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The Coombs test is indirect. What is the purpose of the Coombs test? Possible complications from the Coombs test

- a study that helps determine the content of incomplete anti-erythrocyte antibodies in the blood. This antiglobulin test allows you to detect antibodies to in pregnant women.

In addition, it makes it possible to diagnose hemolytic anemia in newborns with Rh conflict at the initial stages. This helps prevent the destruction of red blood cells necessary for normal blood formation. This test was created in 1945 by Robert Coombs, which is why it got its name.

The Coombs test is a versatile test that allows for timely diagnosis of hematopoietic disorders in both adults and children.

There are the following types of such tests:

Direct Coombs test– allows you to determine antibodies located on the surface of red blood cells. Typically, such a study is prescribed for suspected hemolysis, autoimmune hemolytic anemia, or other autoimmune diseases. In addition, it is carried out after drug therapy with drugs based on quinine, penicillin or methyldopa, or after a blood transfusion. To obtain more accurate results, you must completely stop taking medications for at least 1 week before the study.
Indirect Coombs test– a test that can be used to detect anti-erythrocyte antibodies in plasma. It is usually performed during pregnancy and before a blood transfusion. Anti-erythrocyte antibodies appear in a person’s blood during a reactive immune system or as a reaction to certain medications. For a more accurate study, several samplings are carried out at once with an interval of 2 hours.

Indications for use

The Coombs test is performed only if there are serious indications. This is an expensive and time-consuming study, which is a specific test.

Typically, the following situations are considered indications for its implementation:

During blood transfusion. The test allows you to determine whether the recipient's blood will take root in the human body, as well as whether donation is possible. In this case, it is necessary to examine the material from both the donor and the recipient. It is important to determine the nature of the antibodies, because if they are incompatible in the body against the background of Rh conflict, the immune system is destroyed. This leads to the development of serious illnesses, and in rare cases, even death.
Before surgery when there is a risk of blood loss. This is done so that the doctor can immediately introduce suitable blood to restore the body.
To detect Rh sensitization. Rhesus is a specific antigen that appears in the body of every woman during pregnancy. If the mother has positive Rh and the father negative, or vice versa, there is no dependence for the child - he can inherit anyone. If the child receives the opposite rhesus from the mother, there is a high risk of sensitization. This phenomenon is characterized by the mixing of the blood of mother and child. This can happen both during gestation and during birth.

If a Rhesus conflict occurs in a pregnant woman’s body, the mother’s immune system begins to perceive her fetus as a foreign body. Because of this, there is a high risk that he will begin to attack him.

As a result of such actions, the baby may develop serious pathologies. Most often, erythroblastosis occurs - a phenomenon in which the child’s body cannot produce a sufficient number of red blood cells.

In addition, due to Rh conflict, fetal death may occur in the womb or immediately after birth. With the right approach to treatment, such serious consequences can be easily avoided.

Deviations from the norm

If the Coombs test is positive, the doctor concludes that there are antibodies to red blood cells in the blood serum. This means that the donor's blood may not be compatible with the patient's blood.

If a positive result is diagnosed in the body of a pregnant woman with Rh-negative blood, then her body contains antibodies to the blood of the fetus.

This indicates a Rh conflict, which requires an extremely careful approach to pregnancy management on the part of the doctor, as well as compliance with all instructions and recommendations from the woman.

If antibodies are present in the child’s blood, hemolytic disease of the newborn is diagnosed. In this case, a repeat study is carried out to determine whether the increase in antibody levels in the blood of the expectant mother occurs or not.

Possible complications from the Coombs test

The Coombs test is a fairly safe test that allows you to diagnose a number of autoimmune diseases in the early stages. It rarely causes complications; usually negative consequences are associated with blood sampling.

They are:

Bleeding or hemorrhages under the skin
Dizziness and fainting
Infectious infection

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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 the 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 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 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, 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.

Antiglobulin principle. Anti-erythrocyte antibodies of incomplete type and complement molecules (C) located on the surface of erythrocytes are detected - a direct test - by their agglutination in contact with animal serum containing antibodies to human antiglobulin (antiglobulin serum). Incomplete antibodies free in the serum are detected - an indirect test - by attaching them to a mixture of normal group 0 erythrocytes, all antigens of which belong to the known Rh system, and are then agglutinated under the influence of antiglobulin serum.

Materials, reagents for the Coombs antiglobulin test: test tubes 10/100 ml; graduated pipettes 1, 2 ml; Pasteur pipettes; tripods; unground glass slides; 8.5‰ NaCl solution; red blood cells. The patient’s red blood cells, as well as those belonging to group 0, will be obtained from freshly drawn blood using an anti-clotting agent (EDTA solution).

Group 0 red blood cells should be selected in such a way that they come from normal individuals and contain all Rh antigens. They can be stored for up to 7 days in autologous plasma at + 4°C. In the absence of group 0 red blood cells, a known antigenic mosaic, a mixture of group 0 red blood cells, Rh-positive and Rh-negative red blood cells can be used.

Serum the patient must be freshly selected.

Antiglobulin serum produced by the Institute. Dr. I. Cantacuzino, is available in lyophilized form in 1 ml ampoules. After dissolution, store the serum at -20°C.

Coombs antiglobulin test technique:
A) Direct Coombs test: Rinse the patient’s red blood cells 3 times with 8.5‰ NaCl solution.
Apply a large drop of antiglobulin serum dilutions onto several glass slides, and next to them, a small drop from the patient’s erythrocyte sediment; mix the drops with the corner of the glass. Leave the prepared material on the table for 5 minutes, then examine for the presence of agglutinates. If the result is positive, determine the maximum agglutinating titer.

b) Indirect Coombs test: erythrocytes of group 0, Rh-positive and Rh-negative, rinse 3 times with 8.5‰ NaCl solution and expose to the patient’s serum at the rate of 2 drops of erythrocytes per 8-10 drops of serum, then incubate for 60 minutes at a temperature of 37° WITH. After this, wash the red blood cells again three times and treat them with antiglobulin serum, according to the instructions for the direct Coombs test.

When it comes about cold active antibodies sensitize group 0 red blood cells for 60 minutes. at a temperature of + 4°C.

Note: 1) Do not perform a direct Coombs test on red blood cells stored for one or several days at + 4°C or room temperature, since the results may be false positive due to the fixation of incomplete cold-active antibodies present in normal serum. 2) In cases of severe hyperproteinemia, wash the red blood cells 4-5 times and check the absence of serum proteins in the last washing liquid using sulfosalicylic acid.

A possible residue of 2 μg IgG/ml in the erythrocyte sediment may neutralize antiglobulin serum. The Coombs test can also be performed using monospecific anti-IgG, -IgM, -IgA -C3 and -C4 sera in order to clarify the type of cells located on the surface of red blood cells, for example, in patients suffering from autoimmune hemolytic anemia.

– 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.

Indications

Preparation for analysis and collection of material

Normal results

Diagnostic value of the analysis

Treatment of abnormalities

The Coombs reaction is an effective method for detecting erythrocyte antigens and anti-erythrocyte antibodies in human blood in an indirect agglutination reaction. The basis of the test is the use of specific monoclonal reagents based on class G immunoglobulins (IgG) for typing erythrocyte antigens, followed by the use of Coombs antiglobulin serum (AGS) in the second stage.

AGS is produced by mixing serum from the blood of laboratory animals immunized with human immunoglobulins G and monoclonal antibodies to one of the components - C3D complement of human serum. AGS causes agglutination of red blood cells sensitized with specific antibodies. In the absence of antibodies on red blood cells under the influence of AGS, a positive reaction does not occur.

The indirect Coombs reaction allows you to determine the presence of:

erythrocyte antigens detected by IgG reagents (“incomplete” antibodies, for example, monoclonal reagents anti-D, anti-F Y a, anti-F Y b);
anti-erythrocyte antibodies of the IgG class.

Indirect antiglobulin test: detection of red blood cell antigens

We present the method of the indirect Coombs reaction using the example of anti-D IgG coliclone.

Label a clean test tube: indicate the name of the person being tested.
Add 2 drops (approximately 0.1 ml) of anti-D IgG zoliclone to the test tube.
Add 2 drops of a 5% suspension of the analyzed erythrocytes, previously washed with physiological solution. Mix the test samples with zoliclon.
Incubate the mixture in a water bath or in a thermostat at 37 °C for 30 minutes.
Add 5 - 10 ml of saline solution to the test tube using a Pasteur pipette.
Centrifuge the tube with a centrifugal acceleration of 1200 g at 18 - 25 ° C for one minute.
Remove the saline solution.
Repeat the procedure of washing red blood cells using centrifugation another 2 - 3 times.
Add 2 drops of antiglobulin serum to the blood cell sediment and mix.
Centrifuge the tube with centrifugal acceleration of 1200 g for a minute at a temperature of 18 - 25 ° C.
Using a dispenser or Pasteur pipette, add 3 to 5 drops of saline solution.
Resuspend the sediment and visually assess for agglutination. Pronounced agglutinates at the bottom of the tube against the background of a clear solution indicate the detection of erythrocyte antigen. An opaque suspension of blood cells indicates the absence of antigen.

Coombs test: screening for isoimmune anti-erythrocyte antibodies

The study is carried out as part of an individual compatibility test for all red blood cell antigens of the donor and recipient. The conclusion about the complete compatibility of the recipient's serum with the donor's red blood cells is made on the basis of the absence of hemolysis and/or agglutination at all stages of the analysis. Signs of hemolysis and/or agglutination at any stage of testing indicate incompatibility of blood samples.

Compatibility assessment using the AB0 system, detection of “cold” antibodies

Prepare a donor blood sample:
- add 0.2 ml of blood into the test tube using an automatic dispenser;
- Wash red blood cells in 5.0 ml saline 3 times;
- resuspend the pellet in 3 to 4 mL of low ionic strength LISS solution.
Label the second clean tube: indicate the name of the recipient and the name of the donor.
Using an automatic dispenser, add 0.1 ml of the recipient's serum into a labeled tube.
Add 2 drops of 5% red blood cell suspension in LISS solution.
Immediately centrifuge the mixture with a centrifugal acceleration of 1200 g at a temperature of 18 - 25 ° C for 15 - 20 seconds.
By gently shaking the test tube, separate the sediment from the bottom. Assess for the presence of agglutinates. The presence of hemolysis and/or agglutinates means:
- incompatibility according to the AB0 system;
- the presence in the patient’s serum of “cold” antibodies of the IgM or IgA class, not specific to AB0 antigens.

Detection of “warm” antibodies

If there is no hemolysis and/or agglutinates, incubate the tube for 10 - 15 minutes at 37 °C.
Centrifuge the tube at 1200 g for 15 - 20 seconds at room temperature.
Shake the tube and check for hemolysis and/or agglutinates in the supernatant. A positive result indicates the detection of “warm” IgM antibodies against donor erythrocyte antigens in the patient’s serum.

Detection of IgG antibodies in the Coombs test

If the result is negative at the previous stage of the study, add 5 ml of 0.9% NaCl solution to the test tube using a Pasteur pipette.
Centrifuge the tube at 1200 g for 15 - 20 seconds at an air temperature of 18 - 25 °C. Use a Pasteur pipette to carefully discard the supernatant.
Repeat the red blood cell washing 2 - 3 more times.
Add 1 - 2 drops of antiglobulin serum to the test tube. Perform thorough mixing.
Centrifuge the tube at 1200 g for 15 - 20 seconds.
Carefully break up the red blood cell sediment and visually assess the reaction result. Detection of agglutination means the presence in the patient’s serum of IgG antibodies against the donor’s erythrocyte antigens.