Coombs test

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Coombs test
MeSH D003298
MedlinePlus 003344

The direct and indirect Coombs tests, also known as antiglobulin test (AGT), are blood tests used in immunohematology. The direct Coombs test detects antibodies that are stuck to the surface of the red blood cells. [1] Since these antibodies sometimes destroy red blood cells they can cause anemia; this test can help clarify the condition. The indirect Coombs test detects antibodies that are floating freely in the blood. [1] These antibodies could act against certain red blood cells; the test can be carried out to diagnose reactions to a blood transfusion. [1]

Contents

The direct Coombs test is used to test for autoimmune hemolytic anemia, a condition where the immune system breaks down red blood cells, leading to anemia. The direct Coombs test is used to detect antibodies or complement proteins attached to the surface of red blood cells. To perform the test, a blood sample is taken and the red blood cells are washed (removing the patient's plasma and unbound antibodies from the red blood cells) and then incubated with anti-human globulin ("Coombs reagent"). If the red cells then agglutinate, the test is positive, a visual indication that antibodies or complement proteins are bound to the surface of red blood cells and may be causing destruction of those cells.

The indirect Coombs test is used in prenatal testing of pregnant women and in testing prior to a blood transfusion. The test detects antibodies against foreign red blood cells. In this case, serum is extracted from a blood sample taken from the patient. The serum is incubated with foreign red blood cells of known antigenicity. Finally, anti-human globulin is added. If agglutination occurs, the indirect Coombs test is positive. [2]

Mechanism

Schematic showing the direct and indirect Coombs tests Coombs test schematic.png
Schematic showing the direct and indirect Coombs tests

The two Coombs tests are based on anti-human antibodies binding to human antibodies, commonly IgG or IgM. These anti-human antibodies are produced by plasma cells of non-human animals after immunizing them with human plasma. Additionally, these anti-human antibodies will also bind to human antibodies that may be fixed onto antigens on the surface of red blood cells (RBCs). In the appropriate test tube conditions, this can lead to agglutination of RBCs and allowing for visualisation of the resulting clumps of RBCs. If clumping is seen, the Coombs test is positive; if not, the Coombs test is negative. [3]

Common clinical uses of the Coombs test include the preparation of blood for transfusion in cross-matching, atypical antibodies in the blood plasma of pregnant women as part of antenatal care, and detection of antibodies for the diagnosis of immune-mediated hemolytic anemias. [4]

Coombs tests are performed using RBCs or serum (direct or indirect, respectively) from venous whole blood samples which are taken from patients by venipuncture. The venous blood is taken to a laboratory (or blood bank), where trained scientific technical staff do the Coombs tests. The clinical significance of the result is assessed by the physician who requested the Coombs test, perhaps with assistance from a laboratory-based hematologist.[ citation needed ]

Direct Coombs test

The direct Coombs test, also referred to as the direct antiglobulin test (DAT), is used to detect if antibodies or complement system factors have bound to RBCs surface antigens. [5] The DAT is not required for pre-transfusion testing [6] but may be carried out by some laboratories.[ clarification needed ] Before transfusion, an indirect Coombs test is often done.

Uses

Algorithm for the main diagnoses in a positive DAT Algorithm in positive direct antiglobulin test (DAT, or direct Coombs test).png
Algorithm for the main diagnoses in a positive DAT

The direct Coombs test is used clinically when immune-mediated hemolytic anemia (antibody-mediated destruction of RBCs) is suspected. A positive Coombs test indicates that an immune mechanism is attacking the patient's RBCs. This mechanism could be autoimmunity, alloimmunity or a drug-induced immune-mediated mechanism. [3]

Examples of alloimmune hemolysis

Examples of autoimmune hemolysis/immunohemolytic hemolysis

Drug-induced immune-mediated hemolysis

  • Methyldopa (IgG mediated type II hypersensitivity)
  • Penicillin (high dose)
  • Quinidine (IgM mediated activation of classical complement pathway and Membrane attack complex, MAC)

(A memory device to remember that the DAT tests the RBCs and is used to test infants for haemolytic disease of the newborn is: Rh Disease; R = RBCs, D = DAT.)

Laboratory

The patient's RBCs are washed (removing the patient's own serum) and then centrifuged with antihuman globulin (also known as Coombs reagent). If immunoglobulin or complement factors have been fixed on to the RBC surface in-vitro, the antihuman globulin will agglutinate the RBCs and the direct Coombs test will be positive. (A visual representation of a positive direct Coombs test is shown in the upper half of the schematic).[ citation needed ]

Indirect Coombs test

The indirect Coombs test, also referred to as the indirect antiglobulin test (IAT), is used to detect in-vitro antibody-antigen reactions. It is used to detect very low concentrations of antibodies present in a patient's plasma/serum prior to a blood transfusion. In antenatal care, the IAT is used to screen pregnant women for antibodies that may cause hemolytic disease of the newborn. The IAT can also be used for compatibility testing, antibody identification, RBC phenotyping, and titration studies.[ citation needed ]

Uses

Blood transfusion preparation

The indirect Coombs test is used to screen for antibodies in the preparation of blood for blood transfusion. The donor's and recipient's blood must be ABO and Rh D compatible. Donor blood for transfusion is also screened for infections in separate processes.[ citation needed ]

  • Antibody screening

A blood sample from the recipient and a blood sample from every unit of donor blood are screened for antibodies with the indirect Coombs test. Each sample is incubated against a wide range of RBCs that together exhibit a full range of surface antigens (i.e. blood types).

  • Cross matching

The indirect Coombs test is used to test a sample of the recipient's serum for antibodies against a sample of the blood donor's RBCs. This is sometimes called cross-matching blood.

Antenatal antibody screening

The indirect Coombs test is used to screen pregnant women for IgG antibodies that are likely to pass through the placenta into the fetal blood and cause haemolytic disease of the newborn.[ citation needed ]

Laboratory method

The IAT is a two-stage test. (A cross match is shown visually in the lower half of the schematic as an example of an indirect Coombs test).[ citation needed ]

First stage

Nonpatient, washed red blood cells (RBCs) with known antigens are incubated with patient serum containing unknown antibody content. If the serum contains antibodies to antigens on the RBC surface, the antibodies will bind to the surface of the RBCs.[ citation needed ]

Second stage

The RBCs are washed three or four times with isotonic saline solution and then incubated with antihuman globulin. If antibodies have bound to RBC surface antigens in the first stage, RBCs will agglutinate when incubated with the antihuman globulin (also known Coombs reagent) in this stage, and the indirect Coombs test will be positive.

Titrations

By diluting a serum containing antibodies the quantity of the antibody in the serum can be gauged. This is done by performing serial dilutions of the serum and finding the maximum dilution of test serum that is able to produce agglutination of relevant RBCs.[ citation needed ]

Coombs reagent

Coombs reagent (also known as Coombs antiglobulin or antihuman globulin) is used in both the direct Coombs test and the indirect Coombs test. Coombs reagent is antihuman globulin. It is made by injecting human globulin into animals, which produce polyclonal antibodies specific for human immunoglobulins and human complement system factors. More specific Coombs reagents or monoclonal antibodies can be used.[ citation needed ]

Enhancement media

Both IgM and IgG antibodies bind strongly with their complementary antigens. IgG antibodies are most reactive at 37°C. IgM antibodies are easily detected in saline at room temperature as IgM antibodies are able to bridge between RBC's owing to their large size, efficiently creating what is seen as agglutination. IgG antibodies are smaller and require assistance to bridge well enough to form a visual agglutination reaction. Reagents used to enhance IgG detection are referred to as potentiators. RBCs have a net negative charge called zeta potential which causes them to have a natural repulsion for one another. Potentiators reduce the zeta potential of RBC membranes. Common potentiators include low ionic strength solution (LISS), albumin, polyethylene glycol (PEG), and proteolytic enzymes.

History

The Coombs test was first described in 1945 by Cambridge immunologists Robin Coombs (after whom it is named), Arthur Mourant and Rob Race. [8] Historically, it was done in test tubes. Today, it is commonly done using automated solid phase or gel technology.

Related Research Articles

<span class="mw-page-title-main">Blood type</span> Classification of blood based on antibodies and antigens on red blood cell surfaces

A blood type is a classification of blood, based on the presence and absence of antibodies and inherited antigenic substances on the surface of red blood cells (RBCs). These antigens may be proteins, carbohydrates, glycoproteins, or glycolipids, depending on the blood group system. Some of these antigens are also present on the surface of other types of cells of various tissues. Several of these red blood cell surface antigens can stem from one allele and collectively form a blood group system.

Rh disease is a type of hemolytic disease of the fetus and newborn (HDFN). HDFN due to anti-D antibodies is the proper and currently used name for this disease as the Rh blood group system actually has more than 50 antigens and not only the D-antigen. The term "Rh Disease" is commonly used to refer to HDFN due to anti-D antibodies, and prior to the discovery of anti-Rho(D) immune globulin, it was the most common type of HDFN. The disease ranges from mild to severe, and occurs in the second or subsequent pregnancies of Rh-D negative women when the biologic father is Rh-D positive.

Serology is the scientific study of serum and other body fluids. In practice, the term usually refers to the diagnostic identification of antibodies in the serum. Such antibodies are typically formed in response to an infection, against other foreign proteins, or to one's own proteins. In either case, the procedure is simple.

<span class="mw-page-title-main">Hemolytic disease of the newborn</span> Fetal and neonatal alloimmune blood condition

Hemolytic disease of the newborn, also known as hemolytic disease of the fetus and newborn, HDN, HDFN, or erythroblastosis fetalis, is an alloimmune condition that develops in a fetus at or around birth, when the IgG molecules produced by the mother pass through the placenta. Among these antibodies are some which attack antigens on the red blood cells in the fetal circulation, breaking down and destroying the cells. The fetus can develop reticulocytosis and anemia. The intensity of this fetal disease ranges from mild to very severe, and fetal death from heart failure can occur. When the disease is moderate or severe, many erythroblasts are present in the fetal blood, earning these forms of the disease the name erythroblastosis fetalis.

<span class="mw-page-title-main">Cross-matching</span> Testing before a blood transfusion

Cross-matching or crossmatching is a test performed before a blood transfusion as part of blood compatibility testing. Normally, this involves adding the recipient's blood plasma to a sample of the donor's red blood cells. If the blood is incompatible, the antibodies in the recipient's plasma will bind to antigens on the donor red blood cells. This antibody-antigen reaction can be detected through visible clumping or destruction of the red blood cells, or by reaction with anti-human globulin. Along with blood typing of the donor and recipient and screening for unexpected blood group antibodies, cross-matching is one of a series of steps in pre-transfusion testing. In some circumstances, an electronic cross-match can be performed by comparing records of the recipient's ABO and Rh blood type against that of the donor sample. In emergencies, blood may be issued before cross-matching is complete. Cross-matching is also used to determine compatibility between a donor and recipient in organ transplantation.

Autoimmune hemolytic anemia (AIHA) occurs when antibodies directed against the person's own red blood cells (RBCs) cause them to burst (lyse), leading to an insufficient number of oxygen-carrying red blood cells in the circulation. The lifetime of the RBCs is reduced from the normal 100–120 days to just a few days in serious cases. The intracellular components of the RBCs are released into the circulating blood and into tissues, leading to some of the characteristic symptoms of this condition. The antibodies are usually directed against high-incidence antigens, therefore they also commonly act on allogenic RBCs. AIHA is a relatively rare condition, with an incidence of 5–10 cases per 1 million persons per year in the warm-antibody type and 0.45 to 1.9 cases per 1 million persons per year in the cold antibody type. Autoimmune hemolysis might be a precursor of later onset systemic lupus erythematosus.

Paroxysmal cold hemoglobinuria (PCH) or Donath–Landsteiner hemolytic anemia (DLHA) is an autoimmune hemolytic anemia featured by complement-mediated intravascular hemolysis after cold exposure. It can present as an acute non-recurrent postinfectious event in children, or chronic relapsing episodes in adults with hematological malignancies or tertiary syphilis. Described by Julius Donath (1870–1950) and Karl Landsteiner (1868–1943) in 1904, PCH is one of the first clinical entities recognized as an autoimmune disorder.

In ABO hemolytic disease of the newborn maternal IgG antibodies with specificity for the ABO blood group system pass through the placenta to the fetal circulation where they can cause hemolysis of fetal red blood cells which can lead to fetal anemia and HDN. In contrast to Rh disease, about half of the cases of ABO HDN occur in a firstborn baby and ABO HDN does not become more severe after further pregnancies.

Cold agglutinin disease (CAD) is a rare autoimmune disease characterized by the presence of high concentrations of circulating cold sensitive antibodies, usually IgM and autoantibodies that are also active at temperatures below 30 °C (86 °F), directed against red blood cells, causing them to agglutinate and undergo lysis. It is a form of autoimmune hemolytic anemia, specifically one in which antibodies bind red blood cells only at low body temperatures, typically 28–31 °C.

Hemolytic disease of the newborn (anti-Kell1) is the second most common cause of severe hemolytic disease of the newborn (HDN) after Rh disease. Anti-Kell1 is becoming relatively more important as prevention of Rh disease is also becoming more effective.

Hemolytic disease of the newborn (anti-Rhc) can range from a mild to a severe disease. It is the third most common cause of severe HDN. Rh disease is the most common and hemolytic disease of the newborn (anti-Kell) is the second most common cause of severe HDN. It occurs more commonly in women who are Rh D negative.

The Kidd antigen system are proteins found in the Kidd's blood group, which act as antigens, i.e., they have the ability to produce antibodies under certain circumstances. The Jk antigen is found on a protein responsible for urea transport in the red blood cells and the kidney. They are important in transfusion medicine. People with two Jk(a) antigens, for instance, may form antibodies against donated blood containing two Jk(b) antigens. This can lead to hemolytic anemia, in which the body destroys the transfused blood, leading to low red blood cell counts. Another disease associated with the Jk antigen is hemolytic disease of the newborn, in which a pregnant woman's body creates antibodies against the blood of her fetus, leading to destruction of the fetal blood cells. Hemolytic disease of the newborn associated with Jk antibodies is typically mild, though fatal cases have been reported.

<span class="mw-page-title-main">Rh blood group system</span> Human blood group system involving 49 blood antigens

The Rh blood group system is a human blood group system. It contains proteins on the surface of red blood cells. After the ABO blood group system, it is the most likely to be involved in transfusion reactions. The Rh blood group system consisted of 49 defined blood group antigens in 2005. As of 2023, there are over 50 antigens among which the five antigens D, C, c, E, and e are the most important. There is no d antigen. Rh(D) status of an individual is normally described with a positive (+) or negative (−) suffix after the ABO type. The terms Rh factor, Rh positive, and Rh negative refer to the Rh(D) antigen only. Antibodies to Rh antigens can be involved in hemolytic transfusion reactions and antibodies to the Rh(D) and Rh antigens confer significant risk of hemolytic disease of the fetus and newborn

Hemolytic disease of the newborn (anti-RhE) is caused by the anti-RhE antibody of the Rh blood group system. The anti-RhE antibody can be naturally occurring, or arise following immune sensitization after a blood transfusion or pregnancy.

Acquired hemolytic anemia can be divided into immune and non-immune mediated forms of hemolytic anemia.

This page is currently under construction.

<span class="mw-page-title-main">Blood compatibility testing</span> Testing to identify incompatibilities between blood types

Blood compatibility testing is conducted in a medical laboratory to identify potential incompatibilities between blood group systems in blood transfusion. It is also used to diagnose and prevent some complications of pregnancy that can occur when the baby has a different blood group from the mother. Blood compatibility testing includes blood typing, which detects the antigens on red blood cells that determine a person's blood type; testing for unexpected antibodies against blood group antigens ; and, in the case of blood transfusions, mixing the recipient's plasma with the donor's red blood cells to detect incompatibilities (crossmatching). Routine blood typing involves determining the ABO and RhD type, and involves both identification of ABO antigens on red blood cells and identification of ABO antibodies in the plasma. Other blood group antigens may be tested for in specific clinical situations.

The Lan blood group system is a human blood group defined by the presence or absence of the Lan antigen on a person's red blood cells. More than 99.9% of people are positive for the Lan antigen. Individuals with the rare Lan-negative blood type, which is a recessive trait, can produce an anti-Lan antibody when exposed to Lan-positive blood. Anti-Lan antibodies may cause transfusion reactions on subsequent exposures to Lan-positive blood, and have also been implicated in mild cases of hemolytic disease of the newborn. However, the clinical significance of the antibody is variable. The antigen was first described in 1961, and Lan was officially designated a blood group in 2012.

<span class="mw-page-title-main">Monocyte monolayer assay</span> Laboratory test for clinically significant alloantibodies

The monocyte monolayer assay (MMA) is used to determine the clinical significance of alloantibodies produced by blood transfusion recipients. The assay is used to assess the potential for intravascular hemolysis when incompatible cellular blood products are transfused to the anemic patient. When donor cells possess substances that are not produced by the recipient, the recipient's immune system produces antibodies against the substance; these are called alloantibodies. Specific white blood cells, called monocytes, are tasked with ingesting foreign material and become activated during certain inflammatory events. These activated monocytes come in contact with antibody-sensitized red blood cells (RBC) and may or may not exhibit phagocytosis (ingestion) and destroy the donor red blood cells. If monocytes destroy the RBC, the antibody attached to those RBC is considered clinically significant.

<span class="mw-page-title-main">Antibody elution</span> Laboratory procedure

An antibody elution is a clinical laboratory diagnostic procedure which removes sensitized antibodies from red blood cells, in order to determine the blood group system antigen the antibody targets. An antibody elution is deemed necessary when antibodies of the immunoglobulin class G (IgG) are found sensitized (bound) to peripheral red cells collected from a blood product transfusion recipient. IgG antibodies are detected using an assay known as the direct antiglobulin test.

References

  1. 1 2 3 "Coombs test: MedlinePlus Medical Encyclopedia". medlineplus.gov. Retrieved 2019-05-06.
  2. ↑ F. Rosen and R. Geha, Case Studies in Immunology, 4th ed., Garland Science, p.173.
  3. 1 2 Zantek, Nicole D.; Koepsell, Scott A. (2012-04-12). "The direct antiglobulin test: A critical step in the evaluation of hemolysis". American Journal of Hematology. 87 (7): 707–709. doi: 10.1002/ajh.23218 . PMID   22566278.
  4. ↑ Theiss, Samuel R. (2023). Coombs Test. StatPearls Publishing LLC. PMID   31613487.
  5. ↑ Reid ME, Lomas-Francis C (23 December 2015). "Chapter 136: Erythrocytes Antigens and Antibodies". Williams hematology (9th ed.). New York: McGraw-Hill. ISBN   9780071833004. OCLC   913870019.
  6. ↑ "13.3: Pre-transfusion testing". Joint United Kingdom (UK) Blood Transfusion and Tissue Transplantation Services Professional Advisory Committee. 4 October 2023.
  7. ↑ Image by Mikael Häggström, MD. Source for conditions: Joe Chaffin (2017-02-27). "028: Who DAT? with Sue Johnson". Citing: C Feldman & J O'Connor.
  8. ↑ Coombs, R. R.; Mourant, A. E.; Race, R. R. (1945). "A new test for the detection of weak and incomplete Rh agglutinins". British Journal of Experimental Pathology. 26 (4): 255–66. PMC   2065689 . PMID   21006651.
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