What is a direct Coombs test? Method for determining blood groups. Indications for the indirect antiglobulin test

Hemolytic anemia, caused by autoimmune bodies that are directed against their own red blood cells, are not precisely understood. However, it is assumed that some factors (for example, a virus, an abnormal protein) change red blood cells in such a way that the body perceives them “as something foreign” and fights them with the help of antibodies. According to another theory, antibodies directed against red blood cells arise almost accidentally during the formation of abnormal plasma protein bodies in some diseases. Such protein bodies, just as “randomly,” can give reactions that can be used to make a diagnosis (for example, viral pneumonia, as is known, gives a positive Wasserman reaction, a positive Paul-Bunnel reaction and a cold agglutination reaction).

There are two main types of autoantibodies for hemolytic anemia, namely: warm antibodies (react at 37°C) and cold antibodies (the reactivity of which increases as the temperature approaches zero). Warm antibodies are more common than cold antibodies. Dacie found that warm hemolysins occur 2 times more often than cold ones. Hemolysins and agglutinins are not fundamentally different antibodies: they differ only in the nature of their action. Agglutinins agglutinate red blood cells, and hemolysins make them more susceptible to the complex process of hemolysis (complement!). Autoantibodies, fixing on erythrocytes, form an erythrocyte-globin complex. This complex is detected using the Coombs antiglobin test.

Coombs test carried out with Coombs serum, for the preparation of which the rabbit is sensitized with human serum, against which antibodies are formed in the rabbit serum. When such a sensitized serum acts on human erythrocytes, their agglutination occurs if the erythrocyte receptors are occupied by blocking antibodies. Since these blocking antibodies are derived from human serum, they agglutinate with rabbit serum sensitized to human plasma and containing precipitins. This reaction is called the Coombs test; for hemolytic anemia due to autoimmune bodies (Lo tit) it is almost specific (for details, see Maier).

In general for hemolytic anemias with a primary disorder of erythrocytes, the Coombs test is negative, and with acquired ones, it is positive. However, there are some exceptions to this rule: a false-positive Coombs test was found during crises of constitutional hemolytic anemia, and to a weak degree, also sometimes after splenectomy, with rheumatic arthritis, sarcoidosis, after frequent blood transfusions and with systemic lupus erythematosus. Naturally, in acquired hemolytic anemia without the formation of autoimmune bodies, it is negative.

Hemolytic anemia caused by autoimmune bodies can be divided into:
a) acute, subacute and chronic forms, as well as
b) idiopathic with unknown etiology and c) symptomatic [viral pneumonia (cold agglutinins only), chronic lymphatic leukemia, reticulosarcoma, lymphosarcoma, systemic lupus erythematosus (mainly warm, less often cold agglutinins), syphilis (cold agglutinins), ovarian tumors (Miescher with employees)).
c) symptomatic [viral pneumonia (cold agglutinins only), chronic lymphatic leukemia, reticulosarcoma, lymphosarcoma, systemic lupus erythematosus (mainly warm, less often cold agglutinins), syphilis (cold agglutinins), ovarian tumors (Miescher and co-workers)).

Clinic of hemolytic anemia, developing under the influence of autoimmune bodies, is very diverse, and therefore it is hardly possible to draw their general clinical picture. Persons of all ages and both sexes are equally affected. Still, idiopathic forms seem to be more often observed in women (Sacks and Workman).

Clinical picture of the idiopathic form varies depending on the severity of the disease. In chronic cases, the onset is gradual, the disease lasts for many years with frequent exacerbations. The severity of anemia varies depending on the degree of hemolysis. Drops in hemoglobin up to 10% are observed; in other cases, hemoglobin remains at 50-60% for a long time. The intensity of reticulocytosis and icteric coloration of the skin and serum corresponds to the degree of hemolysis. Bilirubin is very rarely found in urine, since it does not pass through the kidneys, but hemoglobinuria is observed. In chronic cases, the spleen is often enlarged and can even reach a very significant size, but in other cases it can still be felt. The liver is rarely not enlarged.

In the blood in most cases macrocytosis is observed, in acute stages there are also many microcytes, normoblastosis and polychromasia are rarely absent, leukocytosis can reach 30,000, platelets are normal. In some cases, however, there is severe thrombocytopenia. Evans explains these cases by the simultaneous presence of antibodies against platelets, so that there is both hemolytic anemia and thrombocytopenia due to the action of autoimmune bodies - Evans syndrome. Osmotic resistance is slightly reduced, but not to the same extent and not as permanently as in constitutional globular cell anemia. Heat resistance test (Hegglin-Maier) after 6 hours can also give slight hemolysis (own observation), but to a lesser extent than for Marchiafava anemia. Hemosiderin is also found in urine (own observation).

Coombs test

Coombs test– 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.

The Coombs test is a clinical test of 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 various natures, the presence or absence of which is used to determine the blood type and Rh factor. There are many types of antigens; in medical practice, agglutinogens A and B, which determine blood group, and agglutinogen D, the Rh factor, are of greatest practical importance. With a positive Rh factor, D antigens are detected on the outer membrane of erythrocytes, but with a negative factor, they are not.

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 are specific immunoglobulins that are produced in Rh-negative blood when red blood cells with agglutinogens D enter it. This can happen when the blood of the fetus and the 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 red blood cells caused by autoimmune, tumor or infectious diseases, as well as taking medications, 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 a negative Rh factor, provided that the future father of 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. The child in such couples always has Rh-negative blood; an immunological conflict with the mother is impossible. In 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 laboriousness 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 biomaterial. 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 normal.

Preparation for analysis and collection of material

The material used to perform the Coombs test is venous blood. There are no special requirements for the time of the blood sampling procedure and for the preparation of the patient. As with any study, it is recommended to take a break after eating for at least 4 hours, and in the last 30 minutes, stop smoking, exercise, and 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 the cubital vein, or less often from a vein on the back of the hand. 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 steps. 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. The normal result of 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 diet 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, by 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 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.

There are a large number of antigens on the surface of red blood cells. Depending on the type of these antigens, blood groups are distinguished; the most studied groups are ABO, Rh, Kell, Duffy and many others...

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Description of the study

Preparing for the study: Blood is drawn from a vein and then serum (blood plasma without fibrinogen) is obtained by natural clotting or by fibrinogen precipitation. Test material: Taking blood

There are a large number of antigens on the surface of red blood cells. Depending on the type of these antigens, blood groups are distinguished; the most studied groups are ABO, Rh, Kell, Duffy and many other systems. Normally, there are antibodies to antigens of another group in the blood, but during blood transfusion, pregnancy, autoimmune diseases, etc. antibodies to their own antigens are detected. Incomplete antibodies to red blood cells

Method

The indirect Coombs reaction is based on the detection of agglutination (clumping) of red blood cells that have incomplete antibodies on the surface, which appears when antiglobulin serum is added.

At the first stage, donor red blood cells (O(I) group, Rh+) ​​and the test serum are added into one test tube. If incomplete antibodies to red blood cells are present in the test serum, then they are fixed on the surface of donor red blood cells.

At the second stage, donor red blood cells with antibodies (if any) and standard antiglobulin serum with antibodies to human immunoglobulins are applied to the glass. If at the first stage, antibodies to red blood cells are fixed on the surface of red blood cells, then when standard serum is added, the red blood cells stick together due to the interaction of antibodies.

Reference values ​​- norm
(Indirect Coombs test (antiglobulin test, detection of incomplete anti-erythrocyte antibodies), blood)

Information regarding the reference values ​​of indicators, as well as the composition of the indicators included in the analysis, may differ slightly depending on the laboratory!

Norm:

Normally, there should be no antibodies to one’s own red blood cells; when the Coombs test is performed, red blood cell aggregation does not occur.

Indications

Study of humoral specific immunity in cases of suspected autoimmune reactions in the body, Rh conflict between mother and fetus, determination of blood compatibility between donor and recipient

Increasing values ​​(positive result)

Antibodies to red blood cells are detected when:

1. Autoimmune hemolytic anemia

2. Hemolytic disease of the newborn

3. Systemic connective tissue diseases

4. Chronic active hepatitis, etc.

Or dissolved in plasma.

Synonyms: antiglobulin test, Coombs test, AGT, indirect/direct antiglobulin test, IAT, NAT, DAT, indirect/direct Coombs test

Coombs test is

an analysis that detects immunization and antibodies to red blood cells.

Types of Coombs test

• direct Coombs test– detects antibodies fixed on the surface of red blood cells. It is carried out if autoimmune hemolytic anemia is suspected, hemolysis in autoimmune diseases, after taking medications (methyldopa, penicillin, quinine), after blood transfusion and hemolytic disease of the newborn.

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, performed before blood transfusion and during pregnancy.

Anti-erythrocyte antibodies are a type of autoantibodies, i.e. antibodies against your own tissues. An autoantibody occurs when the immune system reacts abnormally to certain drugs, such as high doses of 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 next to this the blood is divided into several groups - according to AB0 system, Rhesus, Kell, Lewis, Kidd, Duffy. The most famous and significant in the work of a doctor are AB0 and the Rh factor (Rh).

AB0 system

Rhesus affiliation a person 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 is not present, then they speak of Rh-negative Rhd.

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

Indications for use direct antiglobulin test

• primary autoimmune hemolytic anemia
• hemolytic anemia in autoimmune, tumor, infectious diseases
• drug-induced autoimmune hemolytic anemia
• post-transfusion hemolytic anemia (days - months)
• hemolytic disease of the newborn (incompatibility of one of the blood groups)

Indications for use indirect antiglobulin test

• before blood transfusion
• during pregnancy of an Rh-negative woman

Autoimmune hemolytic anemia

Autoimmune hemolytic anemia (primary)– a classic autoimmune disease with unknown causes. It is assumed that there is a disruption in the interaction process at the level of the immune system, which leads to the perception of one’s own red blood cells as foreign. The cells produce antibodies of the IgG class (react at t 37°C) and/or IgM (at t 40°C), attaching to the surface of the erythrocyte, triggering a number of enzymes (the complement system) and “perforating” the wall of the erythrocyte, which leads to its destruction - hemolysis.

Symptoms of hemolytic anemia

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

Positive result straight 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 autoimmune hemolytic anemia and a positive Coombs test can occur in the following diseases:

• systemic lupus erythematosus
• Evans syndrome
• Waldenström's macroglobulinemia
• paroxysmal cold hemoglobinuria
• chronic lymphocytic leukemia
• lymphomas
• Infectious mononucleosis
• Mycoplasma pneumoniae infection
• syphilis

A positive antiglobulin test for these diseases does not serve as a diagnostic criterion, but is one of the symptoms of the disease.

Hemolytic disease of the newborn

Cause hemolytic disease of newborns - incompatibility of blood groups in 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, childbirth, abortions, and incompatible blood transfusions. 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
• anemia
• pallor 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 direct Coombs test- after it, if post-transfusion hemolysis is suspected, i.e. if you have symptoms such as fever, chills, 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 of a hemolytic reaction after a blood transfusion

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

Decoding the Coombs test

It is worth recalling that the fundamental rules for deciphering direct and indirect antiglobulin tests are the same. The only difference is the location of the antibodies - in the blood or already on the red blood cell - 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 needs to be looked for further and an indirect Coombs test must be performed
• if a positive result of the Coombs test is detected after a blood transfusion, infections, drugs - positivity can last up to 3 months (lifetime of red blood cells is 120 days - 3 months)
• a positive antiglobulin test result for an autoimmune disease can last months or even years

Coombs test standards

• 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.

4 facts about the Coombs test

• 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

Disadvantages of the antiglobulin test

• the strength of binding of an antigen to an antibody cannot be determined
• unknown number of antibodies bound
• does not determine the type of antibody
• a positive Coombs test will not help determine its cause
• 0.5% of healthy people have a positive Coombs test without any signs of hemolytic anemia
• not suitable for monitoring the success of treatment, since it will not indicate the activity of erythrocyte hemolysis

Coombs reaction

Coombs reaction- antiglobulin test to determine incomplete anti-erythrocyte antibodies. The Coombs test is used to detect antibodies to the Rh factor in pregnant women and to determine hemolytic anemia in newborns with Rh incompatibility, which leads to the destruction of red blood cells. This method was proposed by the English physician Robert Coombs in 1945, and therefore was later called the “Coombs reaction.”

Direct Coombs reaction

Antibodies found on the surface of red blood cells can be either static or free in the 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 - antiglobulin serum is added. If incomplete antibodies are present on the surface of red blood cells, red blood cell agglutination occurs.

Indirect reaction

The indirect Coombs reaction occurs in 2 stages. First, it is necessary to artificially sensitize 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.

Notes

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See what the “Coombs reaction” is in other dictionaries:

COMBES REACTION- (Coombs test) a method for determining resusantibodies on the surface of erythrocytes, which cause the precipitation of globulins in the blood serum. This test is used to diagnose hemolytic anemia in infants with Rh incompatibility who... ... Explanatory dictionary of medicine

A method for determining resusantibodies on the surface of erythrocytes, which cause the precipitation of globulins in the blood serum. This test is used to diagnose hemolytic anemia in infants with Rh incompatibility who have... ... Medical terms

- (antiglobulin test) a method for detecting incomplete Abs naturally or artificially associated with cells (usually erythrocytes) using antiglobulin ki (A.s.). A. s., precipitating incomplete Abs, includes carrier cells in this process,... ... Dictionary of microbiology

COMBES REACTION- (named after the British immunologist R. R. A. Coombs, born in 1921, synonym - antiglobulin test) - a serological reaction based on the agglutination of erythrocytes by incomplete auto- and isoantibodies in the presence of antiglobulin serum. At… … Encyclopedic Dictionary of Psychology and Pedagogy

TRANSFUSION HEMOLYTIC REACTION- honey Transfusion hemolytic reaction (THR) is hemolysis of red blood cells of the recipient or donor (rarely), which occurs during transfusion of blood and its components. Reactions can be immune or non-immune in nature. Etiology and pathogenesis Immune reactions... ... Directory of diseases

- (R. R. A. Coombs, born in 1921, English immunologist) serological reaction based on the agglutination of erythrocytes by incomplete auto and isoantibodies in the presence of antiglobulin serum; used in transfusiology, forensic medicine, and also... ... Large medical dictionary

COMBES REACTION- Coombs reaction, a serological reaction that allows the detection of incomplete antibodies fixed on the surface of red blood cells or contained in blood plasma. K.r. is based on the use of antiglobulin serum obtained by hyperimmunization... Veterinary encyclopedic dictionary

COMBES REACTION- Used for the detection of monovalent or so-called partial antibodies, in particular for the analysis of blood groups of pigs and some other animals... Terms and definitions used in breeding, genetics and reproduction of farm animals

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