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 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).

The more commonly used test, the Direct Coombs test, is used to test for autoimmune hemolytic
anemia.

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
agglutination of RBCs, the direct Coombs test is positive, a visual indication that 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 antigenicity. If agglutination occurs, the indirect Coombs test is positive.[1]

Contents
[hide]

• 1 Mechanism
• 2 Direct Coombs test
o 2.1 Examples of diseases that give a positive direct Coombs test
 2.1.1 Examples of alloimmune hemolysis
 2.1.2 Examples of autoimmune hemolysis
 2.1.3 Drug-induced immune-mediated hemolysis
o 2.2 Laboratory method
• 3 Indirect Coombs test
o 3.1 Examples of clinical uses of the indirect Coombs test
 3.1.1 Blood transfusion preparation
 3.1.2 Antenatal antibody screening
 o 3.2 Laboratory method
 3.2.1 First stage
 3.2.2 Second stage
 3.2.3 Titrations
• 4 Coombs reagent
• 5 Enhancement media
• 6 History of the Coombs test
• 7 References

• 8 External links

[edit] Mechanism

Schematic showing the direct and indirect Coombs tests.

The two Coombs tests are based on the fact that anti-human antibodies, which are produced by
immunizing non-human species with human serum, will bind to human antibodies, commonly
IgG or IgM. Animal anti-human antibodies will also bind to human antibodies that may be fixed
onto antigens on the surface of red blood cells (also referred to as RBCs), and in the appropriate
test tube conditions this can lead to agglutination of RBCs. The phenomenon of agglutination of
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 transfusion in
cross-matching, screening for atypical antibodies in the blood plasma of pregnant women as part
of antenatal care, and detection of antibodies for the diagnosis of immune-mediated haemolytic
anemias.
Coombs tests are done on serum from venous blood samples which are taken from patients by
venepuncture. 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.

[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 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.

[edit] Examples of alloimmune hemolysis

• Hemolytic disease of the newborn (also known as HDN or erythroblastosis fetalis)

o Rh D hemolytic disease of the newborn (also known as Rh disease)
o ABO hemolytic disease of the newborn (the indirect Coombs test may only be
weakly positive)
o Anti-Kell hemolytic disease of the newborn
o Rh c hemolytic disease of the newborn
o Rh E hemolytic disease of the newborn
o Other blood group incompatibility (RhC, Rhe, Kidd, Duffy, MN, P and others)
• Alloimmune hemolytic transfusion reactions

[edit] Examples of autoimmune hemolysis

• Warm antibody autoimmune hemolytic anemia

o Idiopathic
o Systemic lupus erythematosus
o Evans' syndrome (antiplatelet antibodies and hemolytic antibodies)

• Cold antibody autoimmune hemolytic anemia

o Idiopathic cold hemagglutinin syndrome
o Infectious mononucleosis
o Paroxysmal cold hemoglobinuria (rare)

[edit] 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.)

[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] Examples of clinical uses of the indirect Coombs test

[edit] Blood transfusion preparation

Main articles: blood transfusion and 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.

• 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 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 fetal 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] 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] 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.

[edit] Enhancement media

Both IgM and IgG antibodies bind 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. 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.

[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.[2] Historically, it was done in test tubes.
Today, it is commonly done using microarray and gel technology.

[edit] References
1. ^ F. Rosen and R. Geha, Case Studies in Immunology, 4th ed., Garland Science, p.173.
2. ^ 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.
• MeSH Coombs'+Test