Coombs test
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Coombs test (also known as Coombs' test, antiglobulin test or AGT) refers to two clinical blood tests used in haematology and immunology. These two tests are based on the concept that antihuman antibodies, which are produced by immunized non-human species, will bind to human antibodies such as IgG which are bound to RBC’s or free in the serum. Common clinical uses of the Coombs test include cross-matching of blood for transfusion, screening for atypical antibodies in pregnant women, and detection of autoimmune hemolysis.
The two Coombs tests are:
- Direct Coombs test, also direct antiglobulin test or DAT.
- Indirect Coombs test, also indirect antiglobulin test or IAT.
Coombs test can refer to either the direct or indirect form.
Blood samples for Coombs tests are taken by venipuncture from the patient by a phlebotomist, nurse or physician. The blood samples are taken to a laboratory, where Coombs tests are done by trained scientific technical staff. The clinical significance of the result is assessed by the physician who requested the Coombs test, perhaps with assistance from a laboratory-based hematologist.
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[edit] Direct Coombs Test
The direct Coombs test (also known as the direct antiglobulin test or DAT) is used to detect if antibodies or complement system factors have bound to RBC surface antigens in vivo. The DAT is not currently required for pre-transfusion testing but may be included by some laboratories.
[edit] Examples of diseases that give a positive direct Coombs test
The direct Coombs test is used clinically when immune-mediated haemolytic anaemia (antibody-mediated destruction of RBCs) is suspected. A positive Coombs test indicates that an immune mechanism is attacking the patient's own RBC's. This mechanism could be autoimmunity, alloimmunity or a drug-induced immune-mediated mechanism.
[edit] Examples of alloimmune haemolysis
- Hemolytic disease of the newborn (also known as HDN or erythroblastosis fetalis)
- Rhesus D hemolytic disease of the newborn (also known as Rh disease)
- ABO hemolytic disease of the newborn (the indirect Coombs test may only be weakly positive)
- Anti-Kell hemolytic disease of the newborn
- Rhesus c hemolytic disease of the newborn
- Other blood group incompatibility (RhC, Rhe, RhE, Kid, Duffy, MN, P and others)
- Alloimmune haemolytic transfusion reactions
[edit] Examples of autoimmune haemolysis
- Warm antibody autoimmune hemolytic anemia
- Idiopathic
- Systemic lupus erythematosus
- Evans' syndrome (antiplatelet antibodies and haemolytic antibodies)
- Cold antibody autoimmune hemolytic anemia
[edit] Drug-induced immune-mediated haemolysis
- Methyldopa
- Penicillin (high dose)
(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.)
[edit] Laboratory method
The patient's red blood cells (RBCs) are washed (removing the patient's own serum) and then incubated with antihuman globulin (also known as Coombs reagent). If immunoglobulin or complement factors have been fixed on to the RBC surface in-vivo, 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).
[edit] Indirect Coombs test
The indirect Coombs test (also known as the indirect antiglobulin test or 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.
[edit] Titrations
By diluting a serum containing antibodies the quantity of the antibody in the serum can be gauged. This is done by using doubling dilutions of the serum and finding the maximum dilution of test serum that is able to produce agglutination of relevant RBCs.
[edit] Examples of clinical uses of the indirect Coombs test
[edit] 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 Rhesus D compatible. Donor blood for transfusion is also screened for infections in separate processes.
- 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 (ie blood types).
- Cross matching
The indirect Coombs test is used to test a sample of the recipient's serum against a sample of the blood donor's RBCs. This is sometimes called cross-matching blood.
[edit] 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 foetal blood and cause haemolytic disease of the newborn.
[edit] 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).
[edit] First stage
Washed test red blood cells (RBCs) are incubated with a test serum. If the serum contains antibodies to antigens on the RBC surface, the antibodies will bind onto the surface of the RBCs.
[edit] Second stage
The RBCs are washed three or four times with isotonic saline 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.
[edit] 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. Coombs reagent contains animal antibodies specific for human immunoglobulins and human complement system factors. More specific Coombs reagents or monoclonal antibodies can be used.
[edit] Enhancement Media
Both IgM and IgG antibodies react strongly with their 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. RBC’s 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.
[edit] History of the Coombs test
The Coombs test was first described in 1945 by Cambridge immunologists Robin Coombs (after whom it is named), Arthur Mourant and Rob Race.[1] Historically, it was done in test tubes. Today, it is commonly done using microarray and gel technology.
[edit] References
- ^ Coombs RRA, Mourant AE, Race RR. A new test for the detection of weak and "incomplete" Rh agglutinins. Brit J Exp Path 1945;26:255-66.
[edit] External links
- Coombs testing - Institute for Transfusion Medicine.
- Coombs’ test - direct - Medlineplus.org.
- Coombs’ test - indirect - Medlineplus.org.
- Acute Anemia - emedicine.com
- Drugs that cause haemolytic anemia - Merck Manual.
