Wassermann's discovery was announced in these columns (JOURNAL OF 'l'he ROYAL ARMY MEDICAL CORPS, vol. viii., June, 1907, p. 568), it was recognised that a modification of the test in which a vivisection licence was not essential would be necessary for the widespread practice of serum diagnosis of syphilis in Great Britain and Ireland. It was suggested that human complement. human red blood corpuscles and a rabbit serum hffillolytic to them might be substituted for the respective components of Wassermann's reaction. ';I'schernogubow (Berliner klinische W ochenschrift, November 22nd, 1908, p. 2107) was the first to practise such a method. He placed in 'a tube one part of the fresh blood withdrawn from a finger with ten parts of physiological saline fluid. In another tube he mixed one volume of fresh blood with ten of the dilution of. extract. After incubating at 38 C., for one hour he added two and a half volumes of rabbit v. man serum to each tube and returned them to the incubator. Control experiments with normal and syphilitic blood were made at the same time. A positive reaction would be shown by lysis in the tube containing blood and saline fluid, but no lysis in that in which the extract was placed. I have followed his instructions in twenty-two cases. In ten readings could not be obtained on account of absence of laking in the control saline fluid tubes. Of the rest the blood of a patient in an early stage of the infection, strongly positive to Wassermann's test, gave a negative result. The method was therefore unreliable. The absence of lysis in the salt tubes was to be expected, since the time allowed for the first incubation-one hour-is generally insufficient for the elaboration of complement. It was attempted to remedy this defect 29

2 416 A Simple Modification of Wasse1'mann's Reaction by allowing the tubes to stand for six to twenty-four hours. Here,' again, the addition of fifty or sixty doses of hocmolytic amboceptor represented in the two and a half volumes of rabbit v. man serum failed to cause lysis in five out of twenty-seven experiments thus made. It was obvious that the proportion of red blood corpuscles was too large. The small quantity of complement produced could not lake the 9 per cent. blood cells present, though aided to the utmost by the multiple doses of hlbmolytic amboceptor. The natural corollary, evidently. was to preserve the blood for five to twenty-four hours before use in order to permit the complement to attain its maximum, to withdraw the clear serum, and to add to it a small proportion of the corpuscles. This plan, which has been followed in over two hundred cases, has been fomid satisfactory and convenient on account of the stable properties of the substances employed. These are an alcoholic extract of guineapig's heart and a rabbit v. man serum. The extract is easily prepared by pounding a guinea-pig's heart to pulp in a mortar with the aid of broken glass. The paste is put into a Well-corked bottle and is shaken up with a quantity of absolute alcohol equal to five times the weight of the heart. There is no necessity to heat to 60 C. nor to preserve it in the ice-chest, as is recommended by some. In two or tpree days the extract is ready for use and may be pipetted off the surface of the pulp as required. Filtration is unnecessary. By leaving the fluid in contact with the paste the extract increases in strength. The heart extract which is being used at the present time is five months old, and has undergone no deterioration. Besides this, syphilitic liver extracts prepared by Captain L. W. Harrison, and guinea-pig liver, rabbit and ox-heart extracts have been employed. The serum hlbmolytic to human blood was obtained by Major Harrison in November, 1909, from a rabbit to which he had given several intra-peritoneal injections of well-washed human blood corpuscles. Its hlbmolytic property is such that one-twenty-fifth volume added to one volume of a six-hour-old human serum containing 5 per cent. erythrocytes, mixed with four volumes of saline fluid, causes complete lysis in ten minutes. The technique adopted is as follows: The clear serum is withdrawn from the blood after standing for five to twenty-four hours. The pipette is then re-introduced to the bottom of the capsule or tube or into the clot and one-twentieth the quantity of blood corpuscles is drawn up. These are mixed with the serum which will then contain 5 per cent. red blood cells approximately. One volume

3 O. Birt 417 of this mixture of serum and blood corpuscles is added to four volumes of 0'9 per cent. salt solution, and also to four volumes of the various dilutions of extract placed in small tubes (0'6 by 4 cm.). It is important that the saline fluid used for diluting the serum and extract should be made with c1istilled water, since the presence of lime salts in tap-water has an inhibitory effect on hremolysis. These are the details of an experiment:- Tube 1. 0'05 cc. of serum containing 5 % red cells + 0'2 cc. 0'9 % salt solution '2 cc. of 2'5 dilution guinea-pig heart extract '2 cc. of -lo dilution guinea-pig " heart extract. + 0'2 cc. of,h dilution guinea-pig heart extract. + 0'2 cc. of,rh dilution guinea-pig heart extract. The whole are incubated for one hour at 37 C., at the end of " 4. " 5. which it will be observed that no lysis of the red blood corpuscles has occurred. They will be seen lying at the bottom of each tube, leaving the fluid above clear. To each tube is then added one-fifth volume of rabbit v. man serum-that is 0'01 cc., which is equivalent to five hremolytic doses. The tubes are shaken and are returned to the incubator. Since the serum has been diluted five times, each tube of fluid will contain only 1 per cent. red blood' cells. Lysis, accordingly, is usually complete in the control tube within a few minutes of adding the hremolytic serum. If the blood has been derived from a healthy individual, hremolysis will occur in the tubes containing the heart extract, though in tube 2, which has the largest dose, some delay may be observed. Should, on the other hand, the serum be syphilitic, there will be no laking in the extract tubes if the blood be taken from an early untreated case with secondary manifestations.. If the patient has undergone efficient treatment, then tube 5 may show complete lysis, 4 a faint trace in half an hour, but 2 and 3 will remain unlaked. Thus we obtain a quantitative estimate of the power of a serum. A second series of tubes is prepared in which a normal serum takes the place of the suspected, and a third series in which a serum of a florid case of secondary syphilis is substituted for the suspected serum. Such is the test as carried out when the supply of blood, obtained by vene-puncture, is ample. It may be successfully performed with so little as 0'2 cc., but in this case the quantity will allow of a control and one extract tube only. Hence, blood collected in capsules from a prick of the finger will suffice.

4 418 A Simple Modijicat'ion of Wassermann's Reaction Two hundred and three examinations of blood have been made, and the results compared with those given by Wassermann's method and with those depending on the presence in human serum of amboceptors, hmmolytic for sheep and guinea-pig red blood corpuscles. Positive Primary syphilis.. 1 Early secondary syphilis 31 Late secondary syphilis.. 22 Latent syphilis.. 29 Oontrols.. Negative Percentage of positive reactions 6 per cent nil. Most of the cases of primary syphilis were examined immediately after admission to hospital on the day when the Treponema pazzidum had been detected in the chancre. The early period of the infection would thus account for the preponderance of negative reactions. ' The sixty-eight individuals who presented no specific symptoms at the time of vene-puncture had gone through protracted courses of treatment, and from a clinical standpoint were supposed to have recovered. In 162 of these sera, Captain Harrison made examinations by vvassermann's test. Our readings corresponded in; 90 per cent. of,the cases. Our results disagreed seventeen times. In eleven syphilitic sera the human corpuscle method gave a positive response, and Wassermann's a, negative. In six the contrary happened. The reaction introduced by Hecbt and extensively practised by ]'leming, Bassett-Smith, Gibbon, Skelton, and others, in which the hmmolytic action of human blood on sheep's corpuscles is made use of, was also employed seventy-eigbt times to test the above sera. The readings were concordant in forty-nine. Hecht's reaction failed in eleven as there was no lysis in the control tubes. On fourteen occasions Hecbt's method was positive, while the human cell method was negative, and in all of these the Wasser" mann reaction was also negative. On four occasions the results were reversed. A procedure similar to Hecht's, except that guinea-pig corpuscles are substituted for sheep cells, was carried out in 101 instances. The results agreed on seventy-seven occasions. Readings could not be obtained in the guinea-pig blood tubes eleven times on account of the absence of lysis. In six the guinea-pig blood method gave a positive reaction, and the human corpuscle method a negative. In seven the reverse was noted.

5 o. Birt 419 The tests depending on the presence of amboceptors in human blood h:::emolytic to sheep's and guinea-pig's blood corpuscles have given a larger number of failures than when human blood corpuscles have been utilised. In all, 198 examinations have been made, using sheep's cells. In 62, or 31 per cent., satisfactory readings could not be obtained. When guinea-pig cells were employed in place of sheep corpuscles, 23 out of 145 experiments, or 16 per cent., were unsuccessful. Whereas, against the human red cell method are recorded only 14 failures in 203 tests, or in 7 per cent. This might hav~ been expected; for in the former processes there are two variables on which the reaction depends-the complement and the h:::emolytic amboceptor. In the modification now described there is only one variable, the complement. Since it is a well-recognised fact that a minimum dose of complement will produce lysis only in the presence of multiple doses of h:::emolytic amboceptor, it follows that if the quantity of complement in a serum be small, approaching in fact the minimum dose, its h:::emolytic action can still be exerted through the influence of the five doses of rabbit v. man serum added-an influence which would b,e wanting in Hecht's reaction, since the proportion of natural amboceptor in human serum is not so great.., It might be erroneously supposed that human complement would have but a feeble effect on human blood corpuscles treated with rabbit v. man serum; since guinea-pig erythrocytes, rendered sensitive by means of a similar rabbit v. guinea-pig serum, require a larger amount of guinea-pig complement than of complement derived from otper animals to produce lysis in them: This is not the case, however. When blood is tested five to twenty-four hours after abstraction, the laking of the human red cells is almost always rapid. Sometimes, indeed, it occurs immediately after the addition of the rabbit v. man serum. The reaction has been successful as early as three hours and as late as four days after the withdrawal of the blood. It might be urged that errors would arise in the variation of the amount of complement in human sermn. But given a constant number of blood corpuscles, a constant dose of hqlmolytic amboceptor, then the time of lysis varies with the quantity of complement. Thus we have a ready means of measuring the complement in a given serum by examining the control salt solution tube every few minutes. If we find that lysis is complete at approximately the same time in the control tubes which contain normal, syphilitic,

6 420 A Simple Modification of Wassermann's Reaction and the suspected serum, then we know their complement content is the same, and that the readings of the corresponding extract tubes will be strictly comparable. Should a serum have lost its complement the test can be performed by adding to each tube an equal volume of a normal serum which possesses complement. Wassermann's reaction is less delicate than the above-mentioned modifications. Heating the serum to 55 O. for half an hour porduces complementoid which sometimes masks the action and causes some specific sera to give a negative response. To overcome this distur bing effect Wechselmann absorbs the complementoid by treating the heated serum with barium sulphate. Complementoid may be found also in the guinea-pig serum if it has been preserved longer than twenty-four hours. The process of heating the human serum for half an hour introduces a variable which it is desirable to avoid. Harrison has made an important advance by reducing the time of this operation to ten minutes. 'l'herefore in a suspicious case in which the blood has proved negative by Wassermann's method, recourse should be had to these other modifications before a final opinion be pronounced. Hecht's method labours under the disadvantage of requiring newly-shed sheep's blood, to obtain a supply of which is attended with no little difficulty. Many failures have been caused by the use of sheep's blood cells more than twenty-four hours old. In laboratories where the original Wassermann test is being carried out, the clot from the guinea-pig's blood, the serum of which is -used as complement, provides a convenient source of blood corpuscles. If the clot be broken up in o g per cent. salt solution and the washings centrifugalised, a sufficient deposit of cells is obtained. After a second washing they are made into a 10 per cent. emulsion. These cells give more satisfactory readings than sheep's cells, since they are more easily laked.by human serum than are the latter. The amount added should not exceed 1 per cent of the fluid in the tubes. Lysis of the controls must always be complete in reliable experiments. Apart from the greater proportion of successful results which can be attained by the human erythrocyte test, the chief advantage lies in the fact that no perishable materials, no centrifuge, waterbath, or thermostat are required. Seligmann and Pinkus state that incubation at room temperature serves as well as at 3r O. The guinea-pig heart extract and rabbit v. man serum preserve their properties undeteriorated for months. Doubtless the firms which now supply sera including rabbit v. sheep will also provide rabbit v. man serum.

7 o. Birt 421 With the reagents constantly at hand this test is capable of more extended use than when substances so perishable and difficult to procure as fresh sheep or guinea-pig blood cells are required. CONCLUSION. The serum diagnosis of syphilis can be undertaken by employing the complement in the serum, and a small quantity of the red corpuscles of the blood under examination, guinea-pig heart extract, and rabbit v. man serum; both the latter are stable. Compared with Wassermann's reaction this modification has given reliable results, and has proved somewhat more sensitive. C~mpared with the methods depending on the presence of a natural amboceptor hffimolytic to sheep's and guinea-pig's corpuscles it has given a smaller proportion of failures. I am greatly indebted to Captain L. W. Harrison, Royal Army Medical Corps, who has freely placed the resources of his laboratory at, the Military Hospital, Rochester Row, at my disposal, and has supplied me with his clinical notes and the results of his own tests. J R Army Med Corps: first published as /jramc on 1 October Downloaded from on 14 June 2018 by guest. Protected by