Direct Cumbac Sample Methodology. AntiGlobulin test

- Study that helps determine the content of incomplete anti-random antibodies in the blood. Such an anti-globulin test allows you to identify antibodies to pregnant women.

In addition, it allows in the initial stages to diagnose hemolytic anemia in newborns with rhesus conflict. It helps prevent the destruction of the red blood cells needed for normal blood formation. This test was established in 1945 by Robert Kumbs, which is why he received such a name.

Cumbas's test is a versatile study that allows you to diane to diagnose violations in blood formation in both adults and children.

There are the following types of such tests:

1. Direct sample Cumbs- Allows you to define antibodies located on the surface of the red blood cells. Typically, such a study is prescribed in suspected hemolysis, autoimmune hemolytic anemia, other autoimmune diseases. In addition, it is carried out after drug therapy with drugs based on quinine, penicillin or methyldop, or after blood transfusion. For more accurate results, it is necessary to completely abandon the reception of drugs at least for 1 week.
2. Indirect sample Cumbs - A test with which you can identify anti-raspiel plasma antibodies. It is usually carried out during pregnancy and over blood transfusion. Anti-raccitar antibodies appear in human blood in the reactive work of immunity or as a reaction to some drugs. For a more accurate study, several fences are carried out at an interval of 2 hours.

Indications for holding

Cumbac sample is carried out only if there are serious indications. This is expensive and long-term study, which is a specific test.

Typically, the following situations are considered to be indications:

1. When overflowing blood. The test allows you to determine whether the blood of the recipient in the human body takes place, and is it possible to donation. In this case, it is necessary to explore the material both donor and the recipient. It is important to determine the nature of the antibodies, because in case of their incompatibility in the body, an immune system is destroyed against the background of the conflict. This leads to the development of serious diseases, and in rare cases even death.
2. Before surgical interventions, when there is a risk of blood loss. This is done so that the doctor can instantly enter the appropriate blood for the restoration of the body.
3. To detect rhesib-sensitization. Res - a specific antigen, which occurs in the body of each woman during pregnancy. If the mother has a positive reserves, and the father is negative, or vice versa, there is no dependence for a child - it can inherit any. If the child receives the opposite maternal rhesus, the risk of sensitization is great. This phenomenon is characterized by the mixing of the blood of the mother and the child. This can occur both during the tooling of the fetus and in the process of birth.

If a reserves conflict arises in the body of a pregnant woman, then the immunity of the mother begins to perceive his fruit as a foreign body. Because of this, the risk that he will start to attack it.

As a result of such actions, serious pathologies may develop in the kid. Most often, erythroblastosis occurs - a phenomenon in which the child's body cannot produce enough erythrocytes.

In addition, due to the rhesus conflict, the fetus death in the womb or immediately after birth may occur. With the right approach to the treatment of such serious consequences it is easy to avoid.

Deviations from the norm

With a positive result, the Cumbas reaction, the doctor concludes that there are antibodies to red blood cells in serum. This suggests that the blood of the donor cannot be compatible with the patient's blood.

If the positive result is diagnosed in the body of a pregnant woman with rhesse-negative blood, then in its body there are antibodies to the blood of the fruit.

This suggests a rhesus conflict, which requires an extremely attentive approach to making pregnancy by the doctor, as well as the fulfillment of all instructions and recommendations from a woman.

If antibodies are present in the child's blood, hemolytic disease of newborns is diagnosed. In this case, a re-study is carried out, which allows you to determine whether the increase in the zither antibodies in the blood of the future mother or not.

Possible complications from Cumbas test

Cumbas test is a fairly safe study that allows in the initial stages to diagnose a number of autoimmune diseases. It rarely causes complications, usually negative consequences are associated with blood fence.

They conclude:

• Do blood hemorrhage under the skin
• Dizziness and fainting
• Infectious infection

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An anti-globulin test designed to detect incomplete anti-random antibodies was proposed by Cumbsis, Martha, flight in 1945 and received the name of the Cumbas sample. The essence of this method is that antihyglobulin serum containing antibodies to human immunoglobulins, with erythrocytes, sensitized incomplete antibodies, leads to their agglutination.

Depending on whether antibodies are fixed on the surface of the erythrocytes or are in a free state of blood plasma, a direct or indirect Cumbsis test is applied.

The direct Cumbac sample is placed in cases where there is reason to assume that the studied red blood cells are already in vivo. Sensitization with appropriate antibodies, i.e. The first phase of the reaction is fixing the antibodies on the surface of the erythrocytes - occurred in the body and the subsequent addition of antihylobulin serum causes agglutination of sensitized cells.

With the help of indirect Cumbac samples, incomplete antibodies are detected in the serum under study. In this case, the reaction proceeds into two stages. The first stage is the incubation of test erythrocytes with the serum under study, during which fixation occurs on the erythrocyte surface of the antibodies contained in the studied serum sample. The second stage is the addition of antihyglobulin serum.

To date, the Cumbac sample is widely used in laboratory practice for the diagnosis of immunopathological conditions, in particular with autoimmune hemolytic anemia characterized by the destruction of erythrocytes due to the binding of the cell membrane with antibodies and (or) components of the complement system. With it, it is detected by the presence on the erythrocyte membrane Ig G (usually Ig G1 and Ig G3), which can activate the complement, and sometimes complement (C3D). However, in the acute period of the disease due to the destruction of the erythrocytes, which recorded a large amount of antibodies, with a hemolytic crisis, as well as with an insufficient number of antibodies in the chronic course of the disease, a negative direct course of the Cumbess may be marked.

It is necessary to emphasize that the indirect Cumbac sample remains the best method of individual selection of transfusion media, as it allows you to most accurately establish the individual compatibility of the donor and recipient on red blood cell antigens.

Additional execution of a direct antihyglobulin test for the presence of an autoantile agent is recommended for examining all recipients of organs and tissues in the pre-splash period and the recipients of hematopoietic stem cells also after transplantation.

In addition to immunomatology and transfusiology, antihylobulin samples are widely used in the diagnosis of a number of pathological conditions: in hematological diseases, including lymphoproliferative, with systemic diseases of the connective tissue, SHEGREEN disease, chronic active hepatitis, etc.

Cumbac samples are actively used in medical genetics and forensic medicine to determine superficial erythrocyte antigens.

Cumbac sample refers to rather time-consuming research methods requiring special care in fulfillment. When it is used, there are some difficulties associated, in particular, with the interpretation of weakly-positive reactions. It is known that false weakly-bed or negative reactions when performing Cumbac samples may result in insufficiently effective washing of erythrocytes, neutralizing the anti-globulin reagent with sera trails, as well as contact with a poor surface, on which anti-globulin can be fixed, losing its activity. Another lack of Cumbas sample is the instead of an anti-globulin reagent, the receipt and storage of which have certain features, which also makes it difficult to quantify the hemagglutination reaction with anti-globulin serum.

In addition, the studies conducted by A. Holburn, D. Voak et al. It was shown that the cause of false negative results may be excessive shaking during the resuspendence of the erythrocyte suspension. Erroneous results when performing antihyglobulin tests can also be caused by the presence of anti-frequency antibody impurities in the anti-globulin reagent, in particular to C3D-, C3C-, C4C and C4D components of the complement, which are adsorbed on the surface of test erythrocytes during the incubation process and create the visibility of a positive result.

These disadvantages are easily eliminated with a thorough wash of the studied samples and control over the conditions of the reaction.

In the last decade, an isotonic solution with low ionic strengths (LISS) is used to reduce the time of indirect samples of Cumbac and increase its sensitivity (LISS).

The indisputable advantage of anti-globulin samples, according to a number of authors, is their high sensitivity, significantly superior to the resolution of alternative research methods, which are used to detect inflammation antibodies.

We have a comparison of the resolution of methods for studying blood serums for incomplete antibodies using polyglyukin, gelatin and anti-globulin serum. In the course of the study, the titles of incomplete anti-D-antibodies were monitored in 140 samples of blood serum of iswalled donors using gelatin, polyglyukine and indirect anti-globulin tests. The formulation of these methods was carried out in accordance with generally accepted techniques.

It was found that, in its resolution, the methods for the detection of sensitization of erythrocyte anti-D-antibodies are arranged as follows: the most sensitive is the indirect Cumbsis sample, then the gelatin test and the least informative - polyglyukine. The results obtained in this series of experiments fully comply with literary data, which makes it possible to conclude a sufficiently high level of sensitivity of Cumbsy samples, allowing the presence in the body of anti-historic antibodies that do not cause agglutination of red blood cells with a large degree of reliability.

Nevertheless, when setting Cumbac samples in practice, there are cases when incomplete antibodies are not detected, although the clinical picture of the disease or prior immunization indicates their possible presence. In such cases, it can be assumed that the number of antibodies is not enough to precipitate antibodies of anti-globulin serum.

The output is confirmed by its own experiment, in which, using the method of analytic microelectrophoresis of cells, the presence of anti-D-antibodies on test-erythrocytes that are not determined in the indirect sample of the Cumbax was established. In this series of experiments, anti-globulin serum was added to red blood cells, previously incubated with serum obtained from the blood of immunized donors during the antibody period in the course, i.e. In the period when known methods, including Cumbas breakdown, antibodies were not determined.

In conducted proof of the presence of incomplete antibodies on the surface of the erythrocytes, a statistically significant change in the magnitude of the electrophoretic mobility of sensitized red blood cells was served after adding anti-globulin serum. It should be noted that in all immunized donors in the indirect sample of Cumbac in serum, anti-D-antibodies were determined.

Gillerand et al. Also showed that for antihyglobulin tests there is a certain sensitivity threshold: a positive result is noted only when at least 500 IG G G. molecules are recorded on the surface of one erythrocyte

In addition, the literature provides data in favor of the fact that the possible negative result of the Cumbac sample may be associated with low affinity of antibodies, sensitizing erythrocytes, as a result of which they are easily eluted from the surface of red blood cells during the laundering process.

In view of the foregoing, it can be concluded that in some cases the negative result of the Cumbas sample does not yet have proof of the absence of antibodies fixed on the surface of the erythrocytes.

It is known that the Cumbas reactions have high specificity and allow you to detect most of the species of incomplete antibodies. However, as some experimental data show, antihyglobulin samples may be positive and in non-immunological states. E. MUIRHEAD ET AL. On the second day after the introduction of phenylhydrazine, dogs observed a positive Cumbac sample. Such a rapid appearance of a positive reaction testifies to its immunological nature and, rather, due to non-specific protein adsorption on the surface of the erythrocytes.

M. Williams et al. It has established that clavulanic acid may also cause a positive reaction that, according to the authors, is associated with non-specific adsorption of plasma proteins on the erythrocyte surface. The similar effect was observed in the treatment of cephalosporin antibiotics.

The authors of the above studies emphasize the non-immunological nature of the received positive results of the Cumbax samples and insist that these substances are able to cause a modification of the diaphragm of red blood cells, as a result of which the erythrocytes can adsorb proteins (in particular, albumin), in the norm in the blood plasma and not possessing properties of antibodies. In addition, it is possible that precisely xenobiotic, adsorbing on the cell surface, serves as a link between the cell membrane and plasma proteins.

For the correct interpretation of the results of the production of antihyglobulin samples, a quantitative relationship between young and mature red blood cells in peripheral blood should also be taken into account. It was found that the reticulocytes isolated from the body during the reinforced erythron regeneration period can agglutinate anti-globulin serum.

Positive result of direct antihyglobulin dough Options in various pathological conditions, accompanied by impaired immune system, inflammatory processes leading to non-specific adsorption of antibodies of different specificity on erythrocyte membranes. This gives reason to believe that Ig G molecules do not interact with specific anti-hydrocyte antigens, but only fixed on the surface of the cells under study.

It should be borne in mind that when setting the Cumbac sample in cases of diseases characterized by the development of disproteinemia or the appearance of paraproteins, the positive result is due to the presence of proteins on the surface of erythrocytes that do not have antibody properties, which also indicates the insufficient specificity of anti-globulin samples relative to the nature of the protein detected.

Thus, as numerous studies have shown, the positive results of direct and indirect antihylobulin tests are not absolute proof of the presence of antibodies, since positive reactions may be observed in various pathological conditions that are not associated with isosentitibilization or auto-generalsibilization of the body. Therefore, only a comparison of the results of several immunosorological methods with a clinical picture of the disease allows to fully judge the developing pathological process.

A positive indirect anti-globulin test with a negative straight sample usually indicates the presence in the test serum alloant in the free state associated with the preceding blood transfusions or pregnancies.

Cumbas's sample is often positive with the exacerbation of paroxysmal night hemoglobinuria; The positive Cumbas test with anti-C3 and anti-C3dG is a marker of cold agglutinin disease.

In cases where the risk of developing hemolytic diseases of newborns is great, great importance for diagnosis (most often during pregnancy) and, if necessary, the dynamic observation of the appearance and change in the titer of antibodies have the results of direct and indirect anti-globulin tests. Most often, the hemolytic disease of the newborns is associated with the incompatibility of the mother and fetus on the antigen D, less often on the antigens of the AU0 system and even less often - according to other antigens (C, C, K, etc.). Formed with anti la, being, as a rule, incomplete IG G antibodies are clearly detected in an indirect anti-globulin test. With this disease, the established titer and the specificity of the identified antibodies are of great importance, since there is a certain correlation between the level of anti-raspiel antibodies in the blood of a pregnant woman and the possible prognosis of hemolytic disease.

The indirect Cumbas test is also needed in clinical practice in order to ensure safe transfusion therapy. Its implementation is a mandatory component of immunomatological studies of donors and various categories of recipients, as well as planned surveys of all patients of therapeutic and preventive institutions, which may require blood transfusion and its components.

An indirect antihlobulin test applies in the following cases:

For a more accurate establishment of reserves-accessories (antigen D) in fuzzy results of the determination of the rhesv factor by other methods (polyglyukine, gelatin, etc.);

To identify weak erythrocyte antigens (Kell, Duffy, Kidd, Lewis, etc.) and antibodies to these antigens;

To detect and identify alloimmune anti-crochetic antibodies, including antibodies that cause hemolytic post-transfusion reactions;

To determine the presence of immune antibodies of the AU0 system for transfusion hemolytic complications;

As a sample for compatibility with the individual selection of blood flow and its components.

Thus, Cumbac sample is an important diagnostic test used in various fields of medicine (hematology, obstetrics, rheumatology, transfusiology, clinical and laboratory diagnostics, etc.). Knowledge of the features of Cumbac samples will help increase the accuracy of the results obtained and will contribute to the correct interpretation of laboratory data.

Literature

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5. Immunosorology (regulatory documents) / Sost. A.G. Bashlai, S.I. Donskov. - M.: Viniti RAS, 1998.

6. Study of the blood system in clinical practice / ed. G.I. Kozintz, V.A. Makarova. - M.: Triada, 1997.

7. Levin V.I.. To the erythroderosis and erythropoese mechanism during the period of acute postghemorrhagic anemia: author. dis. ... Cand. honey. science - Minsk, 1968.

8. Ragimov A.A., Bayramalibeili I.E. // Fundamentals of diagnosis, prevention and treatment of anemia. - M.: GOU VONMTS MH RF, 2002. - P. 204-209.

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Antihaglobulin test, or Cumbas Reaction It is carried out in order to identify certain antibodies that attack red blood cells (red blood cells).

Antibodies are proteins produced by the immune system. As a rule, antibodies are associated with unauthorized substances, such as bacteria and viruses, and destroy them.

2. Why do you need a test for antibodies?

The antibody test may be carried out in the following cases:

Before blood transfusion

You probably know that a person may have one of the four blood groups. And the anti-globulin test can be carried out to determine the possibility of blood transfusion. If you are doing transfusion, the blood of the donor must fit your type (have the same antigens). If when overflowing antigens differ, the immune system will destroy overfit cells. This may lead to serious illnesses and even death. That is why the search for the right type of blood is such an important factor.

To identify the risk of russia-sensitization

Rus is an antigen. Its full name is a rhesus factor. The Cumbas test is used to detect antibodies to the blood factor in the blood in pregnant women. If a woman with a negative blood factor is pregnant with a child with a positive rear factor (it can be transmitted from his father), there is a risk of rezes-sensitization. Rhow-sensitization occurs when the child's blood is mixed with the blood of the mother during pregnancy or childbirth. If the mother's blood type is incompatible with the child's blood group, its immune system can attack the frozen, perceiving it with an alien object. At the same time, severe disease may develop, referred to as the erythroblastosis of the fetus. In rare cases, if the disease is not treated, the fruit or newborn may die.

A woman with a negative band-factor can be injected with anti-RH-Gammaglobulin (for example, RHOGAM) used to prevent the development of RH-hemolytic disease.

To diagnose autoimmune hemolytic anemia

An autoimmune hemolytic anemia is a rare disease associated with the formation of antibodies to ethitoocyte's own antigens.

3. Types of antihyglobulin sample

There are two types of anti-globulin sample, or the Cumbas reaction: straight and indirect.

Direct Cumbac Reaction, or Direct Anti-Globulin Test Detects antibodies associated with red blood cells. It is used to define anemia. With this disease, red blood cells are destroyed faster than produced.

Indirect Cumbac Reaction, or Indirect Antihlobulin Test It is carried out to search for antibodies that are not related to red blood cells. For sample, blood serum is used, in which antibodies are located. This procedure is quite rare: it is mainly carried out to determine the possibility of blood transfusion or as a stage of examination of pregnant women.

4. Cumbas Reaction Results

Norm:

Negative test result - antibodies are not detected.

• Direct Test Cumbs. The negative result of direct antihyglobulin test means that your blood does not have antibodies associated with red blood cells.
• Indirect Test Cumbs. The negative result of indirect antihylobulin test means that your blood is compatible with the blood of the donor. For a pregnant woman, such a result means that its body did not produce antibodies against the rescue-positive type of blood of his child (the rhesib-sensitization did not happen).

Deviation from the norm:

• Direct Test Cumbs. The positive feedback of the direct antihylobulin test means that your blood has antibodies that fight against red blood cells. This may be caused by the transfusion of incompatible blood or diseases such as hemolytic anemia or hemolytic disease of newborns (GBN).
• Indirect Test Cumbs. The positive result of indirect anti-globulin test means that your blood is incompatible with the blood of the donor. In a pregnant woman, such results mean the presence of antibodies against the positive reserves of the child's blood factor (rezes-sensitization). If the child has a positive band factor, the mother will be carefully observed by the doctor throughout the pregnancy.

Principle of antihyglobulinovoy. Anti-rasty-type anti-racite antibodies and complement molecules (C), on the surface of the erythrocytes are revealed - direct test - their agglutination in contact with animal serum containing antibodies on human anti-globulin (antihyglobulin serum). Incomplete antibodies are detected - an indirect test - by their fixing on the mixture of normal erythrocytes of group 0, all antigens of which belong to the known rhesus system, then agglutinated under the influence of antihlobulin serum.

Materials, Cumbin AntiGlobulin Test Reagents: test tubes for 10/100 ml; Graduated pipettes for 1, 2 ml; Pasteur pipettes; tripods; subject non-polished windows; 8.5 ‰ NaCl solution; Erythrocytes. The erythrocytes of the patient, as well as belonging to the group 0, we obtain from freshly bandaged blood on an antosvetive substance (EDTA solution).

Erythrocytes of group 0 Select with such a calculation so that they proceed from persons in the norm and contained everything antigens Rh Systems. They can be preserved up to 7 days in an autologous plasma, at a temperature of + 4 ° C. In the absence of erythrocytes of group 0, a known antigenic mosaic, a mixture of group grades 0, rhesus-positive and rhesse-negative erythrocytes can be used.

Serum The patient should be fresher.

Antihaglobulin serum produced by the Institute. Dr. I. Cantakuzino, produced in lyophilized form in containing 1 ml ampoules. After dissolving the serum is stored at a temperature of -20 ° C.

AntiGlobulin Test Technique Cumbes:
and) Direct Test Cumbs: The erythrocytes of the patient washed 3 times with 8.5 NaCl solution.
On several subject glasses, apply along a large drop of antihyglobulin serum dilutions, and nearby - in a small drop from the sediment of the patient's erythrocytes; Stir the glass angle drops. The cooked material is left on the table 5 minutes, then explore the presence of agglutinat. In the case of a positive result, determine the maximum agglutinating titer.

b) Indirect Test Cumbs: Erythrocytes of group 0, resuspending and rhesus-negative rinse 3 times with 8.5 ‰ with NaCl solution and expose the serum serum at the rate of 2 erythrocyte drops by 8-10 droplets of serum, then, for 60 minutes, carry out incubation at 37 ° FROM. After that, rinse red blood cells three times and affect them with anti-globulin serum, according to the directions for direct Cumbas test.

When it comes about cold active antibodies Sensibilization of red blood cells of group 0 to spend 60 min. At temperatures + 4 ° C.

Note: 1) Do not conduct a direct Cumbac Test on red blood cells stored one or a few days at a temperature of + 4 ° C or room temperature, since the results may be false positive due to the fixation of the non-optical serum, active in the cold antibodies. 2) In cases of pronounced hyperproteinemia, rinse erythrocytes 4-5 times and check the absence of serum proteins in the last washed fluid, with a sulphosalicylic acid.

Possible residue 2 μg IgG / ml in erythrocyte sediment can neutralize antihyglobulin serum. The Cumbas test can also be carried out using monospecific anti-igg, -igm, -iga -С3 and -С4 serum in order to clarify the species on the surface of erythrocyte deep, for example, in suffering autoimmune hemolytic anemia.

The Cumbas reaction is an effective method for identifying erythrocyte antigens and anti-epic blood cells of human blood in indirect reaction of agglutination. The test of the test is the use of specific monoclonal antigen-based antigen-based immunoglobulins of class G (IgG) with subsequent use at the second stage of the Cumbas anti-globulin serum (AGS) at the second stage.

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

The indirect Cumbac reaction allows you to determine availability:

• antigens of erythrocytes detected by IgG reagents ("incomplete" antibodies, for example, monoclonal anti-D reagents, anti-F y a, anti-f y b);
• anti-random IGG antibodies.

Indirect anti-globulin test: identification of antigens of red blood cells

We present the method of indirect Cumbac reaction on the example of the clerk anti-D igg.

1. Mark the pure test tube: specify the feature of the face under study.
2. Put 2 drops in a test tube (approximately 0.1 ml) of the civocon Anti-D igg.
3. Add 2 drops of 5% of the suspension of pre-washed by the physiological solution of the analyzed erythrocytes. Mix the studied samples with the Cologone.
4. Incubate the mixture on a water bath or in a thermostat at 37 ° C for 30 minutes.
5. Add a Pastern pipette 5 to 10 ml of saline to the test tube.
6. Centrifuge the tube with centrifugal acceleration of 1200 g at 18 - 25 ° C for one minute.
7. Remove physically.
8. Repeat the procedure for washing the erythrocytes using centrifugation 2 more - 3 times.
9. Add 2 drops of antihlobulin serum to the circulation of blood cells, make stirring.
10. Centrifuge the tube with centrifugal acceleration of 1200 g for a minute at a temperature of 18 - 25 ° C.
11. Add 3 to 5 drops of saline using a dispenser or parser pipette.
12. Resuspend the precipitate and visually assess the presence of agglutination. The pronounced agglutinates at the bottom of the test tube against the background of a transparent solution indicate the detection of the erythrocyte antigen. The opaque suspension of blood taurus speaks of the absence of an antigen.

Cumbas Reaction: Screening Isoimmune Anti-Earth Antibodies

The study is carried out as part of the sample for individual compatibility on all anti-genes of blood and recipient blood erythrocytes. The conclusion about the complete compatibility of the serum recipient with the erythrocytes of the donor is based on the lack of hemolysis and / or agglutination at all stages of analysis. Signs of hemolysis and / or agglutination at a stage of testing speak of incompatibility of blood samples.

Assessment of Compatibility on the AU0 system, the detection of "cold" antibodies

1. Prepare a donor blood sample:
   • make a 0.2 ml of blood to the tube of automatic dispenser;
   • 3 times withdrawal erythrocytes in 5.0 ml of saline;
   • resuspend the precipitate in 3-4 ml of a solution with low ionic LISS power.
2. Mark the second clean tube: specify the name of the recipient and the donor name.
3. Make an automatic dispenser 0.1 ml of recipient serum to a marked test tube.
4. Add 2 drops of 5% of the erythrocyte suspension in the LISS solution.
5. Without delay, centrifugize a mixture with centrifugal acceleration of 1200 g at a temperature of 18 - 25 ° C for 15 to 20 seconds.
6. Easy shocking test tubes separate the sediment from the bottom. Evaluate the presence of agglutinat. The presence of hemolysis and / or agglutinatov means:
   • incompatibility on the system AV0;
   • the presence in serum of the patient "cold" antibodies of the IGM or Iga class antibodies, not specific to AV0 antigens.

Identification of "thermal" antibodies

1. In the absence of hemolysis and / or agglutinats, incubate the tube 10 - 15 minutes at 37 ° C.
2. Centrifuge the tube at 1200 g for 15 to 20 seconds at room temperature.
3. Shake the test tube and check the presence of hemolysis and / or agglutinates in the supernatant. A positive result indicates the detection of the patient's "thermal" IgM antibodies against antigen of the erythrocyte donor.

IgG antibody detection in Cumbas test

1. With a negative result, at the last stage of the study, add a 5 ml of 0.9% NaCl solution to the test tube with a Pasteop pipette.
2. Centrifuge the tube at 1200 g for 15 to 20 seconds at an air temperature of 18-25 ° C. Pasteur pipette neatly get rid of the supernatant.
3. Re-follow the laundering of red blood cells 2 more - 3 times.
4. Add 1 to 2 drops of antihlobulin serum to the test tube. Perform a thorough mixing.
5. Centrifuge test tube at 1200 g 15 - 20 seconds.
6. Carefully dismiss the erythrocyte precipitate and conduct a visual assessment of the response result. The identification of agglutination means the presence in serum of patient IgG antibodies against antigen of the donor erythrocytes.