COLLEGE OF HEALTH – HAIL
Medical laboratory Dept.- Second term
THIRD YEAR – Blood banking
Antiglobulin Test
PRACTICE -2
Antiglobulin Test
The antiglobulin test (Coombs test) was introduced by Coombs, Mourant, and
Race in 1945[41] as a method for detecting “incomplete” Rh antibodies (i.e., IgG
antibodies capable of sensitising red cells but incapable of causing agglutination
of the same cells suspended in saline) as opposed to “complete” IgM antibodies,
which do agglutinate saline-suspended red cells.
Direct and indirect antiglobulin tests can be carried out. In the direct antiglobulin
test (DAT), the patient's cells, after careful washing, are tested for sensitisation
that has occurred in vivo; in the indirect antiglobulin test (IAT), normal red cells
are incubated with a serum suspected of containing an antibody and
subsequently tested, after washing, for in vitro–bound antibody.
The antiglobulin test is probably the most important test in the serologist's
repertoire. The DAT is used to demonstrate in vivo attachment of antibodies to
red cells, as in autoimmune haemolytic anaemia alloimmune HDN, and
alloimmune haemolysis following an incompatible transfusion.
Antiglobulin Reagents
Polyspecific (Broad-Spectrum) Reagents
The majority of red cell antibodies are noncomplement–binding IgG; anti-IgG is
therefore an essential component of any polyspecific reagent. Anti-IgA is not
required because IgG antibodies of the same specificity always occur in the
presence of IgA antibodies. Anti-IgM is also not required because clinically
significant IgM alloantibodies that do not cause agglutination in saline are much
more easily detected by the complement they bind.
Monospecific Reagents
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Monospecific reagents can be prepared against the heavy chains of IgG, IgM, and
IgA and are referred to as anti-γ, anti-μ, and anti-α; antibodies against IgG
subclasses are also available. Specific antibodies against the complement
components C4 and C3 and C3 breakdown products can be prepared
Recommended Antiglobulin Test Procedure
The test should be carried out in glass tubes (75 × 10 or 12 mm). Plastic tubes are
not recommended because they may adsorb IgG, which could neutralize anti-IgG
of the antiglobulin reagent.
Detection of Incomplete Antibodies by Means of the Direct Antiglobulin
(Coombs) Test
Principle
The DAT involves testing the patient's cells without prior exposure to antibody in
vitro. For the investigation of cases of AIHA, antiglobulin reagents specific for IgG,
IgM, IgA,C3c, and C3d can be used.
.
* “saline” refers to 9 g/l NaCl buffered to pH 7.0.
Method
Make a 2-5% suspension of red cells that have been washed four times in saline.
Add 1 volume (drop) of the cell suspension to 2 volumes (drops) of antiglobulin
reagent. Centrifuge for 10-60 sec. Refer to reagent manufacturer's instructions for
specific details.
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Examine for agglutination after gently resuspending the button of cells. A concave
mirror and good light help in macroscopic readings. If the result appears to be
negative, confirm this microscopically.
Macroscopic appearances of agglutination in round-bottom tubes or hollow tiles. Agglutination is shown
by various degrees of “graininess”; in the absence of agglutination, the sedimented cells appear as a
smooth round button, as on the extreme right.
Each DAT or batch of tests should be carefully controlled as previously described.
Check negative results with the polyspecific antihuman globulin (AHG) or anti-IgG
reagents by the addition of IgG-sensitized cells and anti-C′ by the addition of
complement-coated cells.
DAT Using Column Agglutination Technology
A card of several microtubes enables multiple sample testing. The microtubes
contain a solid-phase matrix and the antiglobulin reagent to which patient's red
cells are added. During centrifugation, unagglutinated cells pass to the tip of the
tube, but agglutinates fail to pass through the gel, which acts as a sieve. As the
antiglobulin reagent is already present in the microtubes, no washing or addition
of IgG-coated cells to negative tests is required. Refer to individual manufacturer's
instructions for details of methods for performing the tests.
The advantages of column agglutination technology are as follows:
1. Ease of use and reading and can theoretically be performed by relatively
unskilled staff.
2. Less chance of aerosol contamination from infected samples because no cell
washing before IATs.
3. The cards can be kept for up to 24 hours, enabling the results to be
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reviewed by experienced staff.
4. Ease of automation and positive sample identification.
However, the technology is expensive and its performance does not always
compare favourably with the standard LISS–IAT in experienced hands.
* DiaMed, AG, Switzerland.
** Bio Vue, Ortho-Clinical Diagnostics, New Jersey.
False-Negative Antiglobulin Test Results
There are several causes of false-negative test results:
1. Failure to wash the red cells properly—the antisera may then be neutralized
by immunoglobulins or complement in the surrounding serum or plasma
2. Excessive agitation at the reading stage—this may break up agglutinates,
leading to a false-negative result.
3. The use of impotent antisera so that weakly sensitized cells are not
detected.
4. The use of antisera lacking the antibody corresponding to the subclass of
immunoglobulin responsible for the red cell sensitization.
5. The presence of an antibody that is readily dissociable and is eluted in the
washing process.
DAT method in case of HDN
– Wash a 3% suspension of the infant’s red cell four times in physiological saline
Decant the final supernatant fluid.
– Resuspend the packed cells. Add two volumes of AHG reagent and mix.
– Centrifuge slowly for 1 minute. Tilting the tube back and forth, examine for
agglutination. When there is no agglutination, transfer a small number of cells to
a slide and
examine microscopically for agglutination.Ensure that the AHG is active by adding
1drop of IgG AHG control cells to the tube as explained previously for the indirect
AHGtest.
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– A positive DAT indicates antibody coating of the infant’s red cells. In tropical
countries most infants with severe ABO HDN give a positive DAT.
● Check the haemoglobin and serum bilirubin of the infant. Examine
aRomanowsky stained blood film from the infant for features of HDN.
The blood film will show spherocytosis which may be marked, polychromasia (due
to reticulocytosis) and nucleated red cells
Note: ABO HDN is rarely sufficiently severe to require an exchange blood
transfusion. In a situation in which it is indicated, Group O blood (of the same
Rhesus group as the baby) should be selected and crossmatched with the
mother’s serum. In contrast to Rhesus HDN, ABO HDN may occur in the first
pregnancy and may or may not affect subsequent pregnancies depending on the
ABO group of the infant.
HDN due to Rhesus incompatibility
Rhesus HDN is usually caused by immune anti-D and less commonly by other
Rhesus antibodies. It occurs when a Rh negative mother with circulating
IgG anti-D antibody (formed from a previous Rhesus incompatible pregnancy)
becomes pregnant with a Rh positive infant and IgG anti-D passes into the
fetal circulation, destroying fetal cells. The infant can be born severely anaemic
and jaundiced. The severity of disease increases with each Rh positive
pregnancy. Infants with Rhesus HDN are usually more severely affected than
infants with ABO HDN.
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