Coombs test
Coombs test | |
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MeSH | D003298 |
MedlinePlus | 003344 |
The direct and indirectCoombs tests,also known asantiglobulin test(AGT), areblood testsused inimmunohematology.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 causeanemia;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 ablood transfusion.[1]
The direct Coombs test is used to test forautoimmune hemolytic anemia,a condition where the immune system breaks downred blood cells,leading to anemia. The direct Coombs test is used to detect antibodies orcomplement proteinsattached 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 thenagglutinate,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,serumis extracted from a blood sample taken from the patient. The serum is incubated with foreign red blood cells of knownantigenicity.Finally, anti-human globulin is added. If agglutination occurs, the indirect Coombs test is positive.[2]
Mechanism
[edit]![](https://upload.wikimedia.org/wikipedia/commons/thumb/1/1c/Coombs_test_schematic.png/350px-Coombs_test_schematic.png)
The two Coombs tests are based on anti-humanantibodiesbinding to human antibodies, commonlyIgGorIgM.These anti-human antibodies are produced byplasma cellsof non-human animals after immunizing them withhuman plasma.Additionally, these anti-human antibodies will also bind to human antibodies that may be fixed ontoantigenson the surface ofred blood cells(RBCs). In the appropriate test tube conditions, this can lead toagglutinationof 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 fortransfusionincross-matching,atypical antibodies in theblood plasmaofpregnantwomen as part ofantenatal care,and detection of antibodies for the diagnosis of immune-mediatedhemolytic anemias.[4]
Coombs tests are performed using RBCs or serum (direct or indirect, respectively) from venous whole blood samples which are taken from patients byvenipuncture.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 thephysicianwho requested the Coombs test, perhaps with assistance from a laboratory-basedhematologist.[citation needed]
Direct Coombs test
[edit]The direct Coombs test, also referred to as the direct antiglobulin test (DAT), is used to detect if antibodies orcomplement systemfactors have bound toRBCssurface antigens.[5]The DAT is not required for pre-transfusion testing[6]but may be carried out by some laboratories.[clarification needed]Before transfusion, anindirectCoombs test is often done.
Uses
[edit]![](https://upload.wikimedia.org/wikipedia/commons/thumb/4/43/Algorithm_in_positive_direct_antiglobulin_test_%28DAT%2C_or_direct_Coombs_test%29.png/500px-Algorithm_in_positive_direct_antiglobulin_test_%28DAT%2C_or_direct_Coombs_test%29.png)
The direct Coombs test is used clinically when immune-mediatedhemolytic anemia(antibody-mediated destruction of RBCs) is suspected. A positive Coombs test indicates that an immune mechanism is attacking the patient'sRBCs.This mechanism could beautoimmunity,alloimmunityor a drug-induced immune-mediated mechanism.[3]
Examples of alloimmune hemolysis
[edit]- Hemolytic disease of the newborn(also known as HDN or erythroblastosis fetalis)
- Rh D hemolytic disease of the newborn(also known as Rh disease)
- ABO hemolytic disease of the newborn(the direct Coombs test may only be weakly positive)
- Anti-Kell hemolytic disease of the newborn
- Rh c hemolytic disease of the newborn
- Rh E hemolytic disease of the newborn
- Other blood group incompatibility (RhC, Rhe, Kidd, Duffy, Lewis, MN, P and others)
- Alloimmune hemolytictransfusion reactions
Examples of autoimmune hemolysis/immunohemolytic hemolysis
[edit]- Warm antibody autoimmune hemolytic anemia
- Idiopathic
- Systemic lupus erythematosus
- Evans' syndrome(antiplatelet antibodies and hemolytic antibodies)
- Cold antibody immunohemolytic anemia
Drug-induced immune-mediated hemolysis
[edit]- Methyldopa(IgG mediated type II hypersensitivity)
- Penicillin(high dose)
- Quinidine(IgM mediated activation of classical complement pathway and Membrane attack complex, MAC)
(Amemory deviceto remember that theDATtests theRBCsand is used to test infants forhaemolytic disease of the newbornis: RhDisease;R= RBCs,D= DAT.)
Laboratory
[edit]The patient'sRBCsare washed (removing the patient's ownserum) and then centrifuged withantihuman globulin(also known as Coombs reagent). Ifimmunoglobulinor complement factors have been fixed on to the RBC surfacein-vitro,the antihuman globulin willagglutinatethe 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
[edit]The indirect Coombs test, also referred to as the indirect antiglobulin test (IAT), is used to detectin-vitroantibody-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 causehemolytic disease of the newborn.The IAT can also be used forcompatibility testing,antibody identification, RBC phenotyping, and titration studies.[citation needed]
Uses
[edit]Blood transfusion preparation
[edit]The indirect Coombs test is used to screen for antibodies in the preparation ofbloodforblood transfusion.The donor's and recipient's blood must beABOand 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 ofdonorblood 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 theblood donor'sRBCs. This is sometimes calledcross-matchingblood.
Antenatal antibody screening
[edit]The indirect Coombs test is used to screen pregnant women forIgGantibodiesthat are likely to pass through theplacentainto the fetal blood and causehaemolytic disease of the newborn.[citation needed]
Laboratory method
[edit]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
[edit]Nonpatient, washedred blood cells(RBCs) with known antigens are incubated with patient serum containing unknown antibody content. If the serum containsantibodiesto antigens on the RBC surface, the antibodies will bind to the surface of the RBCs.[citation needed]
Second stage
[edit]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 theantihuman globulin(also known Coombs reagent) in this stage, and the indirect Coombs test will be positive.
Titrations
[edit]By diluting a serum containing antibodies the quantity of the antibody in the serum can be gauged. This is done by performingserial dilutionsof the serum and finding the maximum dilution of test serum that is able to produce agglutination of relevant RBCs.[citation needed]
Coombs reagent
[edit]Coombs reagent (also known asCoombs antiglobulinorantihuman globulin) is used in both the direct Coombs test and the indirect Coombs test. Coombs reagent is antihumanglobulin.It is made by injecting human globulin into animals, which producepolyclonal antibodiesspecific for human immunoglobulins and human complement system factors. More specific Coombs reagents ormonoclonal antibodiescan be used.[citation needed]
Enhancement media
[edit]BothIgMandIgGantibodiesbind strongly with their complementary antigens.IgGantibodies are most reactive at 37°C. IgM antibodies are easily detected insalineatroom temperatureas IgM antibodies are able to bridge between RBC's owing to their large size, efficiently creating what is seen asagglutination.IgG antibodies are smaller and require assistance to bridge well enough to form a visualagglutinationreaction. 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 proteolyticenzymes.
History
[edit]The Coombs test was first described in 1945 byCambridgeimmunologistsRobin Coombs(after whom it is named),Arthur MourantandRob Race.[8]Historically, it was done intest tubes.Today, it is commonly done using automated solid phase or gel technology.
References
[edit]- ^abc"Coombs test: MedlinePlus Medical Encyclopedia".medlineplus.gov.Retrieved2019-05-06.
- ^F. Rosen and R. Geha,Case Studies in Immunology, 4th ed.,Garland Science, p.173.
- ^abZantek, 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.PMID22566278.
- ^Theiss, Samuel R. (2023).Coombs Test.StatPearls Publishing LLC.PMID31613487.
- ^Reid ME, Lomas-Francis C (23 December 2015). "Chapter 136: Erythrocytes Antigens and Antibodies".Williams hematology(9th ed.). New York: McGraw-Hill.ISBN9780071833004.OCLC913870019.
- ^"13.3: Pre-transfusion testing".Joint United Kingdom (UK) Blood Transfusion and Tissue Transplantation Services Professional Advisory Committee. 4 October 2023.
- ^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.
- ^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.PMC2065689.PMID21006651.
External links
[edit]- Coombs' test- Medlineplus.org.
- Drugs that cause haemolytic anemia- Merck Manual.
- Coombs'+Testat the U.S. National Library of MedicineMedical Subject Headings(MeSH)