Coombs test
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'''Coombs test''' (also known as '''Coombs' test''', '''antiglobulin test''' or '''AGT''') refers to two [[clinical]] [[blood test]]s used in [[immunohematology]] and [[immunology]]. The two Coombs tests are the '''direct Coombs test''' (also known as '''direct antiglobulin test''' or '''DAT'''), and the '''indirect Coombs test''' (also known as '''indirect antiglobulin test''' or '''IAT''').
In certain diseases or conditions an individual's blood may contain IgG antibodies that can specifically bind to antigens on the red blood cell (RBC) surface membrane, and their circulating red blood cells (RBCs) can become coated with IgG alloantibodies and/or IgG autoantibodies. Complement proteins may subsequently bind to the bound antibodies. The '''direct Coombs test''' is used to detect these antibodies or complement proteins that are bound to the surface of red blood cells; a blood sample is taken and the RBCs are washed (removing the patient's own plasma) and then incubated with antihuman globulin (also known as "Coombs reagent"). If this produces [[biologic agglutination|agglutination]] of RBCs, the direct Coombs test is positive, a visual indication that [[Antibody|antibodies]] (and/or complement proteins) are bound to the surface of [[red blood cells]].
The '''indirect Coombs test''' is used in prenatal testing of pregnant women, and in testing [[blood]] prior to a [[blood transfusion]]. It detects antibodies against RBCs that are present unbound in the patient's [[serum]]. In this case, serum is extracted from the blood, and the serum is incubated with RBCs of known [[antigen]]icity. If agglutination occurs, the indirect Coombs test is positive.<ref>F. Rosen and R. Geha, ''Case Studies in Immunology, 4th ed.'', Garland Science, p.173.</ref>
==Mechanism==
[[Image:Coombs test schematic.png|thumb|350px|Schematic showing the '''direct and indirect Coombs tests'''.]]
The two Coombs tests are based on the fact that anti-human [[antibody|antibodies]], which are produced by immunizing non-human species with human [[Human plasma|serum]], will bind to human antibodies, commonly [[Immunoglobulin G|IgG]] or [[Immunoglobulin M|IgM]]. Animal anti-human antibodies will also bind to human antibodies that may be fixed onto antigens on the surface of [[red blood cell]]s (also referred to as RBCs), and in the appropriate test tube conditions this can lead to [[agglutination (biology)|agglutination]] of [[red blood cell|RBCs]]. The phenomenon of agglutination of [[red blood cell|RBCs]] is important here, because the resulting clumping of RBCs can be visualised; when clumping is seen the test is positive and when clumping is not seen the test is negative.
Common clinical uses of the Coombs test include the preparation of blood for [[Blood transfusion|transfusion]] in [[cross-matching]], screening for atypical antibodies in the [[blood plasma]] of [[pregnant]] women as part of [[obstetrics|antenatal care]], and detection of antibodies for the diagnosis of immune-mediated [[haemolytic anemia]]s.
Coombs tests are done on serum from venous 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]].
== 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.
=== Examples of diseases that give a positive direct Coombs test ===
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 own RBC's. This mechanism could be [[autoimmunity]], [[alloimmunity]] or a drug-induced immune-mediated mechanism.
====Examples of alloimmune hemolysis====
*[[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]]
**[[Hemolytic disease of the newborn (anti-RhE)|Rhesus E hemolytic disease of the newborn]]
**Other blood group incompatibility (RhC, Rhe, Kid, Duffy, MN, P and others)
*Alloimmune hemolytic [[blood transfusion#Complications|transfusion reactions]]
====Examples of autoimmune hemolysis====
*[[Warm antibody autoimmune hemolytic anemia]]
**Idiopathic
**[[Systemic lupus erythematosus]]
**[[Evans' syndrome]] (antiplatelet antibodies and hemolytic antibodies)
*[[Cold agglutinin disease|Cold antibody autoimmune hemolytic anemia]]
**[[Idiopathic cold hemagglutinin syndrome]]
**[[Infectious mononucleosis]]
**[[Paroxysmal cold hemoglobinuria]] (rare)
====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 [[mnemonic|memory device]] to remember that the ''DAT'' tests the RBCs and is used to test infants for ''haemolytic disease of the newborn'' is:<br>
'''''R'''h '''D'''isease''; '''''R''''' = RBCs, '''''D''''' = DAT.)
===Laboratory method===
The '''patient's''' [[red blood cells]] (RBCs) are washed (removing the patient's own [[blood plasma|serum]]) and then incubated with [[Coombs test#Coombs reagent|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 [[Agglutination (biology)|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).
==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 [[cross-match|compatibility testing]], antibody identification, RBC phenotyping, and titration studies.
=== Examples of clinical uses of the indirect Coombs test ===
====Blood transfusion preparation====
{{main|blood transfusion|cross-matching}}
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|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]].
===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).
====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.
====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 [[Coombs test#Coombs reagent|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 using doubling dilutions of the serum and finding the maximum dilution of test serum that is able to produce agglutination of relevant RBCs.
==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 response|polyclonal]] [[polyclonal antibodies|antibodies]] specific for human [[immunoglobulins]] and human [[complement system]] factors. More specific Coombs reagents or [[monoclonal antibodies]] can be used.
==Enhancement Media==
Both [[IgM]] and [[IgG]] [[antibodies]] bind strongly with their [[antigen]]s. [[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 of the Coombs test==
The Coombs test was first described in [[1945]] by [[Cambridge]] [[immunology|immunologists]] [[Robin Coombs]] (after whom it is named), Arthur Mourant and Rob Race.<ref>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.</ref> Historically, it was done in [[test tube]]s. Today, it is commonly done using [[protein microarray|microarray]] and gel technology.
==References==
{{reflist}}
==External links ==
*[http://www.itxm.org/TMU2000/tmu10-2000.htm Coombs testing] - Institute for Transfusion Medicine.
*[http://www.nlm.nih.gov/medlineplus/ency/article/003344.htm Coombs’ test - direct] - Medlineplus.org.
*[http://www.nlm.nih.gov/medlineplus/ency/article/003343.htm Coombs’ test - indirect] - Medlineplus.org.
*[http://master.emedicine.com/EMERG/topic808.htm Acute Anemia] - emedicine.com
*[http://www.merck.com/mrkshared/mmg/tables/69t2.jsp Drugs that cause haemolytic anemia] - Merck Manual.
*{{MeshName|Coombs'+Test}}
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{{transfusion medicine}}
{{Immunologic techniques and tests}}
[[Category:Transfusion medicine]]
[[Category:Hematology]]
[[Category:Immunology]]
[[Category:Medical tests]]
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