Causes of red blood cell agglutination, its types and antiglobulin test. Cold agglutinin disease (hub)

Blood diseases

Cold agglutinins

Cold agglutinins, or cryoproteins, are antibodies that interact with red blood cell membrane antigens. In this case, the complement system can be activated, causing an autoimmune reaction leading to aggregation and destruction of red blood cells, but only when low temperatures Oh. This pathology occurs mainly in patients over 50 years of age. Antibodies can be represented by IgM, IgG or IgA; in the first case, agglutination is significantly expressed. There are laboratory methods for diagnosing cold agglutinins and determining the temperature at which agglutination begins. Usually the cause of cold agglutinins is an infection, most often viral. Their number may gradually decrease, so if they are detected, surgery under conditions of hypothermia and using cold cardioplegia is best postponed if possible. You can also determine the titers of cold agglutinins when different temperatures blood, and when perfusion, avoid cooling to a temperature at which agglutination may occur. If, however, deeper hypothermia is still necessary, plasmapheresis is required to remove the antibodies contained in the serum. With high titers of cold agglutinins, cooling can lead to serious complications: perioperative myocardial infarction, renal failure, hemolytic anemia, thrombosis. Planning of surgery in such patients should be especially careful, aimed at avoiding cooling the patient. Hemodilution during CPB reduces the risk of agglutination to some extent, but this effect is not predictable enough, so the patient should not be cooled before, during, or after CPB. It is advisable to use a heated water mattress; IR should be performed at normothermia, preheating the solution filling the system. If red blood cells are administered during CPB, they must also be prewarmed. If cold cardioplegia is necessary, it should be crystalloid, not containing blood. In this case, they begin with the introduction of 200 - 300 ml of a warm solution, which washes out the blood from the coronary arteries, then a cold solution is introduced. The temperature of a cold cardioplegic solution is such that it will almost certainly lead to agglutination, so cardioplegia containing blood is administered only warm. Immediately before removing the clamp from the aorta, it is advisable to inject a warm cardioplegic solution so that the blood entering the coronary arteries, did not cool.

Sickle cell anemia

Normally, human erythrocytes contain mainly hemoglobin A, sickle cell anemia is caused by the content of abnormal hemoglobin S in erythrocytes. In patients homozygous for this trait, erythrocytes contain predominantly hemoglobin S, clinically this manifests itself as sickle - cellular anemia. In heterozygous patients, hemoglobin S is less than 45% of the total; they are carriers of the disease gene. In sickle cell anemia, red blood cells have a characteristic sickle or crescent shape, are less mobile, tend to aggregate, and are destroyed more quickly. When oxygen levels are low, sickled red blood cells may precipitate. In patients with sickle cell anemia, in addition to anemia itself, intravascular thrombosis is noted. During a crisis (or crisis - I’m not sure, I don’t remember) occlusion occurs blood vessels accompanied by pain, shortness of breath and convulsions.

Perfusion tactics should be aimed at avoiding a crisis with all its consequences. It is necessary to maintain a high level of oxygen saturation and not cool the patient. In patients with a predominant content of hemoglobin S, the oxygen saturation of erythrocytes should be at least 85%, in patients with a partial content of hemoglobin S - at least 40%. In patients with partial hemoglobin S levels, during CPB it is necessary to maintain a high level of oxygen saturation and avoid acidosis, which contributes to destruction. sickle erythrocytes. By following these rules, complications can usually be avoided. Cooling also contributes to sickle cell dysfunction, so it is best to avoid cooling and use warm or crystalloid cardioplegia in the same way as for cold agglutinins. Hemodilution during perfusion also plays a positive role. To prevent vascular obstruction, vasodilators should be used.

A laboratory test aimed at identifying autoantibodies that cause agglutination and hemolysis of red blood cells at low temperatures.

Synonyms Russian

Cold agglutinins, study of total cold agglutinins.

English synonyms

Cold agglutinins blood test, Cold Autoantibodies, Cold-Reacting Antibodies.

Research method

Agglutination reaction.

What biomaterial can be used for research?

Venous blood.

How to properly prepare for research?

Do not smoke for 30 minutes before the test.

General information about the study

Autoimmune hemolytic anemia (AIHA) occurs as a result of a breakdown due to a number of reasons of immunological tolerance and the production of antibodies against one’s own red blood cells. There are warm and cold autoantibodies. Heat ones bind most effectively to erythrocyte antigens at a temperature of 37 °C, and cold ones - at 4-18 °C. Depending on the effect that autoantibodies have on red blood cells in a test tube, hemolysins (destroy cells) and agglutinins (cause red blood cells to stick together) are released.

Autoimmune hemolytic anemia with cold agglutinins - a relatively rare form of immune hemolytic anemia (according to some data, 20% of all cases of AIHA). It can be either idiopathic (the cause is unknown) or symptomatic. The idiopathic variant is more common in elderly and old age(60-80 years), while symptomatic can occur in childhood and adolescence, complicating the course of mycoplasma pneumonia, infectious mononucleosis, legionellosis, as well as systemic autoimmune diseases(systemic lupus erythematosus, rheumatoid arthritis). In older adults, AIHA with cold agglutinins is often associated with lymphoproliferative diseases such as chronic lymphocytic leukemia and Waldenström's macroglobulinemia.

Cold agglutinins are most often IgM, less often they are represented by a mixture of immunoglobulins different classes. They bind to the red blood cell membrane at low temperatures and attach complement, a family of serum proteins that destroy antibody-labeled cells. After the addition of complement, a membrane-damaging complex is formed on the erythrocyte membrane, the formation of which leads to the formation large quantity pores in the cell membrane, its swelling and destruction.

As a laboratory marker of autoimmune hemolysis caused by cold agglutinins, it is advisable to use the detection of antibodies in the patient’s blood serum that lead to agglutination of red blood cells during incubation at low temperatures.

The modern method of testing for cold agglutinins is the gel agglutination reaction. A suspension of donor red blood cells is added to microtubes containing a neutral gel. Then the test serum is added and incubated at a temperature of 2-8 °C. If there are cold agglutinins in the test serum during incubation at low temperatures, they bind to erythrocytes and cause their agglutination. The test result is assessed after centrifugation, during which agglutinated and non-agglutinated red blood cells are separated. Non-agglutinated erythrocytes have a size comparable to the size of gel particles and freely pass through them under the influence of centrifugal force, forming a compact red sediment at the bottom of the microtube, and agglutinated erythrocytes due to large sizes linger on the surface of the gel or in its thickness.

What is the research used for?

To detect anti-erythrocyte antibodies in the patient’s blood serum that cause hemolysis at low temperatures.

When is the study scheduled?

If autoimmune hemolytic anemia with cold agglutinins is suspected: characteristic feature diseases – poor tolerance cold, when the main symptoms appear.

What do the results mean?

Reference values: not detected.

A negative result - the cells form a compact sediment at the bottom of the microtube - indicates the absence of cold agglutinins in the test serum.
A positive result - agglutinated cells form a red layer on the surface or in the thickness of the gel - indicates the presence of cold antibodies in the test serum.

What can influence the result?

In low titers, cold agglutinins can also be detected in healthy people.

Important Notes

In autoimmune hemolytic anemia with cold agglutinins, autoagglutination of red blood cells occurs at room temperature, which creates problems and leads to incorrect results when determining blood group and calculating parameters peripheral blood(erythrocyte count and erythrocyte indices). This agglutination is reversible at 37 °C, so when the blood is heated to body temperature, problems during research are eliminated.

Red blood cell agglutination is biochemical process aggregation, clumping and precipitation of red blood cells, occurring in vitro or in vivo.

The term "Agglutination" is translated from the Latin "agglutinatio" meaning "sticking". IN biological systems or laboratory analysis This is the gluing and aggregation of organic particles (bacteria, sperm, blood cells) having agglutinogen antigens on their surface when interacting with specific agglutinin antibodies. The resulting agglomerate is called agglutinate.

Even normally, antibodies and antigens may be present in human blood that do not cause adhesion. These are components of the ABO antigen system, which correspond to the blood group, antibodies that appear as an immune response when certain bacteria or other pathogens of infectious diseases (dysentery, typhoid fever) enter the body.

Red blood cell agglutination

The agglutination reaction mechanism can be direct (active) or indirect (passive). The effect of direct agglutination occurs in an organism or sample when the structural membrane antigens of red blood cells begin to interact with the plasma's own antibodies or components of bacterial cells.

Direct agglutination is used when clinical studies, determining blood type or the presence of the Rh factor. The effect of passive adhesion is widely used to make a diagnosis of infectious diseases (bacterial, viral).

Why does red blood cell agglutination occur?

Agglutination of the erythrocyte mass becomes a consequence of the biochemical interaction of antigen molecules localized in the structure of the blood cell membrane with antibodies found in the plasma. This reduces the natural negative charge of red blood cells and brings them closer together. Agglutinin molecules that do not correspond to the blood type can form “bridges” between red blood cells. As a result, a blood clot forms, hemolytic disease develops, and even death.

The adhesion of red blood cells (hemagglutination reaction - HRA) is caused by various factors, which depend on the nature of the agglutinating agent on the surface of the formed element or in the plasma:

Cold agglutinins. They can be detected in the blood during diseases caused by viruses and bacteria, some neoplasms and hypothermia, causing symptoms of intravascular hemolysis. Cold agglutinins can be found in low titers in healthy people without causing noticeable hemolytic manifestations. By chemical nature These are, as a rule, immunoglobulin proteins (most often IgM). They are activated when the temperature drops below 37°C, for example, when blood enters the upper or lower limbs or other areas of the body prone to hypothermia. Cold agglutinins, depending on the type, can exhibit activity and be localized differently: act in a wide or narrow temperature range, when the temperature is restored, remain fixed on the surface of the erythrocyte or be in the plasma.
Erythrocyte antigens. Today, more than 400 antigen systems have been identified, the combination of which is individual for a person. Most of them have weak antigenic properties and do not cause noticeable agglutination of red blood cells. The most critical during blood transfusion are the ABO and Rh systems, incompatibility of which can cause adhesion blood cells followed by transfusion shock.
Hemagglutinogens that determine blood group. In the structure of erythrocyte membranes there are specific markers-antigens of a glycoprotein nature (agglutinogens A and B), and in the plasma there are specific immunoglobulin substances-antibodies (agglutinins alpha and beta). One of four possible combinations of these antigens and antibodies determines the blood type, which is genetically determined and cannot change throughout life. Agglutinogens and agglutinins of the same name cannot be present in the human body at the same time, otherwise red blood cells will stick together, followed by hemolysis. This is one of the genetically developed reactions of the body, aimed at preserving antigenic individuality and basic principle blood transfusions.

Rh antigens . Rh antigens (Rh) are lipoproteins by chemical nature. Antigens of the Rh system are represented by several types (C, E, D), the strongest of them is the D type. People who have such an antigen are called Rh-positive, the rest are called Rh-negative. Normal plasma does not contain antibodies to Rh antigens. They appear due to violation of the rules of blood transfusion and in case of Rh conflict during pregnancy.
Viral and bacterial agglutination of erythrocytes. Aggregation of red blood cells due to certain viral or bacterial diseases may manifest itself due to direct interaction of a virus or bacteria with the surface structural molecules of erythrocytes or due to a reaction with the titer of immunized serum of erythrocytes specifically sensitized with the desired antigen (in vitro). Agglutination of blood cells occurs after the adsorption of the virus on the surface of the red blood cell. In most viruses, hemagglutinin is structural component virion.

Specific markers - antigens of glycoprotein nature (agglutinogens A and B)

Laboratory techniques based on agglutination reactions

Agglutination reactions have diagnostic value. These are serological techniques for detecting and studying antibodies or antigens present in the serum titer of patients based on immunological reactions, identification of antigen markers of bacteria and viruses, determination of the antigenic structure of a microbial pathogen.

Indirect or passive hemagglutination reactions (IPHA or RIHA) are the basis for methods for identifying certain antigens or antibodies in the patient’s blood. This method is used to identify the pathogen infectious disease, qualitative and quantitative determination of gonadotropic hormone in case of suspected pregnancy. The equipment used includes glass slides, sterile test tubes, and plastic plates with well-cells.

The main reagent is the so-called erythrocyte diagnosticum(ED), which can be manufactured according to two principles:

antigenic ED (used more often);
antibody ED.

Depending on the type of diagnosticum, the identified antigen or antibody is adsorbed on the surface of the erythrocyte cell, which, upon subsequent reaction with the corresponding antibodies or antigens of the patient’s blood serum, provokes adhesion shaped elements and the formation of a scallop-like sediment uniformly covering the bottom of the tube or cell. If the sample turns out to be negative, the sediment at the bottom of the test tube will be of a different type.

For RPHA, a diagnostic kit is made from human or animal blood cells (rabbit, sheep, rat, horse), which are treated with formaldehyde or other reagents for preservation. To increase their adsorption capacity, the red blood cells themselves are sensitized special drugs(tannin, chromium chloride, rivanol).

The reverse process is the hemagglutination inhibition reaction

Some viruses (influenza, rubella, measles, adenoviruses, plague cattle) can provoke agglutination of formed elements. Diagnostic techniques for such types are based on reactions that stop this process. viral diseases. Antiviral antibodies in pre-immunized serum counteract viruses, causing them to lose their ability to cause red blood cells to clump together.

Antiglobulin test - Coombs test

The Coombs test is performed to identify incomplete antibodies that are localized in the structure of the erythrocyte membrane and cause agglutination when a special antiglobulin serum is added. There are direct and indirect Coombs reactions. A direct antiglobulin test is performed if there is suspicion of the presence of such incomplete antibodies on the surface of the red blood cell.

Indirect sample Coombs is performed with preliminary sensitization of the erythrocyte with a suitable antibody and subsequent introduction of an antiglobulin component. Performed during diagnosis hemolytic disease autoimmune course or in newborns, to establish a Rh conflict between the mother (Rh-negative) and the child (Rh-positive).

The indirect Coombs reaction is widely used by transfusiologists, as it makes it possible to high accuracy determine compatibility donor material with the recipient's blood based on erythrocyte antigens.

Prescription of immunoglobulin for Rhesus conflict, indications and contraindications

There are two forms of this disease: cold agglutinin disease and paroxysmal cold hemoglobinuria. In both cases, intravascular immune hemolysis can be either primary or secondary. In the latter case, it develops against the background of viral infections (for example, infectious mononucleosis), malaria, mycoplasma pneumonia, hemoblastosis or collagenosis. Autoimmune hemolytic anemia, caused by primary intravascular hemolysis, usually occurs chronically.

3. Cold agglutinin disease, caused by secondary intravascular hemolysis,occurs in many diseases: infections (mycoplasma pneumonia, Infectious mononucleosis, measles, parotitis and others viral infections, trypanosomiasis and malaria), collagenosis (rarely), hemoblastosis (non-Hodgkin's lymphoma, Waldenström's macroglobulinemia, chronic lymphocytic leukemia), Kaposi's sarcoma. It should be emphasized that in these diseases cold agglutinins are detected much more often than hemolysis is observed. In all listed cases First of all, the underlying disease is treated. Otherwise, treatment is the same as for cold agglutinin disease caused by primary intravascular hemolysis.

4. Paroxysmal cold hemoglobinuria - a rare form of autoimmune hemolytic anemia. Hemolysis in this disease occurs as a result of a two-phase reaction involving IgG, which are called Donath-Landsteiner antibodies. In the first phase, IgG binds to red blood cells at low temperatures and fixes complement. In the second phase, at a temperature of 37°C, complement activation occurs, leading to hemolysis. Donath-Landsteiner antibodies are specific to the P-antigen of erythrocytes.

A. Clinical picture.Paroxysmal cold hemoglobinuria often occurs in patients with syphilis, especially congenital, and can complicate viral infections (measles, mumps, chicken pox, infectious mononucleosis, influenza), sometimes it is primary. Intravascular hemolysis develops when the patient warms up after hypothermia. The prognosis is favorable. Patients usually recover on their own. Occasionally, the disease lasts for a long time with periodic hemolytic crises.

b. Laboratory research. Laboratory diagnostics Paroxysmal cold hemoglobinuria is based on the detection of Donath-Landsteiner antibodies. To do this, 1) the patient’s serum is mixed with normal red blood cells of group 0; 2) incubate for 30 minutes at 4°C (to fix antibodies and complement on erythrocytes); 3) incubate for 30 minutes at 37°C (to activate complement). As a negative control, use: 1) a sample with heat-inactivated (to remove complement) patient serum; 2) a sample with the patient’s serum incubated with group 0 red blood cells in the reverse order (first at 37°C, then at 4°C).

V. Treatment. Patients should avoid hypothermia.Corticosteroids and splenectomy are ineffective. If red blood cell transfusion is necessary, it is heated. Be sure to treat the underlying disease.

A cold agglutinin test is a blood test that measures the levels of these antibodies in the body. Cold agglutinins are produced by our body in response to infection. They cause red blood cells to clump together in cold temperatures. In healthy people, as a rule, low level cold antibodies in the blood. But lymphoma and some infections (SARS) increase the level of cold agglutinins.

A little increased level cold agglutinins usually do not cause serious problems. Sometimes cold agglutinins collect red blood cells into groups, which get stuck in the vessels located close to the cooled skin. This may cause the skin to become pale and numb. In advanced cases (with extremely prolonged hypothermia), this can lead to gangrene.

Why measure cold agglutinins?

Cold agglutinin testing is done to:

Find out if they are cold antibodies cause of autoimmune hemolytic anemia;
Diagnose atypical pneumonia.

Our clinic has subject matter experts on this issue.

(4 specialists)

2. How to prepare and how is the analysis carried out?

How to prepare for a cold agglutinin test?

There is no preparation required before the cold antibody test.

How is the cold agglutinin test performed?

The level of cold antibodies is measured after drawing blood from a vein. Blood sampling is carried out according to a standard procedure.

3. What are the risks and what may affect the analysis?

What are the risks of cold agglutinin testing?

Possible risks of a blood test for cold antibodies can only be associated with the blood collection itself. In particular, the appearance of bruises at the puncture site and inflammation of the vein (phlebitis). Warm compresses several times a day will relieve you of phlebitis. If you are taking blood thinning medications, you may bleed at the puncture site.

What can interfere with cold agglutinin testing?

The cold agglutinin test result will be inaccurate if you are taking antibiotics, especially penicillin and cephalosporin.

What's worth knowing?

More than half of people suffering from atypical pneumonia, have high levels of cold antibodies. However, other more reliable tests are often used to diagnose atypical pneumonia.

If bound red blood cells are found during general analysis blood, the doctor may require a cold agglutinin test.

Blood type is more difficult to determine if agglutinin levels are high.

If a person has high content cold agglutinins, and he has not been exposed to low temperatures, then he needs to keep himself warm. High level cold agglutinins can lead to anemia, frostbite or Raynaud's disease.