False negative test - indirect Coombs test. Coombs test: direct and indirect

– an antiglobulin test aimed at identifying in Rh-negative blood incomplete anti-erythrocyte antibodies to the Rh factor - a specific protein that is located on the surface of the erythrocytes of Rh-positive blood. There are two types this test: direct – detection of antibodies on the surface of red blood cells, indirect – detection of antibodies in blood serum. Direct testing is carried out in the diagnosis and monitoring of treatment of blood diseases: hemolytic anemia, hemolytic disease newborns and others. An indirect test is performed to assess the compatibility of the blood of the donor and recipient during transfusion, as well as to determine the presence and risk of Rh conflict when planning and managing pregnancy. The material for the Coombs test is venous blood, the study is carried out using methods based on the agglutination reaction. Normally, both tests give negative result. The analysis is completed within one day. In total, 87 addresses were found in Moscow where this analysis could be done.

– an antiglobulin test aimed at identifying in Rh-negative blood incomplete anti-erythrocyte antibodies to the Rh factor - a specific protein that is located on the surface of the erythrocytes of Rh-positive blood. There are two types of this test: direct - detection of antibodies on the surface of red blood cells, indirect - detection of antibodies in blood serum. Direct testing is carried out in the diagnosis and monitoring of treatment of blood diseases: hemolytic anemia, hemolytic disease of newborns and others. An indirect test is performed to assess the compatibility of the blood of the donor and recipient during transfusion, as well as to determine the presence and risk of Rh conflict when planning and managing pregnancy. The material for the Coombs test is venous blood; the study is carried out using methods based on the agglutination reaction. Normally, both tests give a negative result. The analysis is completed within one day.

Coombs test – clinical trial Rh-negative blood, aimed at detecting antibodies to the Rh factor. The test is used to identify the risk of developing Rh conflict and hemolytic reactions. In each person, the surface of red blood cells contains a certain set of antigens or agglutinogens - compounds of different nature, by the presence or absence of which blood type and Rh factor are judged. There are many types of antigens, including medical practice greatest practical significance have agglutinogens A and B, which determine the blood group, and agglutinogen D, which determines the Rh factor. At positive Rh factor D antigens are detected on the outer membrane of erythrocytes, but not if they are negative.

The Coombs test, also called the antiglobulin test, is aimed at detecting incomplete anti-erythrocyte antibodies to the Rh factor system in the blood. Antibodies to the Rh factor - specific immunoglobulins, which are produced in Rh-negative blood when erythrocytes with agglutinogens D enter it. This can occur when the blood of a fetus and a pregnant woman is mixed, during blood transfusions carried out without prior blood typing. The Coombs test exists in two versions - direct and indirect. When performing a direct Coombs test, antibodies attached to the surface of red blood cells are detected. The study is used to determine the cause of the hemolytic reaction. The indirect Coombs test is aimed at detecting anti-erythrocyte antibodies in blood plasma. It is necessary to determine the compatibility of blood between donor and recipient or mother and fetus, and helps prevent the development of Rh conflict and subsequent hemolysis of red blood cells.

Blood for both versions of the Coombs test is taken from a vein. The analysis is performed by agglutination using antiglobulin serum. The results of the study are used in hematology to identify the causes of hemolytic reactions, in surgery and resuscitation when conducting blood transfusions, in obstetrics and gynecology when monitoring pregnancies in women with Rh-negative blood.

Indications

The direct Coombs test, which detects antibodies attached to the surface of red blood cells, is prescribed for hemolytic reactions (destruction of red blood cells) of various origins. The study is indicated for primary autoimmune hemolytic anemia, post-transfusion hemolytic anemia, hemolytic disease of the newborn, hemolysis of erythrocytes caused by autoimmune, tumor or infectious diseases, as well as taking medicines, for example, quinidine, methyldopa, procainamide. The indirect Coombs test, which determines antibodies in blood plasma, is used to prevent the development of Rh conflict. It is indicated for patients in preparation for blood transfusions, as well as for pregnant women with negative Rh factor provided that future father the child has a positive Rh factor.

To determine Rh compatibility, the Coombs test is not prescribed to patients with Rh-positive blood. In these cases, there are already antigens on the surface of red blood cells; the production of antibodies cannot be provoked by blood transfusion or the entry of fetal blood into the bloodstream of the pregnant woman. Also, the study is not indicated for pregnant women if both parents have a negative Rh factor - an inherited recessive trait. A child in such couples always has Rh negative blood, an immunological conflict with the mother is impossible. At hemolytic pathologies The antiglobulin test is not used to monitor the success of therapy, since the results do not reflect the activity of the process of destruction of red blood cells.

A limitation of the Coombs test is the complexity of the research procedure - to obtain reliable results it is necessary to comply with temperature and time conditions, rules for the preparation of reagents and biomaterials. The advantages of the Coombs test include its high sensitivity. In hemolytic anemia, the results of this test remain positive, even if the hemoglobin, bilirubin and reticulocyte levels are normalized.

Preparation for analysis and collection of material

The material used to perform the Coombs test is venous blood. Special requirements There is no time for the blood sampling procedure and for preparing the patient. As with any study, it is recommended to take a break after eating for at least 4 hours, and stop smoking in the last 30 minutes. physical activity, avoid emotional stress. It is also worth discussing with your doctor in advance the need to stop taking medications - some drugs can distort the results of the Coombs test. Blood is taken using a syringe from ulnar vein, less often from a vein on back side brushes Within a few hours, the material is delivered to the laboratory.

When performing a direct Coombs test, antiglobulin serum is added to the patient's blood serum. After some time, the mixture is examined for the presence of agglutinates - they are formed if there are antibodies on the red blood cells. If the result is positive, the agglutinating titer is determined. The indirect Coombs test consists of more stages. First, antibodies present in the serum are fixed on the injected red blood cells during incubation. Then antiglobulin serum is added to the sample, after some time the presence and titer of agglutinates is determined. The analysis period is 1 day.

Normal results

Normally, the result of the direct Coombs test is negative (-). This means that there are no antibodies associated with red blood cells in the blood, and they cannot cause hemolysis. Normal result The indirect Coombs test is also negative (-), that is, there are no antibodies to the Rh factor in the blood plasma. When preparing for blood transfusion for the recipient, this means compatibility with the donor’s blood; when monitoring pregnancy, this means the absence of Rh sensitization of the mother, a low risk of developing an immunological conflict. Physiological factors, such as dietary habits or physical activity, cannot affect the test result. Therefore, if the result is positive, a doctor’s consultation is necessary.

Diagnostic value of the analysis

A positive Coombs test result is expressed qualitatively, from (+) to (++++), or quantitatively, with titers from 1:16 to 1:256. Determination of the concentration of antibodies on red blood cells and in blood serum is performed in both types of samples. If the result of the direct Coombs test is positive, antibodies are detected on the outer membrane of red blood cells, which lead to the destruction of these blood cells. The cause may be blood transfusion without prior typing - post-transfusion hemolytic reaction, as well as erythroblastosis of the newborn, hemolytic reaction due to the use of drugs, primary or secondary autoimmune hemolytic anemia. Secondary destruction of red blood cells can be caused by systemic lupus erythematosus, Evans syndrome, Waldenström macroglobulinemia, paroxysmal cold hemoglobinuria, chronic lymphocytic leukemia, lymphoma, infectious mononucleosis, syphilis, mycoplasma pneumonia.

A positive result of the indirect Coombs test indicates the presence of antibodies to the Rh factor in the plasma. In practice, this means that Rh sensitization has occurred, and there is a possibility of developing Rh conflict after infusion of donor blood during pregnancy. To prevent pregnancy complications, women with a positive Coombs test result are placed on a special register.

Treatment of abnormalities

The Coombs test refers to isoserological studies. Its results make it possible to identify a hemolytic reaction, as well as determine the compatibility of the blood of the donor and recipient, mother and fetus, in order to prevent the development of Rh conflict. If the test result is positive, then you need to seek advice from your attending physician - obstetrician-gynecologist, hematologist, surgeon.

Antibodies, located on the surface of erythrocytes, can be either in a static or free state in blood plasma. Depending on the state of the antibodies, a direct or indirect Coombs reaction is performed. If there is reason to believe that antibodies are fixed on the surface of red blood cells, a direct Coombs test is performed. In this case, the test takes place in one stage - adding antiglobulin serum. If incomplete antibodies are present on the surface of red blood cells, agglutination red blood cells

Indirect reaction

The indirect Coombs reaction occurs in 2 stages. First you need to artificially implement sensitization red blood cells To do this, red blood cells and the blood serum being tested are incubated, which causes antibodies to fixate on the surface of the red blood cells. After which the second stage of the Coombs test is carried out - the addition of antiglobulin serum.

Precipitation reaction - RP (from the Latin praecipilo to precipitate) is the formation and precipitation of a complex of soluble molecular antigen with antibodies in the form of a cloudiness called precipitate. It is formed by mixing antigens and antibodies in equivalent quantities; an excess of one of them reduces the level of formation of the immune complex. The precipitation reaction is carried out in test tubes (ring precipitation reaction), in gels, nutrient media, etc. Varieties of the precipitation reaction in a semi-liquid agar or agarose gel, double immunodiffusion by Ouchterlony, radiap immunodiffusion, immunoepectrophoresis etc.

Ring precipitation reaction. The reaction is carried out in narrow precipitation tubes: a soluble antigen is layered onto the immune serum. With an optimal ratio of antigen and antibodies, an opaque layer is formed at the border of these two solutions. precipitate ring. If boiled and filtered tissue extracts are used as antigens in the reaction, then this reaction is called the first thermoprecipitation reaction (the reaction in which anthrax hapten is detected).
Ouchterlony double immunodiffusion reaction. To set up the reaction, melted agar gel thin layer poured onto a glass plate and after hardening, holes are cut out in it. Antigens and immune sera are placed separately into the wells of the gel, which diffuse towards each other. At the meeting point, in equivalent proportions, they form a precipitate in the form of a white stripe. In multicomponent systems, several lines of precipitate appear between the wells with antigens and antibodies; in identical AGs, the precipitate lines merge; in non-identical AGs they intersect.
Radial immunodiffusion reaction. Immune serum with molten agar gel is poured evenly onto the glass. After solidification in the gel, wells are made into which the antigen is placed in various dilutions. The antigen, diffusing into the gel, forms ring precipitation zones around the wells with antibodies. The diameter of the precipitation ring is proportional to the antigen concentration. The reaction is used to determine immunoglobulins of various classes, components of the complement system, etc. in blood serum.
Immunoelectrophoresis- a combination of the method of electrophoresis and immunoprecipitation: a mixture of antigens is introduced into the wells of the gel and separated in the gel using electrophoresis, then immunoserum is added into the groove parallel to the electrophoresis zones, the antibodies of which diffuse into the gel and form precipitation lines at the site of “meeting” with the antigen.
Flocculation reaction(according to Ramon) (from Latin f1oecus - wool flakes) - the appearance of opalescence or flocculent mass (immunoprecipitation) in a test tube during the reaction toxin - antitoxin or toxoid - antitoxin. It is used to determine the activity of antitoxic serum or toxoid.

HLA typing- study of the human major histocompatibility complex - HLA complex. This formation includes a region of genes on chromosome 6 that encode HLA antigens involved in various immune responses.

Tasks for HLA typing can be very different - biological identification (HLA type is inherited along with parental genes), determination of predisposition to various diseases, selection of donors for organ transplantation - this involves comparing the results of HLA typing of donor and recipient tissues. Using HLA typing, it is determined how similar or different spouses are in terms of histocompatibility antigens in order to diagnose cases of infertility.

HLA typing suggests HLA polymorphism analysis and is carried out by two methods - serological and molecular genetic. The classic serological method of HLA typing is based on a microlymphocytotoxic test, and the molecular method uses PCR (polymerase chain reaction).

Serological HLA typing carried out on isolated cell populations. Major histocompatibility complex antigens are carried mainly by lymphocytes. Therefore, a suspension of T lymphocytes is used as the main carriers of class I antigens, and a suspension of B lymphocytes to determine HLA class II antigens. To isolate the required cell populations from whole blood, either centrifugation or immunomagnetic separation is used. It is believed that the first method can lead to false-positive data, since this results in the death of some cells. The second method is recognized as more specific - more than 95% of the cells remain viable.

But the basis for performing a lymphocytotoxic test HLA typing is a specific serum containing antibodies to various allelic variants of HLA class I and II antigens. A serological test can determine HLA type by examining which sera react with lymphocytes and which do not.

If a reaction occurs between cells and serum, the result is the formation of an antigen-antibody complex on the surface of the cell. After adding a solution containing complement, cell lysis and death occurs. A serological HLA typing test is assessed using fluorescence microscopy to assess positive (red fluorescence) and negative (green fluorescence) reactions, or phase-contrast microscopy to evaluate nuclear staining dead cells. The result of HLA typing is derived taking into account the specificity of the reacted sera and cross-reacting groups of antigens, and the intensity of the cytotoxicity reaction.

Disadvantages of serological HLA typing are the presence of cross-reactions, weak antibody affinity or low expression of HLA antigens, the absence of protein products in a number of HLA genes.

More modern, molecular methods HLA typing they use already standardized synthetic samples that react not with antigens on the surface of leukocytes, but with DNA and directly indicate which antigens are present in the sample. Molecular methods do not require living white blood cells; any human cell can be studied, and a few microliters of blood are enough to work, or you can limit yourself to scraping from the oral mucosa.

Molecular genetic HLA typing uses the PCR method, the first step of which is to obtain pure genomic DNA (from whole blood, leukocyte suspension, tissues).

The DNA sample is then copied - amplified in vitro using primers (short single-stranded DNA) specific to a specific HLA locus. The ends of each primer pair must be strictly complementary to the unique sequence corresponding to a specific allele, otherwise amplification will not occur.

After PCR, during repeated copying, it turns out large number DNA fragments, which can be assessed visually. To do this, reaction mixtures are subjected to electrolysis or hybridization, and whether specific amplification has occurred is determined using a program or table. The result of HLA typing is presented in the form of a comprehensive report at the gene and allelic levels. Due to the standardization of the samples used, molecular HLA typing more precisely serological. In addition, it provides more information (more new DNA alleles) and more high level its detailing, since it makes it possible to identify not only antigens, but also the alleles themselves, which determine which antigen is present on the cell.

Immune lysis reaction. The reaction is based on the ability of specific antibodies to form immune complexes with cells, including erythrocytes and bacteria, which leads to activation of the complement system along the classical pathway and cell lysis. Of the immune lysis reactions, the hemolysis reaction is most often used and the bacteriolysis reaction is rarely used (mainly in the differentiation of cholera and cholera-like vibrios).

Hemolysis reaction. Under the influence of a reaction with antibodies in the presence of complement, a cloudy suspension of red blood cells turns into bright red clear liquid- “lacquer blood” due to the release of hemoglobin. When setting up a diagnostic complement fixation reaction (CFR), the hemolysis reaction is used as an indicator: to test the presence or absence (fixation) of free complement.

Local hemolysis reaction in the gel(Erne reaction) is one of the variants of the hemolysis reaction. It allows you to determine the number of antibody-forming cells. The number of cells secreting antibodies - hemolysins - is determined by the number of hemolysis plaques that appear in an agar gel containing red blood cells, a suspension of cells of the lymphoid tissue being studied and complement.

Immunofluorescent method

(RIF, immunofluorescence reaction) is a method for detecting specific Ags (Abs) using Abs (Ags) conjugated to a fluorochrome. It has high sensitivity and specificity. Used for express diagnosis of infections. diseases (identification of the pathogen in the research material), as well as for the determination of Ab and surface receptors and markers of leukocytes (immunophenotyping) and other cells. Direct I. m. consists of processing a section of tissue or a smear from pathological material or microbial crust containing specific Abs conjugated with fluorochrome; the preparation is washed to free it from unbound Abs and examined under a fluorescence microscope. In positive cases, a glowing light appears around the periphery of the object. immune complex. Control is necessary to exclude nonspecific luminescence. At indirect. I. m. at the first stage, a tissue section or smear is treated with a non-fluorescent specific agent, at the second - with a luminescent agent against -globulins of the animal that was used at the first stage. In the positive case, a luminous complex is formed, consisting of Ar, At to it and At against At (sandwich method). In addition to a fluorescent microscope, RIF is used to take into account the phenotyping of cells. laser cell sorter .

Flow cytometry- a method of optical measurement of the parameters of a cell, its organelles and the processes occurring in it.

The technique involves detecting the scattering of light from a laser beam as a cell passes through it in a stream of liquid, and the degree of light dispersion allows one to get an idea of ​​the size and structure of the cell. In addition, the analysis takes into account the level of fluorescence of chemical compounds that are part of the cell (autofluorescence) or added to the sample before flow cytometry.

The cell suspension, pre-labeled with fluorescent monoclonal antibodies or fluorescent dyes, enters the fluid stream passing through the flow cell. The conditions are selected in such a way that the cells line up one after another due to the so-called. hydrodynamic focusing of the jet in the jet. At the moment a cell crosses the laser beam, detectors record:

light scattering at small angles (from 1° to 10°) ( this characteristic used to determine cell size).

light scattering at an angle of 90° (allows us to judge the nucleus/cytoplasm ratio, as well as the heterogeneity and granularity of cells).

fluorescence intensity through several fluorescence channels (from 2 to 18-20) - allows you to determine the subpopulation composition of the cell suspension, etc.

Apply 1 large drop of serum O(I), A(II), B(III) onto a plate or glass slide using pipettes (different!). After noting the time, use a clean glass rod or a clean corner of a glass slide to combine drops of serum with drops of blood. The determination lasts 5 minutes, shaking the plate, then add 1 drop of saline solution to each mixture of drops and evaluate the results. It is better if the serum comes in 2 different series. The blood group results must match in both serum lots.

Evaluation of isohemagglutination results:

isohemagglutination. If the reaction is positive, tiny red grains of adhesive red blood cells appear in the mixture. The grains merge into larger grains, and the latter into flakes. The serum is almost discolored;

if the reaction is negative, the mixture remains uniformly colored for 5 minutes pink and no grains are found;

When working with 3 sera of groups O(I), A(II), B(III), 4 combinations of reactions are possible:

1. if all 3 sera gave a negative reaction, that is, the mixture is uniformly colored pink - this is O(I) blood type;

   if only serum of group A(II) gave a negative reaction, and sera O(I) and B(III) gave a positive reaction, that is, grains appeared - this is A(II) blood group;

   serum of group B(II) gave a negative reaction, and sera of group O(I) and A(II) gave a positive reaction - this is B(III) blood group.

all 3 sera gave positive reactions - the tested blood was AB(IV) group. In this case, a study is carried out with AB(IV) group serum.

Pay attention! Drops of the blood being tested should be 5-10 times smaller than drops of serum.

Isohemagglutination errors.

Failure to perform agglutination where it should be and presence of agglutination where it should not be. This may be due to a weak serum titer plus poor red blood cell agglutination.

Presence of agglutination where there should not be any- This is pseudoagglutination, when piles of red blood cells form “coin columns”. Shaking the plate or adding saline destroys them.

Panagglutination, when serum sticks together all red blood cells, including those of its own blood type. By the 5th minute, signs of agglutination disappear.

There is also the so-called cold panagglutination, when red blood cells stick together due to low air temperature (below 15 ° C) in the room.

In all these cases, either a repeated reaction is carried out, or using standard red blood cells.

Determination of Rh blood

To determine Rh status, i.e., to detect the presence or absence of Rh system antigens in people’s blood, standard anti-Rh sera (reagents) are used, varying in specificity, i.e., containing antibodies to various antigens of this system. To determine the Rh 0 (D) antigen, anti-Rhesus serum is most often used with the addition of a 10% gelatin solution, or a standard anti-Rhesus reagent prepared in advance with a 33% polyglucin solution is used. To obtain more accurate research results, as well as to identify antigens of other serological systems, the Coombs test is used (it is also very sensitive in determining the compatibility of transfused blood). For research, native blood or blood prepared with some preservative is used. In this case, the blood should be washed from the preservative with a tenfold volume of isotonic sodium chloride solution. When determining Rh status- Rh 0 (D) two samples of serum or anti-Rhesus reagent of two different series should be used and at the same time standard red blood cells obtained from blood from Rh-positive (Rh +) and Rh-negative (Rh -) individuals should be used for control. When determining other isoantigens, control red blood cells that contain or lack the antigen against which the antibodies in the standard serum are directed should be used accordingly.

Partial heat agglutinins are the most common type of antibodies that can cause the development of autoimmune hemolytic anemia. These antibodies belong to IgG, rarely to IgM, IgA.

COOMBS TEST

Coombs test: introduction. The Coombs test is a laboratory diagnostic method based on the hemagglutination reaction.

The main method for diagnosing autoimmune hemolytic anemia is the Coombs test. It is based on the ability of antibodies specific to immunoglobulins (especially IgG) or complement components (especially S3) to agglutinate erythrocytes coated with IgG or S3.

The binding of IgG and C3b to erythrocytes is observed in autoimmune hemolytic anemia and drug-induced immune hemolytic anemia. Direct Coombs test. The direct Coombs test is used to detect antibodies or complement components fixed on the surface of red blood cells. It is carried out as follows:

To obtain antibodies to human immunoglobulins (antiglobulin serum) or complement (anticomplementary serum), the animal is immunized with human serum, immunoglobulins or human complement. The serum obtained from the animal is purified from antibodies to other proteins.

The patient's red blood cells are washed with saline to completely remove serum, which neutralizes antibodies to immunoglobulins and complement and can cause a false negative result.

If antibodies or complement components are fixed on the surface of red blood cells, the addition of antiglobulin or anti-complement serum causes agglutination of red blood cells.

The direct Coombs test is used in the following cases:

Autoimmune hemolysis.

Hemolytic disease of newborns.

Drug-induced immune hemolytic anemia.

Hemolytic transfusion reactions. Indirect Coombs test. The indirect Coombs test detects antibodies to red blood cells in serum. To do this, the patient's serum is incubated with group 0 donor red blood cells, and then a direct Coombs test is performed.

The indirect Coombs test is used in the following cases:

Determination of individual compatibility of donor and recipient blood.

Detection of alloantibodies, including antibodies that cause hemolytic transfusion reactions.

Determination of surface erythrocyte antigens in medical genetics and forensic medicine.

Confirmation of identical twins during bone marrow transplantation.

To conduct a biological test, blood begins to be transfused as quickly as possible (preferably in a stream). After transfusion of 25 ml of blood, the system tube is clamped with a clamp. Then there is a pause for 3 minutes, during which the recipient’s condition is monitored. To perform a biological test, 25 ml of blood is injected three times. At the end of the test (after transfusion of the first 75 ml of blood in fractional doses of 25 ml at intervals of 3 minutes), the system is adjusted to required speed transfusion. When transfusing more than one bottle of blood to a patient, it is necessary to remove the needle from the vein. In this case, the needle is removed from the test tube of the vial in which the blood has run out and inserted into the next vial. The system tube (rubber or plastic) is clamped at this moment with a clamp. If during a blood transfusion it becomes necessary to administer any other drug intravenously to the recipient, this is done by piercing the rubber tube of the system. Punctures of the plastic tube are unacceptable, as they do not fall off. After each blood transfusion, the patient must be monitored to identify and promptly eliminate possible complications, including allergic reactions. 2 hours after the end of the blood transfusion, body temperature should be measured. If it increases, the measurement must be repeated every hour for the next 4 hours. Equally important is monitoring urination and urine composition, which makes it possible to establish the presence of a toxic post-transfusion reaction. The onset of oliguria and anuria after blood transfusion, the presence of blood cells and protein in the urine are a direct indication of the development of post-transfusion hemolysis.

Coombs test– an assay to detect antibodies attached to a surface or dissolved in plasma. It is used to detect immunization and antibodies to red blood cells. Second title antiglobulin test. It can be direct or indirect.

At direct antiglobulin test detects antibodies fixed on the surface of red blood cells. It is carried out in case of suspicion, other autoimmune diseases, after taking medications (methyldopa, penicillin, quinine), etc.

Red blood cells have been sensitized in vivo - antibodies are already firmly attached to them, and the addition of antiglobulin serum (anti-IgG) causes the sensitized cells to stick together, which is visible to the naked eye.

Indirect Coombs test detects anti-erythrocyte antibodies in blood plasma, it is performed before blood transfusion and during.

Anti-erythrocyte antibodies are a type of autoantibodies, i.e. antibodies against your own tissues. Autoantibody occurs during pathological reactivity immune system for some drugs, for example high doses penicillin.

Red blood cells on their surface contain various chemical structures(glycolipids, saccharides, glycoproteins and proteins), in medicine called antigens. A person inherits from his parents a specific map of antigens on each red blood cell.

Antigens are combined into groups and then the blood is divided into several groups - according to the AB0, Rh, Kell, Lewis, Kidd, Duffy system. The most famous and significant in the work of a doctor are AB0 and the Rh factor (Rh).

AB0 system

A person's Rh status is determined by the presence of these antigens. A particularly important antigen of erythrocytes is antigen D. If it is present, then they speak of Rh positive blood RhD, and if it’s not there – oh Rh negative Rhd.

If the corresponding antibody attaches to the erythrocyte antigens, the erythrocyte is destroyed - hemolysis.

Indications

The main indication for directantiglobulin test- suspicion of hemolytic anemia. Most often it is performed for primary autoimmune hemolytic anemia, hemolysis in rheumatic, tumor, infectious diseases, drug-induced hemolysis.

If anemia appears several days or months after a blood transfusion or during long-term jaundice A direct Coombs test is also performed on the newborn.

Indirectantiglobulin test is performed before blood transfusion and during pregnancy of an Rh-negative woman.

Autoimmune hemolytic anemia

Autoimmune hemolytic anemia (primary)– a classic autoimmune disease with unknown causes. The interaction within the immune system is disrupted, which leads to the perception of one’s own red blood cells as foreign. In the lymph nodes, antibodies of the IgG class (react at t 37°C) and/or IgM (at t 40°C) are synthesized, which, when attached to the surface of the erythrocyte, trigger a number of enzymes (the complement system) and “perforate” the wall of the erythrocyte, which leads to its destruction – hemolysis.

The first symptoms are caused by both the destruction of red blood cells and a decrease in hemoglobin. Among them:

• fatigue, general weakness, irritability
• dyspnea
• abdominal and chest pain, nausea
• dark urine color
• back pain
• icteric discoloration of the skin and mucous membranes
• decrease in the number of red blood cells and

Positive result direct Coombs tests 100% confirms the diagnosis of autoimmune hemolytic anemia, proving its autoimmune origin. At the same time, a negative result does not make it possible to remove the diagnosis.

Secondary hemolytic anemia

Secondary autoimmune hemolytic anemia and a positive Coombs test can occur in the following diseases:

• Evans syndrome
• pneumonia infection

A positive antiglobulin test for these diseases is one of the symptoms, and not a criterion for diagnosis.

Hemolytic disease of the newborn

Cause hemolytic disease of newborns - incompatibility of the blood group of mother and fetus, in most cases according to the Rh system, in single cases - according to the AB0 system, casuistically - according to other antigens.

Rh conflict develops if the fetus of a Rh-negative woman inherits Rh-positive blood from the father.

The disease develops in a newborn only if the mother has already developed antibodies to the corresponding antigens, which happens after previous pregnancies, abortions, transfusions incompatible blood. The most common reason for triggering the synthesis of antibodies to antigens of the erythrocyte membrane is childbirth (feto-maternal bleeding). The first birth generally takes place without complications, but subsequent ones are fraught with hemolytic disease of the newborn in the first days after birth.

Symptoms of hemolytic disease of the newborn:

• yellowness of the skin
• , and mucous membranes
• enlarged liver and spleen
• breathing problems
• whole body swelling
• excitation and gradual depression of the central nervous system

Anemia after blood transfusion

Indirect Coombs test carried out before blood transfusion to assess compatibility, and a direct Coombs test - after it if post-transfusion hemolysis is suspected, i.e. in the presence of symptoms such as fever, watering (read below). The purpose of the analysis is to identify antibodies to transfused red blood cells that have bound to the red blood cells of the recipient and are the cause of post-transfusion hemolysis, as well as premature removal of donor red blood cells from the blood circulation of the recipient (the one who received the blood).

Symptoms:

• increase in body temperature
• skin rash
• back pain
• red
• nausea
• dizziness

Decoding

It is worth recalling that the fundamental rules for decoding direct and indirect antiglobulin tests are the same. The only difference is the location of the antibodies - in the blood or on the red blood cell.

• If direct Coombs test is negative– this means that the antibody does not “sit” on the red blood cells and the cause of the symptoms should be sought further and an indirect Coombs test should be performed
• If positive result Coombs test detected after blood transfusion, infections, drugs - positivity lasts up to 3 months (lifetime of red blood cells 120 days - 3 months)
• positive antiglobulin test result with autoimmune disease lasts months and even years

Norm

• direct Coombs test - negative
• indirect Coombs test - negative

A qualitatively positive result is measured in the number of pluses from one to four (+, ++, +++, ++++), and quantitatively in digital form - 1:16, 1:256, etc.

Yes. Your doctor should definitely know that you have received a blood transfusion, since it affects the correct interpretation of test results now. When receiving someone else's (albeit tested many times) blood, there is always the possibility that your body will develop antibodies against the transfused blood. It is these antibodies that will provide negative influence on health status. For subsequent blood transfusions, the doctor must know that you have already received transfusions, which means there has been time for the synthesis of antibodies. For pregnant women, this information is even more relevant.

3. If there is a mismatch in the Rh factor between mother and child, will all children be sick?

Depends on whether the child is Rh positive or negative (RhD). Carriers of blood groups I, II, III and IV can be either Rh positive or negative. In a situation where the mother is Rh negative and the child is Rh positive, antibodies will be produced already with the first pregnancy, but only after the first birth (or termination of pregnancy) will there be direct contact between the blood of the mother and the child. The hemolytic effect of antibodies will be realized only during the second and next birth, which will lead to hemolytic disease in the newborn.

Every woman with a negative Rh factor should be carefully examined during pregnancy, and after childbirth preventive treatment to prevent the appearance of antibodies and further complications.

4. During pregnancy, is it necessary to know my husband’s blood type before performing a Coombs test?

You need to not only know, but also check the blood type of the child’s biological father during pregnancy.

Facts

• first proposed in Cambridge in 1945
• sensitivity threshold - at least 300 fixed antibody molecules on one red blood cell
• the number of antibodies triggering hemolysis - individually for each person (from 16-30 to 300)
• the dynamics of other laboratory indicators of hemolytic anemia (hemoglobin, bilirubin, reticulocytes) may normalize, and the Coombs test will remain at the same level

Coombs test was last modified: March 16th, 2018 by Maria Bodyan

Normally, there are no antibodies to red blood cells in the blood.

The direct Coombs test is an antiglobulin test (agglutination in a gel that allows the detection of complete divalent antibodies), which detects IgG class antibodies and the C3 component of complement on the surface of red blood cells. Typically, antibodies detected by the direct Coombs test have a broad specificity that is not associated with a well-established antigen. A positive direct Coombs test clearly indicates that the patient has hemolytic anemia, although not all patients with a positive direct antiglobulin test suffer from this disease. In approximately 10% of patients, antibodies or complement components on the red blood cell membrane cannot be determined by the direct Coombs test (the test is negative), but nevertheless they suffer from autoimmune hemolytic anemia. To clarify the specificity of antibodies in such cases, tests with their elution are used. A direct Coombs test, positive only for complement, usually refers to cold antibodies of the IgM type. In this case, IgM antibodies are not present on red blood cells when basal temperature bodies. However, due to the fact that IgM antibodies actively fix complement, and complement remains on red blood cells, with this form of autoimmune hemolytic anemia (cold aggutinin disease), the Coombs test will be positive only for complement.

The direct Coombs test is positive for autoimmune hemolytic anemia caused by warm antibodies, autoimmune drug-induced anemia (when taking methyldopa, up to 20% of patients have a positive reaction), drug-adsorption type of hemolytic anemia, immune complex type of hemolytic anemia (the test is positive only for C3), with autoimmune hemolytic anemia caused by cold antibodies - cold agglutinin disease (the test is positive only for C3). In paroxysmal cold hemoglobinuria, the direct Coombs test is negative.

Indirect Coombs test - an indirect antiglobulin test (detects incomplete antibodies) allows you to identify atypical antibodies in the blood, including alloantibodies to foreign erythrocyte antigens. It got its name (indirect) due to the fact that it occurs in 2 stages. Initially, the patient's blood serum, containing incomplete antibodies, interacts with the added corpuscular Ag diagnosticum without visible manifestations. At the second stage, the added antiglobulin serum interacts with incomplete antibodies adsorbed on antigens, with the appearance of a visible precipitate. Transfusion of homologous (allogeneic) red blood cells or pregnancy are the most common reasons formation of these anti-erythrocyte antibodies. The combination of a positive indirect Coombs test with a negative direct test does not provide anything for the diagnosis of autoimmune hemolytic anemia. A positive indirect Coombs test causes certain difficulties when selecting blood for transfusion and conducting a cross-test for compatibility with preserved blood, but has no other diagnostic value.