Editing Coombs test

{{Short description|Blood test used in immunohematology}}
{{Infobox diagnostic |
  Name        = Coombs test |
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  ICD10       = |
  ICD9        = |
  MeshID      = D003298 |
  MedlinePlus = 003344 |
  OPS301      = |
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The direct and indirect '''Coombs tests''', also known as '''antiglobulin test''' ('''AGT'''), are [[blood test]]s used in [[immunohematology]]. The direct Coombs test detects antibodies that are stuck to the surface of the red blood cells.<ref name="Medline 3344">{{Cite web|url=https://medlineplus.gov/ency/article/003344.htm|title=Coombs test: MedlinePlus Medical Encyclopedia|website=medlineplus.gov|language=en|access-date=2019-05-06}}</ref> 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.<ref name="Medline 3344"/> These antibodies could act against certain red blood cells; the test can be carried out to diagnose reactions to a [[blood transfusion]].<ref name="Medline 3344"/>

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 system|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 [[biologic agglutination|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 (blood)|serum]] is extracted from a blood sample taken from the patient. The serum is incubated with foreign red blood cells of known [[antigen]]icity. Finally, anti-human globulin is added. 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==
[[File:Coombs test schematic.png|thumb|350px|Schematic showing the direct and indirect Coombs tests]]
The two Coombs tests are based on anti-human [[antibody|antibodies]] binding to human antibodies, commonly [[Immunoglobulin G|IgG]] or [[Immunoglobulin M|IgM]]. These anti-human antibodies are produced by [[plasma cell]]s 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 [[antigen]]s on the surface of [[red blood cell]]s (RBCs). In the appropriate test tube conditions, this can lead to [[agglutination (biology)|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.<ref name="Zantek et al. 2012">{{cite journal |last1=Zantek |first1=Nicole D. |last2=Koepsell |first2=Scott A. |date=2012-04-12 |title=The direct antiglobulin test: A critical step in the evaluation of hemolysis |journal=American Journal of Hematology |volume=87 |issue=7 |pages=707–709 |doi=10.1002/ajh.23218 |pmid=22566278 |doi-access=free }}</ref>

Common clinical uses of the Coombs test include the preparation of blood for [[Blood transfusion|transfusion]] in [[cross-matching]], 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 [[hemolytic anemia]]s.<ref name="Theis and Hasmhmi 2020">{{cite book |last=Theiss |first=Samuel R. |author-link= |title=Coombs Test |year=2023 |url=https://www.ncbi.nlm.nih.gov/books/NBK547707/ |location= |publisher=StatPearls Publishing LLC |page= |pmid=31613487 |isbn=}}</ref>

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]].{{cn|date=November 2021}}

== 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 [[red blood cell|RBCs]] surface antigens.<ref>{{Cite book|title=Williams hematology|vauthors=Reid ME, Lomas-Francis C|publisher=McGraw-Hill|isbn=9780071833004|edition=9th|location=New York|chapter=Chapter 136: Erythrocytes Antigens and Antibodies|date=23 December 2015|oclc=913870019}}</ref> The DAT is not required for pre-transfusion testing<ref name="JPAC Guidelines">{{cite web |url=https://www.transfusionguidelines.org/red-book/chapter-13-patient-testing-red-cell-immunohaematology/13-3-pre-transfusion-testing |title=13.3: Pre-transfusion testing |author=<!--Not stated--> |date=4 October 2023 |publisher=Joint United Kingdom (UK) Blood Transfusion and Tissue Transplantation Services Professional Advisory Committee}}</ref> but may be carried out by some laboratories.{{clarify|date=October 2015}} Before transfusion, an ''indirect'' Coombs test is often done.

=== Uses ===
[[File:Algorithm in positive direct antiglobulin test (DAT, or direct Coombs test).png|500px|thumb|Algorithm for the main diagnoses in a positive DAT<ref>Image by Mikael Häggström, MD. Source for conditions: {{cite web|url=https://www.bbguy.org/2017/02/27/028/|title=028: Who DAT? with Sue Johnson|author=Joe Chaffin|date=2017-02-27}} Citing: C Feldman & J O'Connor.</ref>]]
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 [[red blood cell|RBCs]]. This mechanism could be [[autoimmunity]], [[alloimmunity]] or a drug-induced immune-mediated mechanism.<ref name="Zantek et al. 2012"/>

====Examples of alloimmune hemolysis====
* [[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]]
** [[Hemolytic disease of the newborn (anti-RhE)|Rh E hemolytic disease of the newborn]]
** Other blood group incompatibility (RhC, Rhe, Kidd, Duffy, Lewis, MN, P and others)
* Alloimmune hemolytic [[blood transfusion#Complications|transfusion reactions]]

====Examples of autoimmune hemolysis/immunohemolytic hemolysis<!--more accurate term than "autoimmune hemolysis," as the former includes extrinsic causative factors like drug exposure see Kumar, et al. Robbins and Cotran Pathologic Basis of Disease, 8th ed., 2010, p. 653-->====
* [[Warm antibody autoimmune hemolytic anemia]]
** Idiopathic
** [[Systemic lupus erythematosus]]
** [[Evans' syndrome]] (antiplatelet antibodies and hemolytic antibodies)
* [[Cold agglutinin disease|Cold antibody immunohemolytic anemia]]
** [[Idiopathic cold hemagglutinin syndrome]]
** [[Waldenström's macroglobulinemia]]
** [[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 [[red blood cell|RBCs]] and is used to test infants for ''haemolytic disease of the newborn'' is:
'''''R'''h '''D'''isease''; '''''R''''' = RBCs, '''''D''''' = DAT.)

===Laboratory===
The patient's [[red blood cell|RBCs]] are washed (removing the patient's own [[blood plasma|serum]]) and then centrifuged 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-vitro]], 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).{{cn|date=November 2021}}

==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 [[cross-match|compatibility testing]], antibody identification, RBC phenotyping, and titration studies.{{cn|date=November 2021}}

=== Uses ===

====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 Rh D compatible.  Donor blood for transfusion is also screened for infections in separate processes.{{cn|date=November 2021}}
* Antibody screening
A blood sample from the recipient and a blood sample from every unit of [[Blood donation|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|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 [[Immunoglobulin G|IgG]] [[antibodies]] that are likely to pass through the [[placenta]] into the fetal blood and cause [[haemolytic disease of the newborn]].{{cn|date=November 2021}}

===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).{{cn|date=November 2021}}

====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.{{cn|date=November 2021}}

====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 [[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 performing [[Serial dilution|serial dilutions]] of the serum and finding the maximum dilution of test serum that is able to produce agglutination of relevant RBCs.{{cn|date=April 2022}}

==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.{{cn|date=November 2021}}

==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 (medicine)|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 (biology)|agglutination]].  IgG antibodies are smaller and require assistance to bridge well enough to form a visual [[agglutination (biology)|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]] [[immunology|immunologists]] [[Robin Coombs]] (after whom it is named), [[Arthur Mourant]] and [[Robert Russell Race|Rob Race]].<ref>{{cite journal |pmid=21006651 |pmc=2065689 |year=1945 |last1=Coombs |first1=R. R. |title=A new test for the detection of weak and incomplete Rh agglutinins |journal=British Journal of Experimental Pathology |volume=26 |pages=255–66 |last2=Mourant |first2=A. E. |last3=Race |first3=R. R. |issue=4 }}</ref>  Historically, it was done in [[test tube]]s.  Today, it is commonly done using automated solid phase or gel technology.

==References==
{{Reflist}}

== External links ==
* [https://medlineplus.gov/ency/article/003344.htm Coombs' test]- Medlineplus.org.
* [https://web.archive.org/web/20060212094351/http://www.merck.com/mrkshared/mmg/tables/69t2.jsp Drugs that cause haemolytic anemia] - Merck Manual.
* {{MeshName|Coombs'+Test}}

{{transfusion medicine}}
{{Immunologic techniques and tests}}
{{Abnormal clinical and laboratory findings for blood}}
{{Authority control}}

{{DEFAULTSORT:Coombs Test}}
[[Category:Transfusion medicine]]
[[Category:Immunologic tests]]
[[Category:Blood tests]]