[PRACTICAL 5] MTEB 2404

DIRECT AGGLUTINATION ASSAY

PRINCIPLE
Direct agglutination assay involves clumping of cells or particulate antigen
by specific antibody with 2 or more receptors linking the particles in
suspension. Such agglutination reaction requires a minimum cell surface
antigen density and access of antibodies to their complementary cell
surface antigens. The most common example of a direct agglutination
reaction is red blood cell aggregation through the addition of ABO
isohemagglutinins.

BLOOD GROUP DETERMINATION

In blood group determination using direct agglutination method,
agglutination takes place when particulate antigen, e.g. red blood cells, is
exposed to the appropriate antibody under suitable conditions. When
these antigens combine with their specific antibody, agglutination of the
particle is seen. If one of the reactants is of known specificity, the
clumping may be used for the identification of the corresponding reactant.

MATERIALS
Reagents
1.Known group A, B, AB and O human erythrocytes, washed and
resuspended in normal saline to make a 3-4% cell suspension
2.Normal saline (NaCI 0.85%)
3.Anti-A, Anti-B, Anti-AB and anti-Rh serum

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EQUIPMENTS

1. Glass microscopic slides 5. Disposable

sterile, stainless steel lancets
2.Disposable Pasteur pipettes 6. Alcohol and swab
3.Glass Pasteur pipettes 7. Applicator sticks or toothpicks
4. Glass test tubes

PROCEDURE

1. Place a drop of Anti-A serum on the left side of the glass slide and
a drop of Anti-B
serum on the right side of the slide.
2.Place a drop of 3-4% cell suspension of known A cells next to each drop
of serum.
3.Mix the 2 drops on each half of the slide with an applicator or
toothpick. DO NOT mix the serum together.
4. Rotate the slide gently while examining it macroscopically.
Record the result observed.
5. Repeat steps 1 to 4 to observe the reaction of known cells of
group B, ANTIBODY
and O in the presence of Anti-A and Anti-B serum.
6.Determine your own blood group. Swab a subject's finger with alcohol.
Using a disposable, sterile lancet, prick the swabbed spot. Allow several
drops of blood to flow directly into a tube containing 1 ml saline.
7.Mix the blood suspension and proceed as in steps 1 to 4. Record the
results.

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Result

Rh Blood
Anti- Anti- Antibo Antibo Anti-
NO Anti-A Contr Grou
B AB dy A dy B D
ol p

A
1 Rh(D)
+ve

B
2 Rh(D)
+ve

AB
3 Rh(D)
+ve

O
4 Rh(D)
+ve

A
Rh(D)
5

- ve

B
Rh(D)
6

- ve

AB
Rh(D)
7

- ve

8 O
Rh(D)

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- ve

: Agglutination

: No agglutination

Discussion:
The basic technique in identification of
the antigens and antibodies of blood groups is the agglutination
test. Agglutination of red cells results from antibody cross-linkages
established when different specific combining sites of one antibody react
with antigen on two different red cells. By mixing red cells (antigen)
and serum (antibody), either the type of antigen or the type of antibody
can be determined depending on whether a cell of known antigen
composition or a serum with known antibody specificity is used.

In its simplest form, a volume of serum containing antibody is added

to a thin suspension (2–5 percent) of red cells suspended in physiological
saline solution in a small tube with a narrow diameter. After incubation at
the appropriate temperature, the red cells will have settled to the bottom
of the tube. These sedimented red cells are examined macroscopically
(with the naked eye) for agglutination, or they may be spread on a slide
and viewed through a low-power microscope.

An antibody that agglutinates red cells when they are suspended in

saline solution is called a complete antibody. With powerful complete
antibodies, such as anti-A and anti-B, agglutination reactions visible to the
naked eye take place when a drop of antibody is placed on a slide
together with a drop containing red cells in suspension. After stirring, the

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slide is rocked, and agglutination is visible in a few minutes. It is always

necessary in blood grouping to include a positive and a negative control
for each test.

An antibody that does not clump red cells when they are suspended
in saline solution is called incomplete. Such antibodies block the antigenic
sites of the red cells so that subsequent addition of complete antibody of
the same antigenic specificity does not result in agglutination. Incomplete
antibodies will agglutinate red cells carrying the appropriate antigen,
however, when the cells are suspended in media
containing protein. Serum albumin from the blood of cattle is a substance
that is frequently used for this purpose. Red cells may also be rendered
specifically agglutinable by incomplete antibodies after treatment with
such protease enzymes as trypsin, papain, ficin, or bromelain.

After such infections as pneumonia, red cells may become

agglutinable by almost all normal sera because of exposure of a hidden
antigenic site (T) as a result of the action of bacterial enzymes. When the
patient recovers, the blood also returns to normal with respect to
agglutination. It is unusual for the red cells to reflect antigenicity other
than that determined by the individual’s genetic makeup. The presence of
an acquired B antigen on the red cells has been described occasionally in
diseases of the colon, thus allowing the red cell to express an antigenicity
other than that genetically determined. Other diseases may alter
immunoglobulins; for example, some may induce the production of
antibodies directed against the person’s own blood groups (autoimmune
hemolytic anemia) and thus may interfere with blood grouping. In other
diseases a defect in antibody synthesis may cause the absence of anti-A
and anti-B antibody.

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Conclusion:
The agglutination reaction can be observed by doing the ABO grouping
test using sera reagent which shown positive by forming clumping within
specific antibody.

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