Direct hemolytic agglutination reaction (DHA)

What is RPGA and what features does it have?

The passive hemagglutination reaction is used to decipher the abbreviation RPGA and RNGA. This is a diagnostic test used to detect the dissolved amount of antibodies in a patient's serum sample against the causative agents of syphilis. To be precise, RPHA helps in the detection of antibodies to the k48 allergen using the indirect hemagglutination method.

The process involves the sensitivity of red blood cells to fragments of T. palladium. When a blood serum sample is exposed, cells collect on the surface of the dish for analysis. The passive hemagglutination test is a specific or treponemal test because it uses real, syphilitic antigens.

The tests are very sensitive, but because they are nonspecific, false positives do occur. This occurs with intravenous drug use, pregnancy, Lyme disease, some types of pneumonia, malaria, tuberculosis, and some autoimmune diseases, including lupus. Studying the characteristics of the immune response in patients with syphilis requires a detailed study of the antigenic structure of the pathogen and its effect on the host organ system.

Types of blood tests for syphilis

A number of methods have been developed to diagnose syphilis, but none of them gives a 100% accurate result. The material for research is most often blood plasma; discharge from skin rashes is also used; in some cases, lymph node puncture is performed. The main types of research include serological tests, which are used for initial diagnosis and to monitor the results of therapy.

Serological tests for syphilis are based on the mechanism of formation in the body, when the causative agent of the disease enters it, antibodies that interact with special antigens. The main serological blood tests for syphilis are as follows:

• Wasserman reaction - during this test, syphilitic antigens are added to the patient's blood serum to determine antilipid antibodies (reagins). Antigens form an immune complex with reagins, which adsorbs and binds complement. When such a complex is formed, the red blood cells are not destroyed, but precipitate, which indicates a positive reaction. If there are no reagins in the blood serum, hemolysis occurs (destruction of red blood cells), in which case the reaction is negative.
• The microprecipitation reaction with cardiolipin antigen (RPR or RMP) is a screening method for diagnosing syphilis, which can be considered as a more modern modification of the Wassermann reaction. The main indications for taking a blood test for syphilis using this method are initial examination, medical examinations, and monitoring the effectiveness of therapy. If a positive result is obtained from a blood test for syphilis using this method, other more accurate tests are prescribed to confirm it.
• Blood test for syphilis ELISA (enzyme-linked immunosorbent assay) is one of the advanced and promising methods of laboratory diagnosis of this disease. The principle of the study is to identify a specific antigen-antibody complex, which is formed by combining syphilitic antigens absorbed on a solid carrier with antibodies from the patient’s blood serum. In modern medical practice, an indirect version of the blood test for syphilis, ELISA, is common, which uses antibodies to human immunoglobulins combined with enzyme markers. Depending on the amount of bound antibodies, the final products of this reaction are stained, the degree of which is used to evaluate the results of the analysis.
• Passive hemagglutination reaction test (RPHA) - during this study, the combination of syphilis pathogens in blood serum with sheep red blood cells processed by special methods is studied. If treponema pallidum is present in the blood serum, agglutination of red blood cells occurs, that is, gluing with subsequent sedimentation. To decipher a blood test for syphilis using the RPHA method, the staining of the sample is assessed, since erythrocyte agglutinate is dark red.
• Immunofluorescence reaction study (RIF) - during this analysis, the intensity of the glow of Treponema pallidum in the preparation is determined, which occurs only if anti-treponema antibodies are present in the blood serum.
• The immobilization test for treponema pallidum (TIRT) is the most specific diagnostic test for syphilis, in which the presence of video-specific antibodies (immobilisins) is determined in the blood serum, which immobilize pallidum treponema in the presence of complement. Under a microscope, the proportion of treponemas that have lost mobility (immobilized) is calculated. This blood test for syphilis is carried out no earlier than 2 weeks after finishing taking antibiotics and other medications.

The RPR test can give a false positive reaction, since antibodies to it can be released not only by syphilis, but also as a result of some other diseases. According to statistics, the Wasserman reaction test gives one false positive result per 10,000 tests. To exclude erroneous diagnosis of syphilis, the result is further clarified using other studies. The most accurate methods are considered to be RIF, RIBT and ELISA, which make it possible to detect syphilis already in the incubation period and at all subsequent stages.

Indications for use

The attending physician prescribes RPGA if there is a possibility of an infectious venereal disease.
In other cases, a doctor may test for antibodies to Treponema pallidum even if there are no symptoms or reason to believe that the disease is present. For example, a test for the presence is given as a routine part of the examination in the case of pregnancy. This is a standard procedure and does not mean that the doctor thinks you have syphilis. Early signs that may occur with this condition include:

• one small painless wound;
• swelling in the lymph nodes near the lesion;
• non-itchy, skin rash.

The study is prescribed when treatment is being carried out for another STI, such as gonorrhea, if you are infected with HIV, or have had sexual intercourse with a high risk of infection. When treatment for syphilis has been given, repeat testing is recommended to ensure that the therapy has worked and the infection has been treated.

How to prepare for the test

The analysis does not require special preparation; all rules correspond to normal venous blood donation.
The patient is required to strictly follow a number of simple rules:

• 72 hours before the procedure, you should follow a diet: you should not eat fatty, fried, salty or spicy foods, alcoholic drinks are also prohibited;
• It is recommended to maintain optimal water balance in the body, and for this you need to drink at least two liters of clean drinking water;
• The venous blood test is carried out on an empty stomach, so at least 10 hours before the procedure you should not eat, drink tea, coffee or other drinks;
• Smoking is prohibited 4 hours before the procedure.

If for health reasons the patient is required to take any pills, it is necessary to notify the attending physician in advance. An hour before handing over the material, it is recommended to avoid physical activity, and before entering the treatment room, you should sit down and rest for 10-15 minutes.

What diseases can be detected during diagnosis?

The Treponema pallidum hemagglutination test (TPHA) is a treponemal test for the serological diagnosis of syphilis, a sexually transmitted infection caused by the spirochete and Treponema pallidum. Based on the principle of passive hemagglutination , the test detects antitreponemal antibodies (IgG and IgM) in serum.

This important diagnostic test is used to detect syphilis. This is a sexually transmitted disease. The disease is characterized by four stages, namely primary, secondary, latent and tertiary. Treatment depends on the stage of infection. It is very important to diagnose the infection as early as possible. This helps in preventing damage to the major organs of the body. Diagnosing its initial symptoms is always difficult, since it only appears in the form of a small, painless sore called a chancre.

Laboratory diagnosis of syphilis

Serological studies are based on immune connections that are observed in blood serum.

Protein compounds merge with antigens, as a result of this interaction, microbial antigens are stopped.

After the introduction of the pathogen (Treponema pallidum), the body begins to produce protective antibodies.

They attack germs and prevent the spread of disease.

For analysis, blood is taken and a suspension of microbes is added to it.

If the reaction does not occur, the person is healthy; if the reaction does occur, the person is sick.

Immunoglobulins for syphilis (immunoglobulins to Treponema pallidum) are protective proteins produced to neutralize it.

They detect infection in the blood and its remains after therapy.

Each of the antibodies to Treponema pallidum has a specific informative status, purpose and configuration.

Immunoglobulins A for syphilis

They control the local immune response - an early defense that responds to the entry of an infectious agent into a certain segment of the body.

They are produced in significant concentrations on the mucous membranes of the intestines, lungs, and oral cavity, and in minor concentrations in the blood.

They are a marker of active infection and are considered nonspecific because they also respond to other bacteria.

Immunoglobulins M for syphilis

Supervise general immunity.

They are characterized by weak specificity.

They promote the evacuation of antigens, prevent their fixation in tissues and the initiation of pathological processes.

Their presence indicates the active stage of the disease; after treatment, these proteins disappear.

Immunoglobulin G for syphilis

The main immunoglobulins of secondary immunity.

Provide a high intensity of the immune response for a long time.

The body is able to produce memory cells of these proteins for several years after taking specialized medications.

Who's at risk

Anyone with signs or symptoms of the disease should be tested for syphilis. Some patients should be screened even if they have no symptoms or know of a sexual partner who is infected. Sexually active people can discuss their risk factors with their doctor and ask about testing for syphilis or other STDs.

Those most at risk include:

• those who have unprotected sex;
• patients with HIV;
• people with multiple sexual partners.

On a note! When a pregnant woman is diagnosed with syphilis, the infection can be passed on to her unborn child. All pregnant women should be tested at their first antenatal visit.

RPGA for syphilis is a method of primary nonspecific laboratory diagnosis. It is the most commonly used screening test worldwide. The test is used because of its simplicity, sensitivity and accessibility. RPGA has quantitative data on antibody titer. This assessment has essential implications for future treatment.

Persons with a positive non-treponemal test, such as RW or the well-known Wassermann reaction, should always undergo a treponemal test to confirm the diagnosis of syphilis. This chain of procedures is considered a classic testing algorithm.

Study of RPGA for the diagnosis of other infections and pathologies

The study of RPHA can be carried out not only to identify the causative agent of syphilis in the body. To diagnose a Salmonella infection, doctors prescribe a Salmonella Diagnostic Test. The sample is sent to a laboratory where bacteria, if present, can be grown. Identification of bacteria in the sample confirms the diagnosis. A negative result indicates the absence of infection. A quantitative titer value from 1:200 to 1:800 indicates an acute phase of infection.

Measuring the patient's serum antibodies to diphtheria toxin before antitoxin administration will help assess the likelihood of diphtheria diagnosis. The method of performing RPGA with a diphtheria marker allows diagnosing the disease. A titer of 1:80 confirms the presence of diphtheria in the body.

Warning! The prozone phenomenon and biological false-positive reaction are two disadvantages of this test.

The doctor may order a RPGA test to check for the presence of the measles virus. A test with a measles marker shows whether the body has antibodies to fight the virus. If sufficient measles antibodies are present, the person is said to have evidence of immunity to measles.

Positive

If the result of the RPGA analysis is positive, then this is not yet a reason to panic. To make an accurate diagnosis, it is necessary to repeat the study and eliminate factors that could give a false positive result.

If the diagnosis is confirmed, the patient is prescribed comprehensive treatment . Most often, the patient is placed in a dermatovenerological clinic, where therapy is combined with monitoring the patient’s condition. The disease is quite serious, so the course of treatment can exceed two years in duration.

Features of the event

Before performing the test procedure, it is necessary to keep the sample, diluent, control and test cells at room temperature (25-30 °C). Each quality test requires a three-well test card. Positive and negative controls should be run with the test serum. With RPGA, red blood cells, the surface of which has adsorbed treponema pallidum antigens, stick to each other, that is, they agglutinated.

A: Serum sample dilution:

1. Add 10 µl of patient serum to the first well A.
2. Then 190 µl of diluent is added.
3. The contents are mixed using a micropipette (the diluted serum is used later).

B: Testing a serum sample for specific antibodies:

1. Add 75 µl of control cells to well B and 75 µl of test cells to well C.
2. Add 25 µl of diluted serum to each well B and C.
3. Gently shake the plate to thoroughly mix the contents.
4. The cells are covered to protect from direct sunlight, heat and any sources of vibration.
5. Incubate for 45-60 minutes at room temperature.
6. Then the results are interpreted.

Preparation and indications for RPGA

A blood test for RPHA is a simple test; a sample of biomaterial is taken by inserting a needle into a vein in the arm. It is later sent to the laboratory for further analysis. In order for the screening results to be reliable, the necessary recommendations should be followed. Most often, analysis results are available within 24 hours; sometimes it may take 2 to 4 days.

Preparing for the study:

1. The test must be taken on an empty stomach (6-8 hours of fasting).
2. It is better to refrain from smoking 30 minutes before the test;
3. You are prohibited from drinking alcohol 24 hours before testing.

Anyone who is concerned they may be infected or have another STI should talk to their doctor and get tested. Early treatment can cure the disease. Many people may not know they are infected. Blood tests are a good idea to detect the disease.

Indications for RPGA:

• after unprotected sex;
• when there are several sexual partners;
• if your sexual partner is diagnosed with a disease;
• with existing symptoms of syphilis.

Note! When the doctor suspects that the syphilis infection has spread to the brain, the doctor may decide to further test the cerebrospinal fluid in addition to the blood.

What can affect the result

Test results can be false positive for many reasons. The results of the analysis are affected by some diseases. These include rickettsial infections such as typhus or Rocky Mountain spotted fever. Results may be inaccurate if screening is done too early after infection. 14–21 days after infection, the body's immune response is detected using a test. Drinking alcohol within 24 hours before the test may also give a false negative result.

Factors influencing the result of the analysis:

• pregnancy;
• intravenous drug use;
• tuberculosis;
• chronic liver diseases;
• recent vaccinations or inflammation of the lining of the heart or valves.

Recommendation! When reading your blood for RPHA, make sure your doctor knows about all the medications, herbs, vitamins and supplements you are taking. This includes drugs that do not require a prescription and any illegal drugs.

Decoding the results

The test value can be deciphered by the intensity of bonding, which can be from – to 4+. Understanding these cases gives a clear idea of ​​the presence of antibodies to the infection.

Laboratory screening can be explained as follows:

1. 4+, if there is a uniform mat of cells covering the entire hole.
2. 3+ if most of the well is covered with a uniform mat of cells.
3. A lower density mat surrounded by a small ring (+2) is another positive analysis scenario.
4. The intensity of the mat is reduced and surrounded by a denser ring with a distinctly open center.

If there is a small, clear center and the cells are located at the bottom, the result can be considered negative. The indicators are within the normal range and do not require further investigation. But in the case of an intermediate positive test, MHA-TP and FTA-ABS studies are prescribed.

Note! Normal ranges may vary slightly between laboratories. Some laboratories use different measurements or test different samples. Talk to your doctor about the meaning of the test results.

Positive result

If the RPGA result is positive, it is likely, but not certain, that the disease is present. In this case, the doctor will prescribe a more specific test to confirm the results. A treponemic test is often used to confirm a positive test. It tests whether the immune system has produced antibodies in response to Treponema pallidum, which causes syphilis.

After making a final diagnosis, it is imperative to undergo full treatment. It is necessary to complete a full course of antibiotic treatment, even if symptoms go away. Failure to do so may cause relapse.

Negative result

A negative test is normal. This means that no syphilis antibodies were detected in the blood sample. Normal ranges may vary slightly between laboratories. Some medical centers use different measurements or test different samples. Talk to your doctor about the meaning of the results.

This test may give a false negative result in early and late stage disease. The test must be confirmed by other screenings to make a definitive diagnosis.

About false positives

Antibodies produced as a result of the infection may remain in the body even after the virus has been treated. In some cases, the body may not produce antibodies even if a person is infected with syphilis. This means that the analysis will be inaccurate.

The following conditions may cause false positive results:

1. HIV.
2. Lyme disease.
3. Malaria.
4. Pneumonia (certain types only).
5. Systemic lupus erythematosus.
6. Intravenous drug use.
7. Tuberculosis.

The RPHA test is not perfect, but it is a reliable test that can be the first step in helping determine whether a person is infected. In a blood test, syphilis can be detected as early as 1-2 weeks after infection. The greatest accuracy can be expected within three months, and non-accurate results are possible any time during the first 90 days after infection.

People who have suffered from a syphilis infection in the past may also have a false positive result due to antibodies lingering in the blood. If RPHA testing returns a negative result 90 days or later after exposure, infection is considered absent.

Note! Other diseases such as yaws and yaws (two other types of skin diseases) can also result in a positive FTA-ABS. Sometimes there can be a false positive result, most often in women with lupus.

What the analysis shows

In most cases, a positive RPGA result indicates congenital or acquired syphilis. Testing can also help detect the following pathologies:

• AIDS virus;
• hepatitis;
• relapsing typhoid fever;
• pulmonary tuberculosis;
• diphtheria;
• whooping cough;
• inflammatory lesions of the mucous membranes of the oral cavity and genitals;
• formation of neoplasms of malignant origin;
• mononucleosis of infectious nature;
• diffuse connective tissue lesions;
• venereal diseases.

Many people are interested in the question of why, if syphilis is suspected, the RPGA analysis is prescribed. The popularity of such testing in diagnosing this dangerous disease is due to its reliability. Doctors claim that the analysis shows the correct result in 99% of cases.

In the absence of Treponema pallidum antibodies, the agglutination reaction will not occur, which indicates that the person is healthy.

It is worth considering that the sensitivity of such a test is very high, so it will be possible to detect the causative agent of syphilis even at the latent stages of the disease.

A sample for such testing is also prescribed if other diseases are suspected, for example, whooping cough or yersiniosis. The demand for the analysis is due to the fact that, compared to other serological tests, it is more sensitive, which increases the likelihood of obtaining a reliable result.