What blood tests are taken for syphilis and how are they interpreted? Agglutination reactions Serological tests

Serological research (tests)— laboratory research methods based on the detection of antibodies or antigens in the patient’s biological material. Most often, blood is used for analysis, less often - urine, saliva, purulent discharge or tissue samples taken during a biopsy.

Scope of application

• Determination of blood group.
• Identification of specific tumor proteins - tumor markers (for example, in case of suspected cancer of the ovaries, prostate, bladder, stomach, etc.).
• Diagnosis of viral, bacterial, fungal, protozoal infections (HIV, syphilis, toxoplasmosis, chlamydia, rubella, herpes, helminthiasis, tick-borne encephalitis, etc.).
• Determination of hormones, enzymes and drugs contained in the studied biomaterial in minor concentrations (less than 10−10 g/l).

The essence of the method is serological tests

Serological tests differ in the technique used, but they are all the result of the interaction of antigens (foreign compounds) with corresponding antibodies. The study consists of two successive phases. The first phase is characterized by the interaction between antigens and antibodies with the formation of immune complexes (positive reaction). In the second phase, external signs appear that confirm the presence of these same complexes (depending on the type of reaction, this may be cloudiness of the test solution, a change in its color, the loss of flakes, etc.). The absence of visible physical phenomena is regarded as a negative test result.

Preparation for serological studies

Depends on the type of research. A medical specialist should tell you about the specifics of taking a specific test when you sign up for the procedure.

You can take the necessary serological test at the Spectra clinic. We order analyzes from the best laboratories in the capital, working according to European standards, which guarantees fast and reliable results. Our doctors will help you decipher the conclusion and give recommendations for further diagnosis.

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Serological blood test is a method for laboratory testing of certain antigens or antibodies ( specific proteins) in the patient's blood serum, based on the body's immune responses. This method is used when diagnostics infectious diseases to detect the presence of antibodies in the patient’s blood to a certain type of virus or bacteria, as well as to determine the blood group.

Material under study

First of all, for serological analysis, biological material collected from the patient is used:

• blood serum
• saliva
• fecal matter

In some cases, material isolated from certain environmental objects is examined:

• soil

Method of analysis or blood sampling

This analysis does not require special preparation of the patient. Blood sampling is carried out in the morning on an empty stomach and is performed in treatment rooms of medical institutions, according to generally accepted hematological methods. For serological testing, blood is collected using two methods: venous blood is taken from the patient’s ulnar vein, and capillary blood is taken from the ring finger. The blood is placed in sterile sealed tubes.

Features of blood transportation and serum storage

Immediately after blood collection, it is transported to a special laboratory, where serum is prepared on the same day. Whey can be stored for no more than 4–6 days in a refrigerator at a temperature of 2–4 degrees. If longer storage is required, the whey is frozen. To avoid compromising the quality of the whey, it is allowed to freeze it once and, accordingly, defrost it. During long-term storage, antibodies lose their properties and become partially inactive; immunoglobulins of the class are most sensitive to freezing M. After thawing the serum, it is necessary to thoroughly mix it until smooth, which helps restore the concentration of antibodies contained in this serum.

What are serological tests used for?

Serological laboratory diagnostic methods are used to identify diseases such as echinococcosis, trichinosis, toxocarosis, opisthorchiasis, cysticercosis, toxoplasmosis, amebiasis, giardiasis, to determine the effectiveness of the course of treatment and, finally, to detect recurrent disease after the patient has fully recovered.

Basic methods of serological diagnosis

Immunofluorescence reaction (RIF)

This reaction is an indirect version of a serological test, that is, it is carried out in two stages. At the first stage, the required antibody is identified in the circulating antigen-antibody complex using antiglobulin, which is similar in structure to immune serum proteins. Identification of the desired antibody is also possible by studying a pre-prepared antigen preparation under a fluorescent microscope, without the use of antiglobulins.

The immunofluorescence reaction is a very labor-intensive process that requires a lot of time and responsibility from a specialist. The reaction begins with the preparation of solutions, then the sera and their control samples are titrated ( a process that allows you to determine the content of a certain substance by gradually mixing the test solution with a controlled amount of the reagent). Previously prepared dilutions and their control samples are carefully applied to glass slides with the antigen. Then the preparations are incubated, followed by washing and air drying. A thin layer of antiserum is applied to the slides with the antigen, after which a secondary incubation of the preparations is performed and the entire previous chain of actions is repeated, ending with the drying of the preparation. As a result, the preparation on a glass slide is treated with glycerol and examined under a fluorescent microscope.

The results of the reaction are assessed on a four-point scale, which is characterized by the intensity of the surface yellow-green glow of antigen cells:
+ very faint glow of the cell, noticeable only at its periphery
++ faint glow of the cell periphery, but with a clearly noticeable green tint
+++/++++ bright green glow of the cell periphery
The reaction titer is considered to be a serum dilution where at least 50% of the antigen cells exhibit a clear surface glow, that is, the result of the reaction +++ or ++++ . The reaction range value is from 1/80 to 1/100.

The intensity of the reaction depends on the number of precipitated red blood cells at the bottom of the formed holes:
– a negative reaction, which is characterized by the deposition of red blood cells to the bottom of the wells in the form of a small ring with smooth edges or a “button”
+ low intensity, this reaction is characterized by small single deposits at the bottom of the hole. Non-agglutinated red blood cells form a “small ring” in the center of the well
++ medium intensity, it is characterized by the formation at the bottom of the hole of a “wide dense ring” of non-agglutinated erythrocytes
+++ intense reaction, agglutinated erythrocytes form so-called “umbrellas”, in the center of which rings formed by settled non-agglutinated erythrocytes are clearly visible
++++ a sharply intense reaction in which all red blood cells agglutinate and, forming “umbrellas,” line the bottom of the wells.
The titer of this reaction is considered to be the last serum dilution at which obvious agglutination of erythrocytes is detected at least +++ , that is, with an intense or sharply intense reaction.

The reliability and quality of serological diagnostic methods depend on the organization of internal and external laboratory control, consisting of several independent procedures designed to assess the quality of the results of the analysis.

The Wassermann test (RW) is the most popular immunological test used to diagnose syphilis since its discovery in 1906. RW belongs to the group of complement fixation reactions (FFR) and is based on the ability of the blood serum of a syphilis patient to form a complex with the corresponding antigens. Modern RSC methods used to diagnose syphilis differ significantly in their antigens from the classical Wassermann reaction, however, the term “Wassermann reaction” is traditionally retained for them.

Antibodies produced by the immune system appear in the blood of an infected person. The causative agent of the disease, Treponema pallidum, contains the antigen cardiolipin, which causes the production of antibodies detected by RW. A positive Wasserman reaction just indicates the presence of such antibodies in a person’s blood, and on this basis a conclusion is made about the presence of the disease.

The hemolysis reaction is an indicator of the result of the study in the RSC. The reaction involves two components: sheep red blood cells and hemolytic serum. Hemolytic serum is obtained by immunizing a rabbit with sheep red blood cells. It is inactivated for 30 minutes at a temperature of 56°C. The results of RSC are evaluated depending on the presence or absence of hemolysis in the test tubes. The presence of hemolysis is explained by the fact that if there are no syphilitic antibodies in the test serum, then the antigen-antibody reaction does not occur, and all the complement goes to the sheep erythrocyte-hemolysin reaction. And if there are specific antibodies, the complement is completely spent on the antigen-antibody reaction and hemolysis does not occur.

All ingredients for the Wasserman reaction are taken in the same volume - 0.5 or 0.25 ml. For strong fixation of complement on a specific complex, a mixture of the test serum, antigen and complement is placed in a thermostat at a temperature of 37° for 45-60 minutes. (phase I reaction), after which a hemolytic system consisting of sheep erythrocytes and hemolytic serum is added (phase II reaction). Next, the tubes are again placed in a thermostat for 30-60 minutes until hemolysis occurs in the control, in which the antigen is replaced with physiological solution, and instead of the test serum, physiological solution is added. Antigens for the Wasserman reaction are produced in finished form, indicating the titer and dilution method.

The maximum positivity of the Wasserman reaction is usually indicated by the number of crosses: ++++ (strongly positive reaction) - indicates a complete delay in hemolysis; +++ (positive reaction) - corresponds to a significant delay in hemolysis, ++ (weakly positive reaction) - evidence of a partial delay in hemolysis, + (doubtful reaction) - corresponds to a slight delay in hemolysis. Negative RW is characterized by complete hemolysis in all test tubes.

However, sometimes false positive results are possible - this is due to the fact that cardiolipin is also found in some quantities in the cells of the human body. The human immune system does not create antibodies against its “own” cardiolipin, but there are exceptions to this rule, due to which a positive Wasserman reaction occurs in a completely healthy person. This is especially often observed after severe viral and other diseases - pneumonia, malaria, liver and blood diseases, during pregnancy, i.e. in moments of severe weakening of the immune system.

If a doctor suspects a patient has a false positive result for the Wasserman reaction, he can prescribe a number of additional tests that are usually used in diagnosing sexually transmitted diseases.

Diseases and cases in which a doctor may prescribe a blood test for RW

Carrying out the procedure for taking a blood test for RW

Blood for RW is donated only on an empty stomach. The last meal should be no later than 6 hours before the test. The medical worker sits the patient down or places him on the couch and takes 8-10 ml of blood from the cubital vein.

If an analysis needs to be done on an infant, the sample is taken from the cranial or jugular vein.

Preparing for a blood test for RW

You should stop drinking alcohol 1-2 days before the test. It is also not recommended to eat fatty foods - they can distort the results. During the period of preparation for the analysis, you should refrain from taking digitalis medications.

Contraindications

The analysis result will be false if:

• the person is sick with an infectious disease or has just recovered from it,
• a woman is menstruating,
• pregnant in the last weeks before giving birth,
• the first 10 days after birth,
• the first 10 days of a baby's life.

With primary syphilis, the Wasserman reaction becomes positive at 6-8 weeks of the disease (in 90% of cases), and the following dynamics are noted:

• in the first 15-17 days after infection, the reaction in most patients is usually negative;
• at the 5-6th week of the disease, in approximately 1/4 of the patients the reaction becomes positive;
• at 7-8 weeks of the disease, RW becomes positive in the majority.

In secondary syphilis, RW is always positive. Together with other serological reactions (RPGA, ELISA, RIF), it allows not only to detect the presence of the pathogen, but also to find out the approximate duration of infection.

With the development of a syphilitic infection in the 4th week of the disease, after the onset of primary syphiloma, the Wasserman reaction passes from negative to positive, remaining so both in the secondary fresh and in the secondary recurrent period of syphilis. In the latent secondary period and without treatment, RW can turn negative so that when a clinical relapse of syphilis occurs, it becomes positive again. Therefore, in the latent period of syphilis, a negative Wasserman reaction does not indicate its absence or cure, but only serves as a favorable prognostic symptom.

With active lesions of the tertiary period of syphilis, positive RW occurs in approximately 3/4 of cases of the disease. When the active manifestations of the tertiary period of syphilis disappear, it often turns negative. In this case, a negative Wasserman reaction in patients does not indicate that they do not have a syphilitic infection.

In early congenital syphilis, RW is positive in almost all cases and is a valuable method for verifying the disease. In late congenital syphilis, its results correspond to those obtained in the tertiary period of acquired syphilis.

Of great practical importance is the study of the Wasserman reaction in the blood of patients with syphilis undergoing treatment. In some patients, despite vigorous anti-syphilitic therapy, the Wasserman reaction does not turn negative - this is the so-called seroresistant syphilis. In this case, it makes no sense to endlessly carry out anti-syphilitic therapy, achieving the transition of positive RW to negative.

From the above it follows that a negative Wasserman reaction is not always a sign of the absence of a syphilitic infection in the body.

A positive Wasserman reaction is possible in people with a number of other diseases and conditions not related to syphilis:

All of the above indicates that a positive result of the Wasserman reaction is not yet unconditional evidence of the presence of a syphilitic infection.

Recovery after testing

After taking a blood test, doctors recommend a proper and balanced diet, as well as plenty of fluids. You can afford warm tea and chocolate. It will be useful to refrain from physical activity and never drink alcohol.

Norms

Normally, hemolysis should be observed in the blood - this is considered a negative reaction to syphilis (Wassermann reaction is negative). If hemolysis is absent, the degree of reaction is assessed, which depends on the stage of the disease (marked with “+” signs). However, you should be aware that in 3-5% of completely healthy people the reaction may be false positive. At the same time, in the first 15-17 days after infection, the reaction in sick people may be false negative.

What are serological tests, why are they carried out, what are the main methods of serological diagnosis?

Serological studies are methods for studying antigens or antibodies in the biological material of patients, based on certain immune reactions. The detection of antibodies to the infectious agent or antigens in biological material makes it possible to establish the cause of the disease.

do not have 100% specificity and sensitivity in diagnosing infectious diseases. Therefore, their results must be assessed taking into account the clinical picture of the disease. This explains the need to use several tests to diagnose infection, as well as the use of methods Western blot, confirming the results of screening methods.

“Immunoblotting (Western blot) is actually the final verification (confirming) method in the chain of immunological studies that allow us to make a final laboratory conclusion. This test is especially important for confirming or refuting an infectious disease (for example, whooping cough, borreliosis, etc.). The Western blot method is usually necessary after detection of positive IgG antibodies of the infectious process, because It is in this combination that a more correct laboratory interpretation of the results and further treatment tactics for the patient occur. To perform a Western blot, special nitrocellulose strips are used, onto which proteins are transferred using horizontal and then vertical immunophoresis in order of increasing molecular weight. Antibodies from the patient’s serum interact with proteins in certain areas of the strip, and then a reaction proceeds similar to an enzyme-linked immunosorbent assay (ELISA),”- explains candidate of medical sciences, medical director of the SYNEVO Ukraine laboratory, Oksana Vladislavovna Nebyltsova.

Biological material used for serological research

• Blood serum
• Saliva
• Fecal matter

What conditions are serological tests used to diagnose?

The purpose of serological studies is to establish the effectiveness of treatment after the patient has recovered, as well as to detect relapse of the disease. In addition, serological methods can detect diseases such as:

• Amoebiasis
• Giardiasis
• Opisthorchiasis
• Trichinosis
• Toxocariasis
• Cysticercosis
• Echinococcosis

Main methods used in serological diagnosis

Immunofluorescence reaction (RIF, Koons method)

Types of method:

Direct: microbes or tissue antigens or microbes are treated with special serums with antibodies, as well as labeled fluorochromes that glow in UV rays. The bacteria thus glow as a green border around the periphery of the cell. Observed under a fluorescence microscope.

Indirect: smears are treated with antibodies from antimicrobial rabbit serum. Antibodies that have not bound to microbial antigens are then washed away. In this way, the antibodies remaining on the microbes are detected. As a result, a complex of microbe + anti-rabbit antibodies + anti-microbial rabbit antibodies, labeled with fluorochrome, is formed. This complex is observed under a fluorescent microscope.

To evaluate the results, there is a four-point scale, which is characterized by the intensity of the surface yellow-green glow of antigen cells:

Very faint cell glow

Faint glow of the cell periphery

+++/++++ bright glow of the cell

The titer of the reaction is considered to be the dilution of the serum with the result of the reaction +++ or ++++.

Indirect hemagglutination reaction (IHR)

The passive or indirect hemagglutination reaction is used for diagnosis of infections caused by protozoa, bacteria and rickettsia.

The technique consists of several stages. First, red blood cells are treated and “washed” with an isotonic solution of sodium chloride, then, if necessary, with a solution of tannin 1: 20,000, and then sensitized with soluble antigens. After treatment with a buffered isotonic sodium chloride solution, the antigen is ready for use. The serum is diluted in test tubes with an isotonic sodium chloride solution, after which an erythrocyte diagnosticum is added to each diluted serum.

The results are assessed by the nature of the erythrocyte sediment:

Low intensity

Medium intensity

Intense reaction

Sharply intense reaction

The result of a reaction with an intense reaction +++ or a sharply intense reaction ++++, in which red blood cells cover the entire bottom of the tube, is considered positive.

Enzyme-linked immunosorbent assay (ELISA)

The method has high specificity and sensitivity, more than 90%. The main advantage is the possibility detection of infection and tracking the dynamics of the process, which is indicated by the level of antibodies.

The test is used to diagnose a wide range of infections, including:

• HIV infection
• Viral hepatitis
• Cytomegalovirus infection
• Herpes infection
• Toxoplasma infection

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The antigen or antibody is fixed on solid plates, then, using an enzyme label, antigen-antibody complexes are detected.

ELISA has a number of advantages over other serological methods. The reaction is the most sensitive, and the tests use universal reagents. The analysis is characterized by its speed and the ability to study immunoglobulins of various classes. Currently, ELISA is one of the main methods of laboratory diagnostics.

Evaluation of ELISA results is carried out automatically using special devices. In some cases, visual recording of reaction results is also allowed. The reliability of serological diagnosis depends on the organization of laboratory control, which consists of several procedures designed to assess the quality of the results.

Serology(from Latin serum - “serum”, logos - “science”) is a branch of immunology that studies the specifics of the interaction of serum antibodies with antigens.

The basis of diagnosis is the detection of specific antibodies that are formed as a reaction to infection of the body with a specific antigen. Depending on what antibodies are found in the blood, a conclusion is drawn about the nature of the infection, and the number of these antibodies indicates the degree of activity of the infectious disease.

The material taken for research as part of a serological blood test is studied for other dangerous diseases - herpes, intestinal infections, rubella, toxoplasmosis, chlamydia, measles, chlamydia. In addition, this study allows you to confirm your blood group and determine the specificity of proteins.

So, serological studies are carried out:

• if a preliminary diagnosis has been made and its confirmation is now required. The study is based on the addition of the corresponding antigen to the blood serum. The response allows us to draw a conclusion about the presence or absence of the disease;
• if the diagnosis cannot be made. As part of the study, antibodies are added to the blood and the type of antigens is determined, which makes it possible to determine a specific disease;
• if necessary .

Thus, a serological blood test helps to make a diagnosis or prescribe the most effective treatment - with minimal time and financial costs.

The advantages of serological studies include:

• the ability to identify pathological microorganisms in the early stages of infection;
• monitoring the development of the disease and the level of effectiveness of the therapy;
• no need for special preparation before taking biomaterial;
• efficiency. The result will be available in two to three hours, which is very important in hospital treatment conditions;
• financial availability of the reagent, which allows samples to be carried out as often as needed;
• no contraindications.

How is serological testing performed?

Blood is drawn from the ulnar vein. An important point is that the blood is taken not with a syringe, but by gravity - a needle is inserted into a vein without a syringe and up to five ml of blood is collected in a test tube. The procedure is carried out in the morning.

Depending on the reactions that underlie the study, several types of procedures are distinguished:

1. neutralization reaction. It is based on the property of immune serum antibodies to react as a neutralizing agent to microorganisms or toxins, preventing their negative effects on the body;
2. agglutination reaction. May be direct or indirect. In the first case, we are talking about studying blood serum for the presence of antibodies (killed microbes are injected into the material, and the reaction is assessed - if there is a precipitate in the form of flakes, the reaction is positive), an indirect reaction is based on the introduction of erythrocytes with antigens adsorbed on them into the material ( scalloped sediment indicates a positive reaction);
3. precipitation reaction. The antigen solution is layered on the immune serum (acts as a liquid medium). A soluble antigen is used. If the antigen-antibody complex precipitates, the reaction is considered positive;
4. reaction involving complement. Area of ​​application: detection of infectious diseases. Complement is activated and reactions are examined;
5. reaction with labeled antigens and antibodies. It is based on the fact that tissue antigens or microbes, processed in a special way, begin to emit light under the influence of UV rays. The method is widely used not only in the diagnosis of antigens, but also for the determination of hormones, enzymes, and drugs.

It is necessary to prepare for the test: four days in advance, the patient must stop taking heart medications; he must also eliminate alcohol in any form, spicy and fatty foods, sweets, limit physical effort, and avoid stress.

If these rules are not followed, the risk of a false positive result increases. Before prescribing a repeat test, the doctor must find out what the patient did the day before the procedure and give recommendations on how to properly prepare for the examination.

Serological analysis: explanation

A serological blood test is a test that allows you to determine/confirm the type of infectious agent and helps a specialist make a diagnosis. This is an indispensable help if the doctor cannot select drug therapy, since the causative agents of various diseases differ in different sensitivity to the action of specific drugs and antibiotics.

After the material collection procedure is completed, laboratory assistants proceed to the next stage - deciphering the indicators. So, if no antibodies are detected in the patient’s blood, we can conclude that there are no infections in the body - the test result in this case is positive.

But this state of affairs is rather an exception to the rule: if there are symptoms of the disease, serological studies identify and prove the presence of a serious pathology in the body.

First, pathogens are found in the body using analysis, then the amount of antibodies is assessed, based on which a conclusion is drawn about how serious the infection is.

Serological testing for hepatitis, HIV, syphilis: features

Syphilis . When conducting an analysis for syphilis, specialists look for proteins that are responsible for the entry of the infectious agent into the human body - we are talking about Treponema pallidum. In this case, blood serum acts as a biological material.

. Viral hepatitis is a serious infectious disease, the danger of which lies in the fact that it can live in the body for quite a long time without manifesting itself. The disease can be detected in the early phase, when it is more treatable, by analyzing markers - markers appear in the blood after an illness or the administration of a vaccine.

Need to understand that identification of the pathogen is possible only 1.5-2 months after infection. If a pregnant woman takes the test, a false positive result is possible.

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If you experience one or more of the symptoms listed below, you should consider getting a serological test:

• vomit;
• poor appetite or lack thereof;
• causeless weakness of the body, overwork;
• yellowish complexion;
• changes in the color of stool and urine.

HIV. If the result is positive, this does not mean that the patient is infected with AIDS. If the infection occurred recently (no more than two months ago), the presence of antibodies does not allow us to determine whether the disease has developed. A repeat study is ordered.

Serological blood test- the most important research method, the main purpose of which is to quickly identify viruses, infections, microbes in the body.

This unique laboratory “tool” allows you to identify any disease that is a consequence of immunosuppression, so do not be lazy, but get tested regularly in order to be able to identify the disease in time and quickly get rid of it.

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