AB red cells and the anti-B antibody agglutinates
type B and type AB red cells. Type O red cells do
not agglutinate with either antibody. The type
assignments are confirmed by reverse typing in
which test serum is mixed with reagent red cells
(Table 9.5). Owing to the strong reactivity of the
reagent antibodies, the results of forward grouping
are usually very clear cut. The agglutination reac-
tions in reverse typing can be more difficult to score
because of variability in the reactivity of antibodies
in patient and donor plasma. The agglutination
induced by weak antibodies can be increased by
using a prolonged room temperature incubation or
by incubating at 4şC.
Typing for the Rh D antigen also uses the
hemagglutination assay. Most DD homozygous and
DD
-
heterozygous red cells agglutinate with the
reagent antibodies currently in use. There are poly-
morphic forms of the D antigen which are not
revealed by routine typing, however, usually
because of reduced levels of expression (Cartron
et
al.
1998) These weak reacting antigens are detected
by the use of a sensitive variant of the hemagglutina-
tion assay called the antiglobulin test (also called the
antihuman globulin test and, formerly, the Coomb’s
test). In the antiglobulin test, antibody or comple-
ment bound to red cells is demonstrated by incubat-
ing the cells with a reagent mixture that contains
antibodies to human IgG and the complement
components C3b and C3d. These antibodies induce
agglutination of red cells even when the antibody
bound to the red cells does not. When the anti-blood
group antibody is a reagent antibody, as in Rh
typing, the antiglobulin test is referred to as indirect.
The antiglobulin test is called direct when the anti-
blood group antibody is made by the patient and the
antibody binding reaction occurs
in vivo
.
A number of automated agglutination techniques
and solid phase, nonagglutination methods for ABO
and Rh typing have been developed and are in use in
many large blood centers (Plapp and Rachel 1992).
Solid-phase methods and a micro-column agglutina-
tion system are replacing the conventional tube
method for performance of the antiglobulin test, both
for Rh typing and for antibody detection (Knight and
de Silva 1996).
Presensitization of a transfusion recipient is
evaluated by an antibody screen in which serum
from the candidate is tested against a panel of type O
red cells that express the blood group antigens giving
rise to the most frequently encountered anti-blood
group antibodies. Here again, sensitive variants of
the hemagglutination assay are used. Typically, the
variations include prolonged incubation time and the
use of enhancement media such as low ionic strength
saline. The enhancement media promote agglutina-
tion chiefly by reducing the cationic charge cloud
that surrounds the negatively charged red cells. The
surface charge of the red cell can also be diminished
by proteolytic enzyme treatment which removes
sialic acid containing glycoproteins. Importantly,
antibody screen incubations are usually carried out at
37şC in order to limit positive results to antibodies
that can react at body temperature; if an antibody
cannot react at body temperature, it is very unlikely
to be clinically significant. Following incubation,
the mixtures are centrifuged and examined for
hemolysis or agglutination. Non-agglutinating panel
red cells are washed with saline and have an indirect
antiglobulin test performed. If there is a positive
result, the specificity of the antibody is determined
(Garratty 1998). Antibody screens are also
performed on donor blood to detect unexpected
antibodies that could react with recipient red cells.
Additional testing for pre-existing recipient
antibodies by the performance of a red cell cross-
match used to be a standard component of pretrans-
fusion testing. A full crossmatch is now no longer
required, unless the recipient is known to have made
antibodies in the past, as it has become recognized
that ABO and Rh typing and modern antibody
screens are adequate to assure red cell compatibility
in almost all cases (Cordle
et al.
1990). A red cell
crossmatch designed to detect ABO incompatibility
is adequate in the usual case. It consists of a hemag-
glutination assay in which a dilute solution of donor
red cells is mixed with serum from the recipient.
The mixture is centrifuged immediately, or after a
Tissue Injury
9-11
Table 9.5 Agglutination Reactions in ABO Blood Group
System Typing (+, agglutination; o, no agglutination)
Type
O A B AB
Forward grouping
reagent antibody
anti-A
o + o +
anti-B
o o + +
Reverse grouping
reagent red cells
A
+ o + o
B
+ + o o
