– 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 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 various natures, the presence or absence of which is used to determine the blood type and Rh factor. There are many types of antigens, including 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 erythrocytes caused by autoimmune, tumor or infectious diseases, as well as reception 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 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 normalized.
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 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 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. Normal result indirect test Coombs 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, 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.
The Coombs test is a method laboratory research, made by influencing hemagglutination. It is based on the susceptibility of antibodies to immunoglobulins and enzyme elements, as well as their ability to agglutinate erythrocytes coated with C3 or Lg.
Direct Coombs diagnosis
Used to detect antibodies or complement components installed on the outside of cells. The direct Coombs test is performed as follows.
The use of such a sample
Direct Coombs diagnosis is used in certain cases, such as:
- transfusion effects;
- autoimmune hemolysis;
- drug-induced hemolytic anemia.
Indirect Coombs test
This diagnosis makes it possible to detect antibodies to cells in serum, which is incubated, as a rule, with donor red blood cells of type 0, and then a direct test is carried out. Apply indirect diagnosis Coombs in the following cases:
How to prepare for analysis
There are some rules for preparing for the examination.
- If the patient is a newborn, parents need to be aware that the test will help diagnose hemolytic disease of the newborn.
- If the patient has suspicions of hemolytic anemia, he should be explained that the analysis will allow him to find out whether it is caused by protective disorders, medications, or other factors.
- The Coombs test, direct and indirect, does not make any restrictions on nutrition or diet.
- It is necessary to notify the patient that the examination will require taking blood from a vein, and also tell him exactly when the venipuncture will be performed.
- You should also be warned about the possibility discomfort during the period of applying the bandage to the arm and the procedure itself.
- Drugs that can affect the sample result should be discontinued.
These medications include:
- "Streptomycin";
- "Methyldopa";
- "Procainamide";
- sulfonamides;
- "Melphalan";
- "Quinidine";
- "Rifampin";
- "Isoniazid";
- cephalosporins;
- "Hydralazine";
- "Chlorpromazine";
- "Levodopa";
- "Tetracycline";
- "Diphenylhydantoin";
- "Ethosuximide";
- "Penicillin";
- mefenamic acid.
Blood sampling is done in the morning on an empty stomach.
How the event is held
The Coombs test is carried out in the following order:
- When performing diagnostics in an adult patient, after venipuncture, blood is taken into tubes with EDTA (ethylenediaminetetraacetate).
- The newborn's blood is drawn from the umbilical cord into a beaker containing EDTA.
- The puncture area is pressed with a cotton swab until bleeding stops.
- If a bruise appears at the venipuncture site, warm compresses are prescribed.
- After blood collection, the patient is allowed to return to taking medications.
- It is necessary to inform the parents of the newborn that secondary analysis may be required to monitor the dynamics of anemia.
Advantages of the Coombs test
Such research has some advantages, namely:
Disadvantages of analysis
The positive Coombs test is a rather labor-intensive examination method, which requires a characteristic accuracy of execution. When using it, you may encounter certain difficulties, especially related to the interpretation of weakly positive effects.
It has been established that erroneous negative or weak positive reactions during the production of Coombs samples can be the consequences of unsatisfactorily active cell washing, weakening of the antiglobulin reagent by serum residues, as well as connections with the non-fat exterior, on which antiglobulin can attach, thereby losing its effectiveness.
The Coombs test has another drawback - the low stability of the antiglobulin reagent, the acquisition and storage of which individual characteristics, which similarly makes it difficult to numerically assess the effect of antiglobulin serum on hemagglutination.
Diseases that can be detected during examination
Coombs diagnostics makes it possible to detect certain types of diseases, such as:
- hemolytic malaise of the newborn;
- various transfusion reactions;
- autoimmune hemolysis;
- drug-induced hemolytic anemia.
Today, the Coombs test is considered a fairly popular blood test system for both adults and newborns. It makes it possible to identify many different diseases.
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:
if all 3 sera gave a negative reaction, that is, the mixture is uniformly colored pink - this is O(I) blood type;
If negative reaction only serum of group A(II) gave, 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.
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 color and no grains are found;
When working with 3 sera of groups O(I), A(II), B(III), 4 combinations of reactions are possible:
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.
Note! 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 the required transfusion rate. 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.
COMBES REACTION(R. R. A. Coombs, English immunologist, born in 1921; synonym: Coombs test, antiglobulin test) - an immunological reaction to detect incomplete antibodies to auto- and isoantigens of erythrocytes.
The reaction was developed in 1908 by S. Moreschi, but received wide application only since 1945 after Coombs demonstrated its role in determining compatibility during blood transfusions, Rh conflict, diagnosis of autoallergic and autoimmune conditions, etc.
The Coombs reaction is based on the use of a specially prepared drug - antiglobulin serum. In the presence of antiglobulin serum, red blood cells loaded with incomplete antibodies agglutinate. Red blood cells that do not contain antibodies on their surface remain non-agglutinated.
K.r. is widely used for: a) establishing the state of isosensitization, i.e., detecting isoantibodies that occur during repeated blood transfusions (see Blood transfusion) or pregnancies (see Pregnancy); b) performing a compatibility test during blood transfusions; c) determination of the type of Rh factor in erythrocytes (see); d) detection of autoimmune antibodies on the red blood cells of patients with acquired hemolytic anemia (see) and other autoallergic diseases (see), as well as in some infections that occur with an allergic component; e) detection of isoimmune antibodies fixed on the red blood cells of children suffering from hemolytic disease of newborns (see). K.r. also used in forensic and anthropological research.
The main material for staging K. r. are serum or citrated plasma and red blood cells of the patient. There are two variants of K. r.: indirect and direct. With indirect K. r. The patient's serum is studied and freely circulating antibodies are determined. With direct K. r. examine red blood cells for the presence of antibodies fixed on these shaped elements blood.
Antiglobulin serum for K. r. obtained by immunization laboratory. animals (rabbits, goats, sheep, etc.) with globulins isolated from human serum by fractionation with ethanol, ammonium sulfate or gel filtration on Sephadex. When obtaining antiglobulin serum, it is necessary to remove heteroagglutinating antibodies resulting from immunization of animals with human globulins. This is achieved by adsorption of immune serum with a mixture of erythrocytes from people with different group blood or diluting it with isotonic sodium chloride solution. In the latter case, the titer of hetero-agglutinins should be low (1: 16 - 1: 32), so that the antiglobulin serum, after dilution, retains good activity in K. r.
Indirect Coombs reaction
The indirect Coombs reaction is carried out in two stages. The first stage is carried out in small test tubes measuring 4 X 0.5 cm specially designed for this purpose. In each test tube, add one drop to three drops of serum (whole, 1: 2, etc.), in which the presence of antibodies is suspected sediment of erythrocytes of known antigenic composition. The contents of the test tube are mixed and placed in a thermostat at t° 37° for 1 hour. Then the red blood cells are washed three times with isotonic sodium chloride solution. The second stage consists of preparing a 5% suspension of washed erythrocytes and combining one drop of erythrocytes with one drop of antiglobulin serum on a white (porcelain) plate with a wetted surface. The results are taken into account up to 10 minutes. Exception false positive results produced by performing control studies. The use of isotonic sodium chloride solution instead of antiglobulin serum should not be accompanied by agglutination of red blood cells. Performing indirect K. r. against erythrocytes of a known isoantigen phenotype allows one to establish the specificity of antibodies. For example, a study of the patient’s serum with red blood cells 0(I), CDE, Kk, Fya; 0(1), CDe, Kk, Fya; 0(I), сDe, Kk, Fya; 0(I), cDE, Kk, Fya; 0(I), cde, kk, Fya showed positive result with a blood sample in cases 1 and 4; other blood samples (cases 2, 3, 5) showed a negative result. The serum contains anti-E antibodies. With the help of indirect K. r. incomplete antibodies against antigens can be detected: C, D, E, c, e; K, k; Fya, Fyb; Lea, Leb; Jka, Jkb, etc. (see Blood groups).
Direct Coombs reaction
The direct Coombs reaction in its technique corresponds to the second stage of indirect K. R.: the patient’s red blood cells (5% suspension), washed three times with isotonic sodium chloride solution, are combined with antiglobulin serum. Direct K.r. performed when there is reason to believe that the red blood cells of the patient being studied are already sensitized with antibodies in vivo. Positive straight line K. r. serves as a diagnostic sign for hemolytic disease of newborns, caused by sensitization of the woman’s body to fetal antigens and the penetration of antibodies through the placenta into the child’s body, as well as acquired hemolytic anemia.
Behind last years K.r. significantly improved. With its help, it is possible not only to ascertain the presence of antibodies on red blood cells, but also to establish the class of immunoglobulins (see). To do this, use serum against certain classes of immunoglobulins: IgG, IgM, IgA. Isoimmune antibodies against Rhesus, Kell antigens, Duffy antigens and other antigens, as well as warm autoimmune antibodies, are usually classified as IgG. Cold autoimmune antibodies, as well as isoimmune antibodies against Le and some other antigens, as a rule, belong to IgM. Only rare autoimmune antibodies are of IgA nature (see Autoantibodies).
Bibliography: Dygin V.P. Autoimmune diseases in the clinic of internal diseases, L., 1970, bibliogr. ; Kassirsky I. A. and Alekseev G. A. Clinical hematology, M., 1970; Kosyakov P. N. Human isoantigens and isoantibodies in health and disease, M., 1974, bibliogr.; Boivin P. e. a. Les anemies hemolytiques, p. 93, P., 1971, bibliogr.; Clinical aspects of immunology, ed. by P. G. H. Geli a. o., Oxford, 1975; Coombs R. R. A., Mourant A. E. a. Race R. R. In-vivo isosensitisation of red cells in babies with haemolytic disease, Lancet, v. 1, p. 264, 1946.
Coombs test is clinical analysis blood test, which is done to detect whether the blood contains certain antibodies that may be unsafe. These antibodies stick to red blood cells and can invade immune system, as well as cause harm in other ways. In medical terminology, this study is also called an antiglobulin test (AGT).
Types of Coombs samples
There are two types of Coombs tests - direct and indirect.
Direct Coombs test, also known as direct (DAT), detects auto-antibodies that attach to the surface of red blood cells. These antibodies are sometimes produced in the body due to certain diseases or when taking certain medications, such as procainamide, methyldopa, or quinidine.
These antibodies are dangerous because they sometimes cause anemia by destroying red blood cells.
This test is sometimes ordered to diagnose the cause of jaundice or anemia.
Normally, the Coombs reaction is negative.
Positive for:
- hemolytic disease of newborns;
- autoimmune hemolysis;
- hemolytic transfusion reactions;
- drug-induced immune hemolytic anemia.
Indirect Coombs test, also known as , is used to detect antibodies to red blood cells found in blood serum (serum is the clear yellow liquid of blood that remains after the red blood cells and coagulant have been eliminated).
The indirect Coombs test is used during blood transfusion to determine whether the donor's blood matches the recipient's blood. This is called a compatibility test and helps prevent any adverse reaction to the donor's blood. This analysis also recommended for pregnant women. Some women have IgG antibodies, which can cross the placenta into the fetal blood and harm the newborn, causing a hemolytic disease known as hemolytic anemia.
Procedure
Blood is taken using a syringe from a vein, usually with back side palm or on the crook of the elbow. Before this, the puncture site is thoroughly disinfected, and after taking a blood test, clean gauze or cotton wool is applied.
The resulting blood is purified in the laboratory and the red blood cells are separated. The sample is then tested sequentially using various serum and Coombs reagents, which are contrasted. If there is no agglutination (clumping of red blood cells), this means a positive result.
However, if the test is negative, it means that there are antibodies in the blood that act against red blood cells. This may indicate various diseases, such as anemia (both natural and caused by taking medications), syphilis, or mycoplasma infection. After receiving the results, the attending physician will prescribe appropriate treatment.
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