Serodiagnosis

SERODIAGNOSIS, making a diagnosis on the basis of examining the properties of blood serum. This diagnostic method is based on the presence of antibodies in blood serum (agglutinins, opsonins, complement-fixing substances, etc.) and is built upon the phenomena of immunity in the sense of specificity. Depending on the nature of the antibodies present in the serum, the methodology of serological studies changes. Formerly, all types of antibodies were regarded as completely independent and autonomous of one another; at present, a unitary view of antibodies prevails, while differences in the manifestation of their action are made dependent on differences in the form of the antigen applied in vitro and the methodology of the experiment. The beginnings of serodiagnosis were laid by the classical experiments of Pfeiffer (1894) (see Pfeiffer's phenomenon), who indicated new ways of identifying bacteria using known antibodies. Gruber and Widal (1896) applied the reverse principle and, using a known antigen, determined unknown antibodies in a diseased organism. To serodiagnosis in a broad sense one can refer the determination of both an unknown antigen by a known antibody and, conversely, the establishment of an unknown antibody by a known antigen. The Gruber-Widal principle of determining the content of an unknown antibody in the test serum is the basis of serodiagnosis in a narrower sense; reactions built on this basis constitute clinical serodiagnosis proper. Antibodies that have arisen in the organism under the influence of a disease or artificial immunization with vaccines persist in the organism for an indefinite time; then their quantity decreases, and they may disappear altogether, yet they can appear again under the influence of various intercurrent factors (infection, trauma), but in a relatively small amount. It is also necessary to reckon with the presence in normal sera of a small amount of normal natural antibodies. Therefore, serodiagnostic reactions acquire clinical significance only with quantitative titration. Thus, for example, the Widal reaction can be considered clinically positive at a serum dilution of 1:50; the Wright reaction in brucellosis, at a dilution of 1:100, whereas agglutination in stronger dilutions of 1/10 to 1/25 cannot be considered clinically positive. Quantitative determinations in serodiagnosis acquire even greater significance in connection with the presence in the serum, alongside the main specific antibodies, of secondary group antibodies for related species of antigen, the titer of which is usually lower than the titer of the main antibodies. Thus, in typhoid fever, one can detect in the serum, alongside typhoid agglutinins, paratyphoid agglutinins as well. Despite the variety of serodiagnostic methodology, certain provisions are common to all types of studies. Thus, the already mentioned quantitative setup with falling doses of serum makes it possible to exclude the influence of group and normal antibodies on the result of the studies. In some cases, in strong dilutions of serum, one can observe the phenomenon of "inhibition"; thus, in the agglutination of the culture Bac. abortus Bang, one can observe the absence of agglutination in dilutions of 1/10 to 1/80, whereas in dilutions of 1/160 to 1/320 and higher the reaction may be positive. All serodiagnostic reactions occur only in the presence of salts, i.e., in an electrolyte medium. Usually, a 0.85% physiological solution of sodium chloride is used. Hypertonic solutions inhibit the reaction. The optimal temperature of the reaction is 37°. Serodiagnostic reactions proceed as colloid-chemical reactions between two colloidal systems. Fundamental in serodiagnostic reactions is the interaction between antigen and antibody. According to the earlier views of Ehrlich, the nature of this interaction is chemical; according to newer views, physicochemical. The antigen used for serodiagnosis in some cases is nonspecific in the immunobiological sense and is not connected with the causative agent of the disease; such are, for example, extracts for the serodiagnosis of syphilis; the culture Proteus X₁₉ in the Weil-Felix reaction in typhus; despite the nonspecificity of the antigen, the seroreactions in these cases are specific in their clinical significance. The importance of serological methods is not limited only to the field of the clinic; in view of the persistence of antibodies in the serum even after recovery, as well as their appearance in latent forms of infection, one can use serodiagnostic reactions to detect foci of former diseases, as well as hidden carriers of infections. Hence the importance of serodiagnosis in epidemiology. Types of serological reactions. 1. Bacteriolysis has not found wide application in vitro; in vivo it is known as the Pfeiffer phenomenon. 2. Precipitation: 1) with a specific antigen—bacterioprecipitation—has not received wide application in clinical medicine; in veterinary practice, it has great application in the recognition of anthrax—Ascoli thermoprecipitation (see Ascoli-Valenti reaction); precipitation with a specific protein is of great importance in forensic medicine (see); 2) with a nonspecific antigen: a) precipitate (sedimentation) reactions (see Precipitation) play a large role in the serodiagnosis of syphilis; b) recently, melanoprecipitation in malaria has been described. 3. Opsonins (see) are of little practical application. 4. Agglutination has found wide application in clinical serodiagnosis in various diseases; it is used: 1) with the causative agent of the disease: a) the Widal reaction in typhoid fever and paratyphoid fevers, b) the Wright reaction in brucellosis, c) the agglutination reaction in tularemia, d) partly in bacillary dysentery, e) in Weil's infectious jaundice, f) in sporotrichosis; 2) paraagglutination with accompanying bacteria: a) the Weil-Felix reaction with Proteus X₁₉. 5. Complement fixation reaction: 1) with a specific antigen: a) in gonorrhea, b) in brucellosis, c) in rhinoscleroma, d) in tuberculosis, e) in echinococcal diseases—the Weinberg reaction, f) in glanders in veterinary practice; 2) with an antigen that is immunologically nonspecific; of the latter, the Wassermann reaction has the greatest clinical significance (see Wassermann reaction). 6. The Abderhalden reaction and its various modifications as methods of dialysis, polarimetry, interferometry, the Lhotte-Mertz reaction for determining the so-called "protective" enzymes (Abwehrfermente) against the cells of the organism. 7. Serodiagnostic methods can also include the determination of blood groups in humans (for details, see individual reactions).

Dr. Borovetskaya. Serodiagnostic determination of the systematic position of organisms. The serodiagnostic method for determining systematic relations between organisms is based on the principle of the species specificity of proteins. An extract (containing proteins) from the tissues of one of the organisms being compared is injected into a guinea pig or rabbit to induce the formation of specific antibodies. The serum of the rabbit thus immunized is mixed with a tissue extract of another of the organisms being compared. The presence of a precipitin reaction (a precipitate) in this case speaks in favor of a relationship between the organisms being compared, while the absence of a precipitate indicates a lack of systematic affinity. The degree of relationship is determined by the amount of precipitate formed. The serodiagnostic method has been applied to both animals and plants. In particular, in this way it is possible to demonstrate the systematic affinity between man and higher apes. For plants, an entire evolutionary tree was constructed using the serodiagnosis method, which in some respects differs sharply from trees constructed on the basis of comparative morphological studies. Along with results confirming systematic affinity discovered by other means, the serodiagnosis method sometimes leads to clearly absurd conclusions. Therefore, along with the continued use of this method, its thorough development and a strictly critical attitude toward the results obtained are necessary. Serodiagnosis in pregnancy. Serodiagnosis of pregnancy is based on identifying the features possessed by the blood serum of a pregnant woman in order to resolve a critically important practical question: the reliable determination of early pregnancy. This task seemed resolved when Abderhalden proposed a serological reaction that now bears his name (see Abderhalden reaction). Even earlier, Veit succeeded in proving that the serum of a pregnant woman breaks down placental villi with the aid of special substances he named syncytiolysins. In order to improve the results of the Abderhalden reaction and eliminate errors in technique, which requires extremely careful execution, various modifications of the original method were proposed. First of all, it seemed necessary to eliminate dialysis thimbles, which, according to the conclusion of some authors, can serve as a source of inaccurate reaction results. In view of this, Nedrigailov proposed impregnating the antigen with carmine and judged protein cleavage by the color of the serum, which, together with the antigen, was placed in a test tube in an incubator for 24 hours. Kottmann, performing the Abderhalden reaction, used an antigen representing a combination of placental protein and iron ("Sorcym"). Upon protein cleavage by the serum, the released iron can be determined in the filtrate by a color reaction with HCl and potassium thiocyanate. According to Zdrodovsky, three samples are placed in the incubator: antigen with serum, serum alone, and antigen with distilled water. The filtrate of each sample is titrated with ninhydrin. The result of the experiment is evaluated by the difference between the titer of the first sample and the sum of the titers of the other two control samples. Since upon cleavage of the antigen by the serum the specific gravity of the latter increases, E. Krasnushkin proposed determining both the specific gravity of the serum that acted on the antigen and the specific gravity of the control serum without the antigen. For this purpose, a drop of the test serum is introduced into a mixture of chloroform and benzene. Depending on the density of the serum, the drop placed in this mixture either rises, sinks, or remains suspended in the middle of the liquid if the specific gravity of the liquid and the serum are equal. Having prepared a mixture of chloroform and benzene of such a composition that the drop of control serum remains in the middle of this liquid, a drop of the serum that has been in contact with the antigen is lowered into the latter. By comparing the position of both drops, changes in the specific gravity of the serum are judged. Finally, Lüthe and Mertz proposed a very successful simplification of the Abderhalden reaction. The test tube with the test serum and placental substrate is placed in an incubator for 24 hours. After this period, a 10-fold amount of 96% alcohol is poured into the test tube. The serum proteins coagulate into a dense mass; then the test tube is boiled for a short time on a gas burner. The contents of the test tube are filtered. The filtrate is boiled for one minute, adding 0.2 cm³ of a 1% alcoholic ninhydrin solution. In the case of a positive result, a violet coloration is obtained. According to Lüthe and Mertz, the reaction gives a correct answer in 98.7% of cases. Other authors working with Kottmann's and Zdrodovsky's methods also obtained faultless results in almost 100% of cases. However, the majority of authors using the original Abderhalden reaction and its modifications did not obtain an unquestionably correct answer. The question of the non-specificity of the reaction remains unresolved. Abderhalden himself notes that sera taken from patients with inflammatory processes are not suitable for performing the reaction, because a positive answer is always obtained with them. It should also not be forgotten that many sera react positively with ninhydrin even without a substrate, and thus the reaction cannot be performed with them. All this significantly reduces the value of the Abderhalden reaction and deprives it of practical value. Hirsch modified the Abderhalden reaction using a Löwe-Zeiss interferometer. With the aid of this apparatus, it is possible to precisely determine the concentration of two liquids and detect the slightest difference. If serum containing a "protective" enzyme is allowed to act on the corresponding substrate, the concentration of the serum should increase due to the transition of cleavage products into solution. This increase in concentration is established by the interferometer when comparing the same serum, but without the substrate. At Hirsch's suggestion, the substrate is prepared in the form of a sterile powder he named "optim" (Pharmagans firm). To 0.5 g of the test serum, 5 mg of placental substrate and a solution of Vucine hydrochloride are added. The test tube is hermetically sealed and placed in an incubator for 24 hours with control tubes containing 0.5 cm³ of the same serum, but without the substrate. Both control sera are compared with each other at the end of the study and must show complete coincidence of interference bands. The serum that is in contact with the substrate is centrifuged, examined interferometrically, and compared with the control serum. A number of erroneous data in the performance of this reaction gave grounds for a critical evaluation of this method. Streck established that the supposed "cleavage" occurs both in the incubator and at room temperature. A number of authors established that a change in concentration is obtained both when using specific and non-specific (non-pregnant) serum, as well as with serum inactivated by heating. Streck also established that the structure of the powdered substrate and the size of its particles exert a enormous influence on the results of the reaction. Fine-grained powder gave phenomena of low cleavage, while coarse-grained powder gave high cleavage. Küster and Koulen, after introducing a number of complex changes into the course of this modification of the Abderhalden reaction, had to admit that these are only ways to improve, rather than eliminate, the shortcomings of this method. Kleesattel points out that the good results obtained by certain authors are explained by the fact that they dealt with few cases where pregnancy was absent; the more such cases are taken, the more failures are revealed. Inaccuracy, expensive equipment, and the complexity of the methodology deprive this method of serious significance. Subsequently, Lüthe and Mertz developed a new serological reaction and proposed using specially prepared extracts from organs, respectively the placenta, instead of a substrate. This is the so-called alcohol-extract reaction (AER), which is performed without an incubator. To 1 cm³ of serum, 1 cm³ of placental extract is added; after thorough shaking, 10 cm³ of absolute alcohol is added. The mixture is filtered; the filtrate is boiled for 1 minute, and 0.2 cm³ of a 1% ninhydrin solution and 1 drop of HCl are added to it. In the case of a positive result, the liquid immediately or after cooling acquires a violet color. According to Lüthe and Mertz, a correct answer is obtained in 98.7% of cases. However, this has not been confirmed by other authors. Attempts to improve this reaction have had almost no success. The essence of the reaction is poorly understood. As Mandelstam points out, the products obtained in the Abderhalden reaction, as well as the extracts used in the alcohol-extract reaction, have low molecular weight, whereas substances of high molecular weight are produced when serum acts on extracts. On this basis, Mandelstam concludes that in the alcohol-extract reaction, it is not the extract that undergoes decomposition, but the serum. This is therefore a special reaction, completely different from the Abderhalden reaction.

A more plausible explanation for the alcohol-extract reaction is given by Schmidt, who believes that the course of this reaction is influenced by the ratio of albumins to globulins in the serum, the cholesterol content, and the ion concentration. Having performed the erythrocyte sedimentation reaction alongside the alcohol-extract reaction, Schmidt established that when the erythrocyte sedimentation rate is less than 30 minutes, the reaction yields more erroneous conclusions. This strongly supports the assumption regarding the importance of the physicochemical composition of the serum in evaluating the results of the alcohol-extract reaction. To an equal degree, this also applies to other reactions, for example, Dienst's reaction, which consists in determining the antithrombin present in the form of metathrombin, i.e., in a state bound to thrombin. The addition of alkali disrupts this bond, and the liberated antithrombin can be detected by the biuret reaction or with the aid of ninhydrin. The work of many authors has shown that this method cannot be used for the diagnosis of pregnancy, because the color indices characteristic of pregnancy according to Dienst are very frequently encountered in non-pregnant women and in men. Similar results were also obtained when testing Vogel's reaction. In this reaction, blood serum is diluted with a standard alkaline solution and boiled with a ninhydrin solution. According to Vogel, in the serum of pregnant women, the protein appears as a suspension and barely noticeable blue flakes. In non-pregnant women, the protein precipitates as coarse gray-white flakes that stand out distinctly in the blue-colored liquid. According to Yakovlev, Vogel's reaction cannot serve for the recognition of pregnancy, since the presence or absence of color and the identical character of the flakes are encountered in both pregnant and non-pregnant women. Wishing to avoid complex and meticulous techniques in performing the alcohol-extract reaction, Merz proposed a new serological reaction for determining pregnancy, which is distinguished by great simplicity and, according to his data, yields very good results. To 1 cm³ of serum is added 1 cm³ of a solution of phosphotungstic acid (2.8 g per 1 liter of water); after ¹/₂ hour, in the case of pregnancy, turbidity is obtained. After another ¹/₂ hour, 2-3 drops of bromphenol blue are added. In the case of pregnancy, a light blue coloration is obtained, passing into green, and the solution does not transmit light; in the absence of pregnancy, the coloration is blue and the solution is transparent. Schmidt obtained a correct result in 92% with this reaction. However, a repeat examination is necessary, since there is no reason to consider this reaction more accurate than, for example, Vogel's reaction, the failure of which has been fully proven. The reaction proposed by Manoilov (adding to 0.3 cm³ of serum 1 cm³ of a 2% diuretin solution and a drop of a 0.2% alcohol solution of Nile blue—in pregnant women after some time a yellow, pinkish-yellow coloration is obtained; in non-pregnant women—a blue, pinkish-blue coloration), according to the author's data, yields a positive result in 92-96%. Check investigations, for example, from Linzenmeier's clinic, recognized this reaction as completely unsuitable for the diagnosis of pregnancy. Almost all the serological reactions just cited are based on the physicochemical features of the serum of pregnant women. Along with this, it is quite clear, as is also confirmed, that all these reactions cannot be strictly specific. In reality, the same physicochemical features of the serum as in a pregnant woman are also observed in diseases that are characterized by increased tissue breakdown and the overloading of the bloodstream with metabolic products. Therefore, these same reactions are so frequently observed with the serum of cancer patients, in acute infectious diseases, in tuberculosis, and in sepsis. Therefore, the ability of the blood serum of a pregnant woman to activate the hemolytic action of cobra venom in relation to horse erythrocytes, and the increase in antitrypsin in the blood serum of a pregnant woman, are also observed in cancer patients. The meiostagmin reaction discovered by Ascoli and used for the diagnosis of cancer also yields a positive result in pregnant women. Abderhalden's reaction stands apart in this respect. Physicochemical features of the serum play no small role in this reaction as well. However, further research and probably a more refined technique are necessary for the full confirmation of Abderhalden's idea. At the present time, not a single one of the proposed serological reactions for the diagnosis of pregnancy can lay claim to accuracy and reliability.