WASSERMAN REACTION ( Wassemann A. , 1866 — 1925, it is mute. the immunologist) — the kind of reaction of binding complement applied to serodiagnosis of syphilis. It is offered in 1906 by A. Wasserman, A. Neicer and Brook (S. to Bruck).
Wassermann reaction (RW) it is based on property of blood serum of patients with syphilis, unlike serum of healthy faces, to form the complex adsorbing a complement with the corresponding antigen. This property is found at addition to ispytuyemy serum of the indicator system consisting of erythrocytes of a ram and hemolitic serum (serum of a rabbit, immunizirovanny erythrocytes of a ram). Lack of hemolysis demonstrates adsorption of a complement which was added to mix of the studied serum and antigen, and, therefore, about their interaction and formation of the corresponding complex (positive reaction). On the contrary, hemolysis indicates existence of the free complement participating in hemolysis, i.e. interaction between serum and antigen does not happen (negative reaction; tsvetn. fig. 4).
Ingredients of reaction
Ingredients of reaction: antigens, ispytuyemy serum, erythrocytes of a ram, hemolitic serum, complement.
Antigens. Originally as antigen used a water suspension of a liver of the dead vnutriutrobno of the fruit (the antigen offered by A. Wasserman) affected with syphilis. Also other antigens were recommended: specific and nonspecific. Carry antigens from a pale treponema to specific:
1) suspensions of apatogenous culture of a pale treponema (water, karbolizirovanny, alkogolizirovanny); the greatest use was received by the suspension of a cultural treponema of a strain of Reuters processed by ultrasound (sonikat);
2) protein fraction (is more sensitive, than polisakharidny and lipoid) from culture of a treponema [D'Alessandro and Puchchinelli (G. D’ Alessandro, V. Puccinelli)];
3) suspensions of a pathogenic fabric treponema from the struck small egg of the rabbit infected with syphilis. The offer to use specific antigens proceeded from idea that RW is based on interaction of specific antibodies with antigens of the activator.
Carry to nonspecific antigens: 1) spirit extract from a muscle of a cor bovinum (1 part of a muscle, 5 parts of alcohol with addition 0,25 — 0,3% of cholesterol); 2) cardiolipin antigen [M.Pangborn, L. S. Reznikova, etc.]; cardiolipin — the purified extract from muscles of a cor bovinum (phospholipid — 0,0175%, lecithin — 0,0875% which show activity at addition of cholesterol — 0,3%); antigen differs in high sensitivity, especially at the latent forms of syphilis; it is widely applied in a serodiagnosis of syphilis around the world (in the USSR its use is obligatory); 3) the so-called artificial lipoid antigens prepared from cholesterol, lecithin and sodium oleate were of little use; the antigens made of plant substances (sitolipina) were not included into practice.
Antigens for RW shall meet the following requirements:
1) not to cause hemolysis of erythrocytes; 2) not to inactivate and not to adsorb a complement; 3) not to form with serum of healthy faces of a complex capable to adsorb a complement; 4) at addition of antigen in an optimum dose to serum of patients with syphilis to form the complex adsorbing a complement.
Ispytuyemy serum by a classical technique of R W previously gets warm in the water bath of 30 min. at t ° 56 ° for an inactivation of a complement and stabilization of globulinovy fractions.
Methodical requirements to statement of the main experience
In view of the fact that results of reaction are considered on absence or existence of hemolysis after addition of hemolitic system, the main methodical requirement of RW is use of precisely titrated dose of a complement so that in case of adsorption of a complement at interaction of ispytuyemy serum with antigen there was no excess of a complement in the quantity sufficient for dissolution of erythrocytes, sensibilized hemolitic serum; on the other hand, the dose of a complement shall not be too small to provide hemolysis of erythrocytes in case of lack of reaction between the studied serum and antigen. Therefore before statement of the main experience the minimum dose of a complement causing hemolysis at addition of hemolitic system ottitrovyvatsya in the presence of antigen and normal serum. In the main experience the complement is applied about a nek-swarm by an extra charge (not less than 15 — 30%). Each of 5 ingredients of reaction (ispytuyemy serum, antigen, a complement, a suspension of mutton erythrocytes and hemolitic serum) is applied in identical volume (e.g., on 0,25 ml). For providing optimal conditions of complement deflection on the formed complex in case of interaction of the studied serum with antigen mix of serum, antigen and a complement maintain in the thermostat within 45 — 60 min. (the 1st phase of reaction) then add hemolitic system and test tubes place in the thermostat (the 2nd phase) again.
At the same time put 3 control experiments in one of which antigen is replaced with isotonic solution of sodium chloride, and in another the studied serum is excluded, in the third it is investigated obviously positive on RW serum. The accounting of reaction is made at approach of hemolysis in control test tubes of the main experience.
Methods of statement of reaction
Depending on character of ingredients of reaction, quantitative ratios between them and conditions of carrying out experience distinguish the following modifications of RW.
1. Quantitative methods: a) statement of experience with constant doses of serum and a complement and with different doses of antigen; b) carrying out experience with the raising doses of a complement; c) carrying out experience with constant doses of antigen and a complement and with different doses of the studied serum: the last method is widely applied in practice.
2. RW during the binding on cold — the first phase of reaction is carried out at t ° 2 — 4 ° during 18 — 20 hours that considerably increases its sensitivity (Kolmer's reaction which received especially big use).
3. Active methods of reaction, which are based on use of a natural complement or hemolysin, or that and another, contained in ispytuyemy serum (N. A. Chernogubov, 1908; N. V. Nartsissov, 1952). The greatest use in the USSR was received by the accelerated active methods of statement of RW — Grigoriev's reaction — Rapoport (see. Grigoryeva-Rapoport reaction ), active modification of Weinstein — Reznikova. The principle of use of a natural complement and hemolysin of the studied serum of the person is the basis for the last. Are necessary for performance of reaction: active (not warmed up at t ° 56 °) the studied serum, no more 24-hour prescription; two antigens of different series (from usually applied in RW) divorced on the trebled caption, and 1% a suspension of not washed defibrinated blood of a ram. In three test tubes measure 0,2 ml of active ispytuyemy serum: in the first and second test tubes — on 0,3 ml of divorced antigens, and in the third (control) — 0,3 ml of isotonic solution of sodium chloride. Test tubes stir up within 3 min., add 1% of a suspension of not washed blood of a ram on 0,75 ml; after 45 — 60 min. standing at the room temperature (18 — 25 °) consider result.
The serodiagnosis of syphilis in the USSR is carried out by means of a complex of reactions (see. Syphilis ). In the republican, regional and large regional centers statement of the complex of standard reactions consisting of RW and two sedimentary reactions — Legislative Assembly — Vitebsk and Cana is necessary (see. Legislative Assembly-Vitebsk reaction , Cana reaction ). In other regional centers where statement of a complex of standard reactions is impossible, statement of the accelerated simplified complex is allowed (active modification + two sedimentary reactions). At a blood analysis of donors, according to the instruction of M3 of the USSR, the accelerated complex of reactions to the syphilis consisting of active modification and two microreactions on glass is applied:
a) with cardiolipin antigen;
b) with cytocholic antigen and in case of emergency — microreaction with a fresh drop of blood.
For an explanation of the mechanism of Wassermann reaction several theories are offered. The Ambotseptorny theory considers R W as true reaction of immunity between antigen and specific antibodies of serum of the patient. On character of the interacting antigens and antibodies various opinions were expressed. Generally it is considered that it is about antigens of a pale treponema and the corresponding antibodies. In antigenic structure of a pale treponema find not less than four antigens: protein, polisakharidny and two lipoid, one of two last is specific to a treponema, another — from among eurysynusic in the nature. In serums of patients it is possible to differentiate three types of specific antibodies (anti-protein, antipolisakharidny, anti-lipoid) and reagins (the proteinaceous and lipoid connections forming in response to lipoid antigen of a treponema, eurysynusic in the nature). The possibility of statement of RW has a talk with various antigens it (specific and nonspecific, water and spirit).
Physical. - the chemical theory explains RW as result of changes of serum at patients with syphilis that is characterized by increase gamma globulinovoy fractions, strengthening of hydrogen ions, increase in surface intention and viscosity, reduction of dispersion of colloids, lability of globulins in view of weakening of communication with the environment and in this regard their easy sedimentation capacity at addition of antigen.
Apparently, the concept is most correct that RW is caused immunological and physical. - chemical factors.
The positive R W which are observed at a number of diseases, at some fiziol, states (pregnancy) and at healthy faces induced to find ways of increase in its specificity. The reactions excluding or, on the contrary, the diseases confirming the syphilitic nature in disputable cases [H. Neurath, were offered 1948; Caen (R. Kahn), 1952, etc.]. However these reactions are technically difficult and not completely differentiate positive RW from false positive.
For confirmation of specificity of RW a number of seroreactions using as antigen of a pathogenic fabric pale treponema is offered. Treat them: 1) reaction of binding complement with antigen which receive by extraction from virulent treponemas 0,2% solution of a dezoksikholat of sodium [The tailor and Magnusson (J. Porthnoy, H. Magnuson), 1955]; 2) an agglutination test, for a cut the virulent treponema is warmed up at t ° 56 ° [Ermann and Nielsen (G. Ehrmann, H. Nielsen), 1955]; 3) reaction of an immobilization of Nelson — Meyer (see. Nelson-Meyer reaction ), edges consists that the virulent treponema loses the mobility in the presence of syphilitic serum and a complement; Nelson's test — Meyer differs in high specificity, but is rather difficult; 4) reaction of Treponema pallidum immune-adherens test which essence is that the virulent pale treponema, sensibilized antibodies of syphilitic serum, sticks around a surface of erythrocytes of the person in the presence of a complement [Nelson (R. Nelson), 1953]; however and these reactions yield sometimes a positive take at healthy faces and negative — at patients with fresh forms of syphilis; 5) reaction of an immunofluorescence (see. Immunofluorescence ); antigen and serum of the patient with syphilis form a complex of pathogenic pale treponemas, add the rabbit luminescing serum against globulins of the person to Krom; at a research in a luminescent microscope serum of the patient with syphilis gives a flavovirent luminescence of treponemas.
Many domestic and foreign works are devoted to standardization of RW. In the USSR the instruction for statement of RW was approved at the XI congress of bacteriologists and epidemiologists in 1928, reconsidered and added in 1956 and in 1973.
Pouring of ingredients. Accuracy of pouring of ingredients has paramount value for the result of reaction. From the offered devices of special attention A. V. Florinsky's equipment deserves. It consists of the following parts.
1) The rubber supports representing rubber levels (9 X 23 cm) with 40 openings into which 40 test tubes (14X65 mm) are densely inserted; each support is expected a research of 10 serums with three antigens, the fourth row is intended for kontroly serums; test tubes, without taking out from a level, wash with a ruff in warm water and dry in a drying oven at t ° 120 — 130 °.
2) Folding and bladed supports can be used for draining and an inactivation of the studied serums, for statement of sedimentary reactions and the quantitative RW method; in a support of 10 levels (they can be rubber, wooden, metal or from plexiglass), all in a support of 100 test tubes (fig).
3) The glass measured devices with pipettes for pouring of ingredients containing 10 pipettes with a capacity of 0,025; 0,05, 0,1; 0,15; 0,25; 0,5; 1,0 ml; after pouring of each ingredient devices wash out isotonic solution of sodium chloride; upon termination of work — at first a water, then distilled water.
During the use of Florinsky's equipment on pouring of 200 serums only 20 min.
Practical value of Wassermann reaction
Preventive actions, questions of diagnosis are required, assessment of results of therapy etc. cannot in a crust, time to decide without statement of serological tests among which R W occupies one of important places. In the USSR as standard reactions the complex of serological tests is applied (RW with two or three antigens and two sedimentary: Cana and cytocholic). Such complex allows to establish the diagnosis more precisely. Repeatedly positive R W in a high caption made m serology are qualified, in most cases points to existence of syphilis, despite the lack of sometimes clinical manifestations. Is of especially great importance of R W: 1) in primary period of syphilis for specification of the diagnosis and establishment of duration of terms of treatment and overseeing by patients, though division of primary syphilis on seronegative and seropositive (see. Syphilis ) conditionally, since at use of more sensitive method, napr, statements of reaction on cold, positive takes it is possible to reveal earlier: 2) at the latent syphilis when clinical manifestations are absent, and only serological tests quite often help to diagnose it; 3) at diseases of internals and a nervous system for an exception of a possible syphilitic infection; 4) at prevention of syphilis (inspection of members of the family of the patient and the persons which were with it in contact, personnel of child care facilities, employees of the food and utility enterprises, donors etc.); 5) at removal from the accounting of patients with syphilis; 6) at all diseases with not clear etiology, suspicious on syphilis; 7) at assessment of efficiency to the carried-out therapy; 8) in court. - medical practice, in particular at a research of cadaveric blood.
RW at patients with primary syphilis in the first 15 — 17 days of a disease is negative, on 5 — 6 weeks positive takes are observed at 25% of patients, to 7 — 8 weeks — at 75 — 80%, at patients with secondary fresh syphilis of R W becomes positive in 100% of cases, at secondary recurrent in 95 — 100% of cases, at patients with the latent syphilis of RW is positive in 40 — 96% of cases depending on duration of a disease, intensity of the previous therapy etc., at patients with tertiary syphilis of R W is positive in 75 — 80% of cases, at patients with syphilis of a brain in 60 — 70%, at a general paralysis — in 100%, at back to tabes — apprx. 60%, at early inborn syphilis of RW is positive about 100%, at late — in 70 — 80% of cases.
However not always positive RW points to existence of syphilis. Positive reaction can be received sometimes and at healthy faces, and also at diseases at which changes in blood serum are close to those at patients with syphilis. Positive takes of RW at the persons who do not have syphilis call false positive or nonspecific. False positive results of serological tests can be observed at the diseases (a typhinia, a frambeziya, bedzhel, pint, a hay fever, etc.) caused by availability of the general antigens at similar activators — so-called group reactions. At these diseases the antibodies similar to those at syphilis are developed. False positive results can be caused by disturbance of exchange (first of all lipidic) and change in globulins of serum. Positive takes of R W at patients with gout, malaria, sapropyra, scarlet fever, smallpox, a pemphigus, an endocarditis are sometimes observed, at lead poisoning, by phosphorus, chloroform, etc. Nonspecific positive takes of reaction sometimes arise because of technical errors at its statement. Also so-called paradoxical reactions when with the same serum at the same time in different laboratories receive contradictory results that is explained by use of various ingredients at statement of reaction are noted. In a crust, time when techniques of production of antigens and statement of reaction are unified, paradoxical reactions meet seldom. Especially big percent of false positive results of R W is observed at patients with a tuberculoid leprosy (N. A. Torsuyev). Positive R W at patients with a system lupus erythematosus, listerellosis, ferruterous fever, tuberculosis, flu, measles, pneumonia, malignant tumors in a stage of disintegration, Plaut's quinsy — Vincent, a venereal ulcer, and also directly after a radio and chloroformic anesthesia is possible. Slabopolozhitelny and doubtful results, especially with blood sera of children can be observed, at went. - kish. frustration, at women during periods, just before and after the delivery. Percent of false positive RW at pregnant women on average apprx. 2%, but some authors consider these results with serums pregnant with an error of laboratory engineering. However false positive results of serological tests with serums of pregnant women are the fact, about the Crimea it is necessary to be considered since it has fiziol, justification.
Nonspecific results of RW are described at capture of blood for a research at in the fever patients, directly after acceptance of plentiful greasy food, alcohol. It is necessary to consider that serums of patients with a subacute septic endocarditis have the self-detaining properties (a delay of hemolysis in control test tubes of the main experience).
The main reactions which give the chance to distinguish false positive reactions from the reactions caused by syphilis are reaction of an immobilization of pale treponemas and reaction of an immunofluorescence. However and they in some cases can be nonspecific. It is necessary to show care at assessment of false positive results of reaction: their revaluation or underestimation can lead to serious diagnostic mistakes. In each case it is necessary to consider the clinical and serological data which besides are carried out repeatedly. It is not necessary to revaluate also values of determination of height of a caption of RW since it has only auxiliary value. In assessment of the carried-out treatment an important efficiency factor of therapy of syphilis is transition of positive serological tests in negative. However if at patients with fresh forms of syphilis of R W rather quickly becomes negative, then at treatment of patients with late syphilis the negativation comes more slowly and quite often does not come at all, despite sufficient treatment (serorezistentny syphilis). In these cases it is necessary to find out the reasons of a serorezistentnost, involving in inspection of sick other specialists (neuropathologists, ophthalmologists, therapists, radiologists, etc.). Except blood serum, the research of cerebrospinal liquid is necessary. If R W remains in them positive, it is necessary to carry out additional treatment using at the same time nonspecific methods of therapy. If after this RW remains positive, treatment is stopped and the patient is left under clinical and serological control.
In the mid-seventies in a number of the countries (the USA, England, etc.) almost do not use original RW, having replaced it with bystry reaction on glass with cardiolipin antigen (reaction of VDRL — the name on initial letters of laboratory, in a cut it is for the first time offered) with the subsequent statement (in case of its positive takes) reactions of an immobilization of treponemas or reaction of an immunofluorescence.
Wasserman reaction at children
Wasserman reaction at children is important at recognition of active forms of the inborn or acquired syphilis, and especially at diagnosis of the latent inborn syphilis. Due to the features of a proteinaceous range of blood serum at chest age 3 — 6% of the children who had active inborn syphilis, can have negative R W (Yu. A. Finkelstein, M. M. Rayts). Negative R W at late inborn syphilis is even more often observed.
Without being strictly specific reaction, RW can be positive at other diseases (scarlet fever, an infectious mononucleosis, eosinophilic pulmonary infiltrate, some forms of pneumonia, a system lupus erythematosus, malaria, at increase in blood of amount of pathological globulins, sometimes after vaccinations or inoculations a pertussoid and diphtheritic and tetanic vaccine). Nonspecific character of R W is proved by negative reaction of an immobilization of pale treponemas (RIT), an immunofluorescence of pale treponemas and careful clinical inspection of children and their mothers.
Nonspecific positive R W can arise in 7 — 10 days after the birth. In such cases the repeated serological research in a month after the birth and careful clinical inspection is necessary.
At some children who were born from mothers with positive RW, enough treated concerning syphilis passive transfer of syphilitic reagins from mother is possible. If the child is healthy, then usually in 2 — 5 months of RW gradually becomes negative.
As a result of treatment by penicillin at the vast majority of the children sick with the active inborn or early latent inborn syphilis, RW gradually becomes negative. Much more slowly this phenomenon occurs at the children sick with the recurrent acquired syphilis or late inborn syphilis. At a part of such children of RW the long time remains with firmness positive.
See also Reaction of binding complement .
Bibliography: Bukharovich M. N. Gray rezistentny sifsh1s (Pathogeny, lshuvan-nya, profshaktika), Kshv, 1971, bibliogr.; Ovchinnikov H. M. Serological researches at syphilis and gonorrhea, M., 1958, bibliogr.; Ovchinnikov H. M and Sazonova of L. V. Serodiagnostik of syphilis, in book: The unified methods a wedge. Laborat, researches, under the editorship of V. V. Menshikov, century 4, page 193, M., 1972; Rayts M. M. Inborn syphilis, Mnogotomn, the management on dermas, and veins., under the editorship of S. T. Pavlov, t. 1, page 308, M., 1959, bibliogr.; Reznikova L. S. Cardiolipin antigens in a serodiagnosis of syphilis, Owls. medical, No. 1, page 40, 1953; Torsuyev N. A. Leprosy, M., 1952; Finkelstein Yu. A. Serodiagnosis of syphilis, venereal and skin diseases and their immunotherapy, M. — L., 1930, bibliogr.; Gertler W. Systematische Dermatologie und Grenzge-biete, Bd 2, Lpz., 1972; Gumpesber-ger G. Die Lues connata, in book: Dermatol. u. Venerol., hrsg. v. H. A. Gott-ron u. W. Schonfeld, Bd 5, T. 2, S. 9 75, Stuttgart, 1965, Bibliogr.; W asser-m a n n A., N e i s s e r A. u. B r u with k C. Eine serodiagnostische Reaktion bei Syphilis, Dtsch. med. Wschr., S. 745, 1906.
L. S. Reznikova; H. M. Ovchinnikov (dermas.), L. A. Shteynlukht (ped.).