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Coombs analysis. Indirect Coombs test (antiglobulin test, detection of incomplete anti-erythrocyte antibodies), blood

Of the many existing antigens in medical practice, the greatest importance is given to three types of blood agglutinogens. One of them is the type responsible for the manifestation of the Rh factor: if it is present on the erythrocyte membrane, the blood group is diagnosed as Rh+, if absent - Rh-. If erythrocytes with Rh+ agglutinogens enter the Rh-negative blood, the body triggers an immune response and begins to produce antibodies to this antigen, which causes pathological conditions.

REFERENCE! The Rh factor is a complex multicomponent system of several dozen antigens. The most common of them are agglutinogens type D (85% of cases), as well as E and C.

The Coombs test is performed only if there is direct evidence. General list of reasons for prescribing the Coombs test:

planning and management of pregnancy (parents have different Rh);
donation and preparation for blood transfusion (blood mismatch according to Rh is no less destructive than mismatch according to the AB0 system);
planned surgical intervention (in case of replenishment of blood loss with blood transfusion);
diagnosis of hemolytic diseases.

More specific indications depend on the type of study being performed.

Direct Coombs test

The direct test detects antibodies on the surface of red blood cells. This is necessary to diagnose existing hemolytic pathologies:

autoimmune (red blood cells and hemoglobin are destroyed as a result of an attack by the body’s own antibodies);
medicinal (the pathological process is triggered by taking certain medications such as quinidine or procainamide);
post-transfusion (if the blood group does not match during transfusion), as well as in the form of Rh conflict during pregnancy (erythroblastosis of newborns).

REFERENCE! Hemolytic anemia is a disease associated with the premature destruction of red blood cells as a result of hemolysis, which leads to insufficient oxygen saturation of the blood and hypoxia of the brain and/or internal organs.

Hemolysis of blood elements is observed in oncological, infectious, and rheumatic diseases, so the direct Coombs test can be used as an additional means of diagnosing a pathological condition. It is worth remembering: a negative test value does not exclude the possibility of hemolysis, but is a reason for additional examination.

Indirect Coombs test

Indirect testing is more often used to prevent pathological situations. It helps to detect antibodies in blood plasma, which is necessary for assessing transfusion compatibility and diagnosing the risks of Rh conflict during pregnancy.

More than 80% of people have a positive Rh factor (Rh+), respectively, just under 20% are Rh negative. If an Rh- mother develops an Rh+ child, her body begins to produce antibodies that attack the fetal red blood cells, causing hemolysis.

Taking into account the fact that the percentage of “different rhesus” marriages reaches 12-15%, the risk of hemolytic disease of newborns should be high, but in reality, only in 1 out of 25 such cases, women experience the phenomenon of sensitization (for every 200 successful births there is 1 example of hemolytic pathology). This is partly due to the fact that the first Rh-positive child usually does not cause open aggression from the mother’s body; The overwhelming majority of cases occur in the second and subsequent children. The same principle applies as with conventional sensitization to a particular allergen.

There is no reaction upon first contact. The body is just getting acquainted with a new antigen, producing antibodies of the IgM class, which are responsible for a rapid immune response, but rarely penetrate the placental barrier into the child’s blood. All pathological reactions manifest themselves upon a second “meeting”, when the body begins to produce IgG class antibodies that easily penetrate the fetal bloodstream, starting the process of hemolysis.

Indirect Coombs test during pregnancy allows you to detect the presence of antibodies in the mother’s body and timely identify the initial stage of sensitization. A positive answer requires registration with a monthly antibody titer test and mandatory hospitalization 3-4 weeks before birth.

REFERENCE! Rh factor incompatibility does not affect the mother's condition in any way; hemolytic disease develops only in the child. In severe cases and in the absence of a timely response, the fetus may die in the womb or immediately after birth.

Preparation for the procedure and its implementation

Venous blood is used for diagnosis. No special long-term preparation is required for the Coombs test. Try to follow a standard set of rules before taking blood from a vein for analysis:

3 days in advance, give up alcohol and medications (if possible);
Plan your last meal later than 8 hours before taking blood for analysis;
within 1 hour, stop smoking, physical, mental and emotional stress;
Before the procedure, drink a glass of clean still water.

The research method is based on the hemagglutination reaction.

When performing a direct test a blood sample is exposed to pre-prepared antiglobulin serum with known indicators, the mixture is kept for some time and checked for agglutinates, which are formed when antibodies are present on red blood cells. The level of agglutinates is diagnosed using the agglutinating titer.

Indirect sample Coombs has a similar technique, but a more complex sequence of actions. Antigenic erythrocytes (with Rh factor) are introduced into the separated blood serum, and only after these manipulations antiglobulin serum is added for diagnosis and agglutinate titer.

Research results

Normally both straight and indirect Coombs test should give a negative result:

a negative direct test indicates that specific antibodies to the Rh factor associated with red blood cells are absent in the blood and cannot cause hemolysis
a negative indirect test shows that there are also no free antibodies to the Rh factor in the blood plasma; this fact indicates the compatibility of the donor’s blood with the recipient’s blood (or the blood of the mother and child) according to the Rh factor.

A positive Coombs test indicates the fact of Rh sensitization of the body, which is the main cause of Rh conflict in the case of blood transfusion or when carrying a child with a different Rh status. In this case, the results remain unchanged for 3 months (the lifespan of red blood cells). If the cause is autoimmune hemolytic anemia, then a positive test can follow the patient for several years (in some cases, throughout his life).

REFERENCE! The antiglobulin test is highly sensitive, but has little information content. It does not record the activity of the hemolytic process, does not determine the type of antibody and is not able to identify the cause of the pathology. To obtain a more complete picture, the attending physician must prescribe additional tests (blood microscopy, general and biochemical analysis, rheumatic tests, ESR, iron and ferritin levels).

The degree of sensitization can be expressed qualitatively (from “+” to “++++”) or quantitatively in the form of a titer:

1:2 - low value, not dangerous;
1:4 - 1:8 - the beginning of the development of an immunological reaction; does not pose a danger, but requires constant monitoring;
1:16 -1:1024 - a strong form of sensitization, immediate action should be taken.

The reason for a positive test may be:

transfusion of untyped blood (or with a typing error), when the Rh factor of the donor and recipient do not match;
Rh conflict during pregnancy (if the composition of blood antigens in the father and mother do not match);
autoimmune hemolytic anemia - both congenital (primary) and secondary, which is a consequence of certain diseases (Evans syndrome, infectious pneumonia, syphilis, cold hemoglobinuria, lymphoma);
drug hemolytic reaction.

None of the above problems can be solved by the patient without medical assistance. In all cases, urgent consultation, registration or emergency hospitalization will be required.

ATTENTION! In rare cases, a false-positive Coombs test is possible. The reason for this can be frequent blood transfusions, as well as a number of diseases: rheumatoid arthritis, lupus erythematosus, sarcoidosis. This phenomenon can also be observed after removal of the spleen, as well as when the reaction is disrupted (frequent shaking of the contents, the presence of contaminants).

Direct Coombs test. This test is used to prove the presence of blocking antibodies fixed in the child’s red blood cells. A positive direct test indicates sensitization and serves as a convincing sign of hemolytic disease of the newborn even before the appearance of other clinical signs. As an exception and only in very severe cases, a direct Coombs test may be negative due to the already occurring, almost complete hemolysis of sensitized red blood cells.

A direct Coombs test is performed as follows: 5 drops of blood taken from the child’s heel are placed in a test tube and 5 ml of saline is added. Stir well and centrifuge for 10 minutes. The clear liquid above the red blood cell sediment is separated. Then add 5 ml of saline again, mix and centrifuge. After mixing with saline three times, the red blood cells are well washed. After the last separation of the supernatant, the erythrocyte sediment in the amount of 0.1 ml is mixed with 0.9 ml of physiological solution. Apply 2-3 drops of this mixture to a glass slide and add one drop of Coombs serum. The presence of agglutination indicates that the reaction is positive (positive direct Coombs test). The study should be carried out at room temperature above 16° to avoid the effect of cold agglutinins.

Indirect Coombs test serves as evidence of the presence of free antibodies in maternal serum and is performed with maternal serum.

Hemolytic disease in a newborn with Rh incompatibility usually manifests itself after the second pregnancy. The first child is born healthy, the second with signs of mild anemia, and only after the third pregnancy are children born with obvious signs of hemolytic disease. Only pre-sensitized women, even during their first pregnancy, can give birth to a child with symptoms of hemolytic disease. In some cases, immunization causes abortions and stillbirths. For the onset and severity of the disease, the condition of the placenta and the duration of exposure of the maternal agglutinins to the fetus are important. When agglutinins appear 10-14 weeks before birth, the child usually experiences subclinical forms. The early appearance of agglutinins, 15-26 weeks before birth, causes severe forms of the disease. In all forms of the disease, the main process is hemolysis. The consequence of the antigen-antibody reaction is hemolysis, damage to the liver and brain capillaries. Depending on which lesion predominates, various forms of the disease are observed. Some anaphylactic phenomena are also dangerous. They lead to the formation of histamine-like substances, causing severe damage to the liver cells and especially to the ganglion cells of the basal ganglia, ammon's horn, medulla oblongata and even the cerebral cortex. When liver cells are damaged, hepatic jaundice is added to extrahepatic jaundice. Children die due to severe symptoms of kernicterus. If they survive, symptoms of damage to the nervous system remain (disorders of the extrapyramidal system with choreoathetotic movements, a peculiar dancing gait, forced movements of the head, sometimes a disorder of coordination of voluntary movements with frequent falls, increased muscle tone, mental retardation, i.e. with signs of such called encephalopathia posticteria infantum).

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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 blood.

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 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 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 decoding 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

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 at basal body temperature. 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 causes of the formation of these anti-red blood cell 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.