 Agglutination (clumping) of the red blood cells is
positive.
 No agglutination is negative.
 It is critical to read the results immediately as false
positives can occur when the mixture begins to dry on
the side.
Clinical Significance
 The ABO blood groups (A, B, AB, and O) represent the
antigens expressed on the erythrocytes (red blood cells,
RBCs) of each group.
 Reagent typing sera contains specific antibodies to A
antigen and B antigen.
 When an unknown patient’s RBCs are mixed with known
antibody A or antibody B, agglutination of the RBCs will
occur if a specific antigen-antibody reaction occurs. This
is called direct blood typing.
 Agglutination, like precipitation, is a two-step process
that results in the formation of a stable lattice network.
 The first reaction, called sensitization, involves antigen–
antibody combination through single antigenic
determinants on the particle and is rapid and reversible.
 The second step, or lattice formation, is the formation of
cross-links that form the visible aggregates. This
represents the stabilization of antigen–antibody
complexes with the binding together of multiple
antigenic determinants.
 Sensitization is affected by the nature of the antigens on
the agglutinating particles. If epitopes are sparse or if
they are obscured by other surface molecules, they are
less likely to interact with antibody.
 Additionally, red blood cells (RBCs) and bacterial cells
have a slight negative surface charge; because like
charges tend to repel one another, it is sometimes
difficult to bring such cells together into a lattice
formation.
 IgM antibodies are strong agglutinins because of their
larger size.
 Achieving visible reactions with IgG often requires the
use of enhancement techniques that vary
physicochemical conditions such as the ionic strength of
the solution, the pH, and the temperature.
 Antibodies belonging to the IgG class agglutinate best at
30°C to 37°C, whereas IgM antibodies react best at
temperatures between 4°C and 27°C. Because naturally
occurring antibodies against the ABO blood groups
belong to the IgM class, these reactions are best run at
room temperature. Antibodies to other human blood
groups usually belong to the IgG class; reactions involving
these must be run at 37°C.These latter reactions are the
most important to consider in selecting compatible
blood for a transfusion because these are the ones that
will actually occur in the body.
 If an agglutination reaction involves RBCs, then it is called
hemagglutination.
 The best example of this occurs in ABO blood group
typing of human RBCs, one of the world’s most
frequently used immunoassays.
 Patient RBCs mixed with antisera of the IgM type can be
used to determine the presence or absence of the A and
B antigens; this reaction is usually performed at room
temperature without the need for any enhancement
techniques. Group A RBCs will agglutinate with anti-A
antibody and Group B RBCs will agglutinate with anti-B
antibody. This type of agglutination reaction is simple to
perform, is relatively sensitive, and is easy to read.
 A titer that yields semiquantitative results can be
performed in test tubes or microtiter plates by making
serial dilutions of the antibody. The reciprocal of the last
dilution still exhibiting a visible reaction is the titer,
indicating the antibody’s strength.
 Interpretation of the test is done on the basis of the cell
sedimentation pattern.
 If there is a dark red, smooth button at the bottom of the
microtiter well, the result is negative.
 A positive result will have cells that are spread across the
well’s bottom, usually in a jagged pattern with an
irregular edge.
Test tubes also can be centrifuged and then shaken to see if
the cell button can be evenly re-suspended.
If it is re-suspended with no visible clumping, then the result
is negative. Positive reactions can be graded to indicate the
strength of the reaction