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CHAPTER 5 Basic Immunologic Principles 59

CD8 recognizes self

HLA Class I

CD8+
CTL
Virus TcR
infected
body cell CD3

Viral Ag in HLA
Class I groove

FIGURE 5-6 Specific cells of the immune response.

CD8+
CTL
Virus TcR
infected
body cell CD3

1. CTL binds target with TcR. Granules move into area. Perforin released.

Perforin
Ca++
Ca++ Ca++
Ca++
Ca++
Ca++
Poisoned,
2. Perforin sets up ion channels which allow unlimited Ca++ to enter dead, virus
Ca++
the cell, poisioning it. infected cell

FIGURE 5-7 CD8 + cytotoxic T-lymphocyte antiviral activity.

Specific anti-tumor Ab

FcR

Killer cells have Fc receptors that bind Ab NK cells bind their tumor targets by an
already bound to the tumor target. unknown mechanism. Kill with perforin.

FIGURE 5-8 Killer and natural killer cells. Large granular lymphocytes that carry neither T- nor B-cell markers. Kill
tumor cells using the perforin mechanism.
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60 UNIT 2 Genetic and Immunologic Principles
B Lymphocytes
The remaining 5% to 15% of peripheral blood lympho-
cytes can be classified as B lymphocytes on the basis of
their surface antigen receptors, which are actually
antibody molecules. After migration from the bone
marrow, B lymphocytes reside in the germinal centers
and medullary areas of the lymph nodes and spleen,
where they lose some cytoplasm and become smaller.
More than 50% of the lymphocytes in the spleen and
tonsils are B lymphocytes. The immature B lymphocyte
may be thought of as the precursor to the antibody-
producing and morphologically recognizable plasma
cell, which is differentiated by exposure to antigen and
interaction with T-helper cells.
If a B cell has recognized its specific antigen
using its specific cell surface antigen receptor (an anti-
body molecule, which may also be called surface
immunoglobulin) and has received a cytokine signal
from the T-helper cell, the B cell divides and differen-
tiates to become a plasma cell. The plasma cells, which
are large and full of protein-producing machinery
(called the endoplasmic reticulum), gush antibody for
3 to 4 days. The antibody produced by the plasma cell
has the same antigen-binding specificity as the origi-
nal B cell’s surface antigen receptor (Fig. 5-9).
Groups of B cells or plasma cells that produce an-
tibody with the same specificity are said to be mem-
bers of the same B cell clone. Not all B cells are
transformed into plasma cells and produce antibody.
Some become memory cells capable of rapid antibody
B “help”
1. B cells surface Ig Ag receptor binds
free Ag or receives Ag from APCs.
FIGURE 5-9 Antibody production: the antigen selection hypothesis.
production on reexposure. The antibodies produced
during this so-called anamnestic response are very
high-quality antibodies said to belong to the im-
munoglobulin G or IgG subclass.
The preceding discussion described the clonal se-
lection theory in its basic form. This theory postulates
that a given B lymphocyte is triggered by contact
with an antigen for which it carries specific cell
surface receptors. The activated cell proliferates
and synthesizes antibody with the same binding
specificity as the surface antigen receptor. The the-
ory also states that all of the progeny of that acti-
vated lymphocyte produce antibody of identical
structure and specificity. Although the cells in the
B-lymphocyte population have many features in
common, they synthesize different classes of im-
munoglobulin—IgM, IgG, IgA, IgD, and IgE. Addi-
tional variation has led to subdivision of the
classes into subclasses, the importance of which
becomes apparent in the section on immunoglobu-
lin subclasses later in this chapter. It is also impor-
tant to realize that antibody synthesis is an
extremely complex process under the control of
genes beyond those that control diversification.
Many aspects are controversial and under intense
investigation.
Keep in mind that most immune responses are a
combination of nonspecific inflammation (caused
predominantly by macrophages, neutrophils, and
mast cells) and specific, acquired immunity (conferred
IL-2 thru 6
et al. T cell B
2. B cells are stimulated to divide and
differentiate by Ag binding and cytokines.
3. Plasma cells gush Ab for 3-4 days. The Ab
specificity is the same as the slg (same
variable regio AA sequence). The Ab
produced helps to destroy the Ag. Thus the
Ag has selected the B cell clone producing
Ab that eventually led to the destruction of
Ag. Immunization, memory.
82043_ch05.qxd 11/11/09 5:51 PM Page 61
Complement
activation
Bacteria &
tissue damage
Chemotaxins. WBC
called to the area
Mast cells PMN
Phagocytosis
and bacterial
killing
FIGURE 5-10 An immune response consists of both nonspecific inflammation and specific acquired immunity.
by lymphocytes). The complex interactions and
cooperation demonstrated by these cell populations
are shown in diagrammatic fashion in Figure 5-10.
This example describes the cells that work together to
fight off a bacterial infection.
IMMUNOGENS VERSUS ANTIGENS
An immunogen is a substance that causes a detectable
immune response. Frequently, the terms “immuno-
gen” and “antigen” are used interchangeably, although
they are not, in the strictest sense of their definitions,
the same thing. An antigen is a substance that is
capable of reacting with the product of an immune
response. Antigen combines with antibody (i.e.,
“antigen–antibody reaction” is used instead of “im-
munogen–antibody reaction”).
Introduction of nonself immunogens present on
human RBCs, white blood cells, and platelets may
elicit antibody production. On a biochemical level, an
immunogen is a substance with a molecular weight
of 10,000 D or more. Substances with a molecular
weight of less than 5,000 D seldom cause antibody
formation. If coupled with larger carrier molecules,
however, these substances, known as haptens, can be-
come immunogenic (able to stimulate an immune re-
sponse). Once an immune response has been initiated
by the hapten–carrier complex, the hapten alone
can react with the product of the immune response
(i.e., antibody). Complex biochemical compounds,
CHAPTER 5 Basic Immunologic Principles 61
Antibodies,
opsonization B cell,
plasma cell,
memory
Cytokines,
growth
factors
Cytotoxic
T cell
memory
T-helper cell, memory
Macrophage antigen presentation
especially those containing proteins or polypeptide–
carbohydrate combinations, are highly immuno-
genic. The more diverse the molecule is, the more im-
munogenic it becomes. As previously indicated, the
structure must be recognized by the cells of the recip-
ient’s immune system to be nonself.
Route of Administration
Intravenous and intraperitoneal injection of immuno-
gen is highly effective in producing an immune
response. Intradermal, subcutaneous, or intramuscu-
lar routes are less effective.
Shape and Charge
It is now recognized that molecular shape and charge
are the most important features affecting immuno-
genicity and antigenicity (the ability of a substance to
react with the product of an immune response). Anti-
gens on human RBCs protrude from the cells’ mem-
brane in a steric (three-dimensional) configuration
and carry ionic charges. These immunogens are pro-
teins coupled with carbohydrate molecules (glycopro-
teins) or lipids (lipoproteins). Although the immunogen
is large, the antigenic determinant, known also as the
epitope, is a small portion that may be composed of as
few as five or six amino acids or sugars. This small
epitopic region is responsible for specificity, meaning
that the region contains the molecular configurations
that allow recognition by the corresponding antibody.
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62 UNIT 2 Genetic and Immunologic Principles
Many epitopes are present on an immunogen and
multiple antibodies that have somewhat different
specificity and reactivity may be formed in response
to a single, large immunogen. Because of the steric
or spatial configuration of these biochemical struc-
tures, some epitopes may be rendered nonfunctional
or unable to combine unless the molecule is struc-
turally altered (such as may occur during deliberate
modification with enzymes or during the combina-
tion of antigen with its antibody).
The chemical composition of the human blood group
substances has been studied for many years. Similar
macromolecular configurations are widely distributed
throughout the biologic world in mammals, birds, am-
phibians, reptiles, bacteria, and plants. Since 1950, when
the carbohydrate composition of the A, B, and H blood
group substances became known, the new science of
immunochemistry has added much knowledge to our
understanding of the structural arrangement of the com-
ponent sugars. The structures of the ABO, Lewis, and H
antigens are better understood than any of the other blood
groups. Also, the Rh antigens have a lipoprotein structure.
Because the task is complicated by the isolation of anti-
gens from the red cell, much remains to be learned about
many of the other blood group antigens.
The lipoprotein immunogens (antigens) are proba-
bly even more complex than the carbohydrate structures.
Antigens expressed on human red cells are developed
under the control of genes. The names for the genes and
the antigens each defines are the same for most of the
blood groups (i.e., A gene codes for the A antigen).
ANTIBODIES
The terms “antibody” and “immunoglobulin” are
commonly used interchangeably, though there is a
semantic difference between the two terms. Immu-
nologists call a molecule an antibody if its binding
specificity is known—for example, an antimeasles
virus antibody. Immunoglobulin refers to a collec-
tion of different antibodies with no single binding
specificity, such as a preparation of intravenous im-
munoglobulin.
Antibodies may be thought of as substances pro-
duced in response to immunogenic stimulation that are
capable of specific interaction with the provoking
antigens. The immunoglobulins that function as anti-
body may be divided into five classes based on their dif-
ferent physical, chemical, and biologic properties: IgG,
IgM, IgA, IgD, and IgE. Each of these classes can bind
antigens. Box 5-1 summarizes some of the properties
of these immunoglobulins, and Figure 5-11 shows the
simplified structure of IgG, dimeric IgA, and penta-
meric IgM.
BOX 5-1 Properties of the Immunoglobulin
Classes
IgG
• Monomer; molecular weight 180,000 D
• Most abundant in serum; 10 immune response,
class shift
• “Maternal Ab”; crosses placenta
• Binds complement to destroy antigens which have
been bound
• Is opsonic; macrophage Fc receptors have high
affinity for IgG Fc regions
IgA
• Dimeric, 400 kD
• Secreted by mucous membranes, in mother’s milk
• Does not bind complement
IgM
• Pentameric, 900 kD
• Primary immune response Ab; binds complement
IgD
• 180 kD
• B cell surface Ig?
IgE
• Monomeric, 180 kD
• In allergy or anaphylaxis, binds mast cell and allergen
Immunoglobulins are the product of plasma cell
protein production, and these protein–carbohydrate
moieties (glycoprotein) bind to their antigens in a highly
specific fashion. Each immunoglobulin molecule is
made of 82% to 96% protein (polypeptide) and 4% to
18% carbohydrate. Human blood group antibodies
are generally of two types, IgM or IgG, although
IgA forms also exist. Within the five classes of
immunoglobulins, there exist differences in each
single type that have resulted in the further division into
subgroups or subclasses. For example, IgG has four
identified subclasses: IgG1, IgG2, IgG3, and IgG4.
Structure and Physiochemistry
Each immunoglobulin molecule may be thought of
as consisting of two identical polypeptide heavy
chains (H) and two identical polypeptide light
chains (L) (Fig. 5-12). Each of the heavy chains con-
tains a sequence of approximately 440 amino acids
with a molecular weight between 50,000 and 70,000 D
that is identical within each molecule. The light
chains are also identical to each other within the
same molecule, although different from the heavy
chains. The light chains contain about 220 amino
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CHAPTER 5 Basic Immunologic Principles 63

J Chain Secretory Piece

IgG Dimeric, Secretory IgA

Pentameric IgM

J Chain

FIGURE 5-11 Simplified structures of IgG, IgA, and IgM.

acids with a weight of 20,000 to 25,000 D. These two bonds holds together loops of folded polypeptide
pairs of chains are held together covalently by chains known as domains. The disulfide bonds also
molecular bridges called disulfide bonds (S–S) and allow the antibody molecule to flex, and to change
noncovalent forces. The interaction of disulfide shape in three-dimensional space. Antigens are

Heavy Chain

Variable Region
Heavy Chain Light Chain
Ag

Variable Region
Light Chain Fab Region

• Fraction Ag binding

• Ag bound here
Constant Region
Light Chain
SS SS

Disulfide bonds SS
Fc Region

• Complement binds
here

Constant Region • Macrophage FcR

Heavy Chain bind here

FIGURE 5-12 Basic structure of the immunoglobulin molecule.

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64 UNIT 2 Genetic and Immunologic Principles
bound in the groove formed by the amino-terminal
ends of the heavy and light chains.
Human immunoglobulins have two types of light
chains, known as kappa and lambda, and both types
can form antigen-binding sites. Human heavy chains
are found in five variations known as gamma, alpha,
mu, delta, and epsilon. All five types can bind anti-
gens. An immunoglobulin (Ig)-containing gamma
heavy chains is called immunoglobulin gamma, or IgG,
whereas a mu-containing immunoglobulin is called
IgM, and so forth.
Variable and Constant Regions
Approximately one fourth of each heavy chain and
one half of each light chain contains regions of amino
acid sequence that vary from one antibody molecule
to another. These regions of varying amino acid
sequence may be found at the amino-terminal end of
the molecule (see Fig. 5-12). The variable regions are
concerned with antigen binding and confer specificity
on the molecule; that is, the particular amino acid
sequence allows the molecule to bind or not bind to a
particular antigen.
There are about 20 different amino acids that are
used to make up human proteins, and each amino
acid has its own unique shape. Therefore, the amino
acid sequence of the variable region determines the
three-dimensional shape of the antibody-binding
site. Thus, this sequence determines which antigen
is bound by that antibody molecule. If the amino
acid sequence of the variable region is changed, the
shape of the antibody-binding site and which anti-
gen will fit in the groove between the H and L chains
are also changed.
Actually, the ability to bind one antigen as
opposed to another is believed to be controlled more
precisely by hypervariable amino acid sequences in
these regions, three in each light chain and four in
each heavy chain. More specifically, the shapes of both
are complementary and dictate the “goodness of fit”
and whether the antibody and antigen will complex.
The analogy of the “lock and key” supplemented with
positive and negative charges is a good description of
antibody–antigen binding.
(The principle of variable and constant regions
described here for the formation of antibodies that
bind their antigen specifically can also be applied to
the T-cell surface antigen receptor, with the exception
that the T-cell receptor is made of two protein chains
with one antigen-binding site. Antibodies are
constructed of four protein chains with two antigen-
binding sites per molecule.)
The sequence of amino acids in the hypervariable
regions of the heavy and light chains is referred to as
the idiotype of the antibody molecule. Anti-idiotype
antibodies can prevent the antibody from complexing
with its antigen by combining with these idiotypic
determinants.
The remaining portions of each heavy and light
chain pair comprise the constant regions. These
regions are so named because the amino acid
sequence is identical for molecules in the same class
or subclass (except for differences due to genetic
polymorphisms). The constant regions function
to control biologic effector mechanisms, such as
macrophage Fc receptor binding and complement
activation.
Heavy chains have at least three constant re-
gions, and these domains are numbered consecu-
tively from the amino-terminal end (as are all of the
amino acids that comprise the molecules) as CH1,
CH2, and CH3 (and CH4 in the case of IgM and IgE
molecules, which possess this additional domain).
Although specific functions for the CL (constant re-
gion of the light chain) and CH1 domains elude
demonstration, it is at least reasonable to suppose
that they help orient the hypervariable sequences
for antigen binding. The CH2 domain has been
shown specifically to activate complement by bind-
ing the Clq molecule. The CH3 domain is the C re-
gion responsible for binding to macrophages. In
addition, the constant region is involved in placen-
tal transfer, which confers passive immunity to the
newborn. IgG constant and variable domains are
shown in Figure 5-13.
These functions of the constant regions of the
heavy and light chains are of vital importance and
should not be considered secondary to the essential
“antigen recognition” function of the amino-terminal
end. The primary interactions of antigen with anti-
body would be insignificant if these effector functions
did not become manifest.
The Hinge Region
The hinge region of the antibody molecule between
the CH1 and CH2 domains is the area that provides
flexibility in allowing the molecule to combine with
antigen (Fig. 5-13). As is apparent from the previous
discussion of the hypervariable sequences, the elec-
tric charges present, with positive and negative
charges attracting and like charges repelling each
other, the molecule must have the ability to conform
three-dimensionally and shape itself spatially to fit
closely or widely separated antigens. It has also been
suggested that the molecule in the unbound state, as
normally occurs in the serum, is in a “T” shape with
the amino-terminal ends maximally distant from
each other. When combined with antigen, these ends
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CHAPTER 5 Basic Immunologic Principles 65

Light chain
+H3N hypervariable L chain

S
S-
regions
VL
+H3N H chain

S
107

S-
VH CL 214 Fab

S-
S
Heavy chain

S
CH1 S

S-
S- 117 hypervariable

S-
regions

S
S S 220
Site of papain cleavage S S Hinge region
Site of pepsin cleavage 235
Complement binding region

S-S
CH2
Carbohydrate
341 Fc
S-S
CH3

446

COO-

FIGURE 5-13 IgG molecule.

flex to become “Y” shaped, and the arms swing in-
ward. This flex change may also allow the CH2 and
CH3 effector domains more accessibility for comple-
ment binding by reducing steric hindrance. The
lengths and flexibility of the hinge have been found
to vary with each of the IgG subclasses, and this vari-
ability is reflected in the ability to trigger the biologic
or effector functions.
Fragment, Antigen-binding and Fragment,
Crystallizable
In 1959, the research of Rodney Porter showed that
the IgG molecule could be split in the hinge region by
the use of proteolytic enzymes. Different products
also are obtained when the enzymes pepsin and
papain are used. When papain is used, the molecule is
split into three fragments, two of which are identical
and capable of antigen binding (although not capa-
ble of causing red cell agglutination or precipitation
reactions). These two fragments are referred to as
fragment, antigen-binding (Fab) because they bind
antigen, and each is composed of one light chain and
roughly half of its heavy chain (Fig. 5-12).
The remaining portion of the molecule split by
papain is referred to as the fragment, crystallizable
(Fc) region. This is composed of the two remaining
carboxy-terminal end halves of the heavy chains.
Enzyme treatment has allowed these two portions
to be studied independently, and the functions of
complement activation (CH2), placental transport, and
macrophage binding (CH3) were assigned to the Fc
fragment. Treatment with pepsin results in a slightly
different type of Fab fragment, called F(ab⬘)2, which
in addition to retaining its antigen-binding ability, is
capable of causing agglutination and precipitation
reactions. F(ab⬘)2 is roughly the top half of the
molecule, including the disulfide bonds holding the
two heavy chains together. Pepsin splits the heavy
chains just below the disulfide bonds: papain splits
them above these bonds. Pepsin also splits the remain-
ing Fc pieces into many small fragments.
IgG Subclass and Function
Immunoglobulins help to protect from disease by
specifically binding with the nonself substance that
elicited their production. After antigen binding, the
immunoglobulin molecule promotes phagocytosis,
because macrophage and other cells have a receptor
on their surface that binds to the Fc region of the
molecule. This function allows for the destruction of
potentially harmful bacteria and the neutralization of
toxic substances. The Fc regions of antibodies, which
have bound their antigen, also activate complement.
For the immunohematologist, these basic functions
extend to and include the nonself antigens present on
allogeneic transfused RBCs, white blood cells, and
platelets. Through the action of antibody, elimination
of transfused cells may occur extravascularly (outside
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66 UNIT 2 Genetic and Immunologic Principles
the blood vessels) in the reticuloendothelial system
(a system of fixed-tissue phagocytic cells in organs
like the liver), or intravascularly (within the blood
vessels) through the mechanism of complement
activation.
Of the total amount of serum immunoglobulin in a
normal adult, fully 80% is constituted by IgG: its level
is approximately 600 to 1600 mg/dL of serum. Within
this class, approximately 60% to 70% is IgG1, 20% to
30% is IgG2, 4% to 8% is IgG3, and 1% to 6% is IgG4. The
level of each is influenced by genetics, age, race, and sex
and may vary considerably from one individual to
another. The main interest in subclasses lies in the differ-
ences in their biologic properties.
In humans, IgG is the only immunoglobulin
class capable of crossing the placenta. IgG also fixes
complement with unequal facility within the
subclasses, in the order IgG3 > IgG1 > IgG2, with IgG4
possibly activating only in the alternate pathway.
This difference in complement-binding ability derives
from differences in subclass hinge region flexibility, as
previously described, but it is influenced by the length
of the hinge region and the number of interchain
disulfide bonds (which varies among each of the sub-
classes).
IgG1 and IgG3 molecules also complex with
macrophages through Fc receptors. At least three
different receptor types have now been studied: FcRI,
FcRII, and FcRIII. The interaction between these recep-
tors and the IgG molecule is integral to the processes of
phagocytosis and immune complex clearance.
Hybridomas and Monoclonal Antibodies
Since the mid-1980s, the use of monoclonal antibodies
has expanded considerably. A monoclonal antibody is
a very specific preparation. All molecules produced
are identical, unlike what would be seen in a normal
immune response. In the normal immune response,
many clones of lymphocytes produce antibody
directed toward many different epitopes. The hetero-
geneous antibodies produced by many lymphocyte
clones are known as polyclonal, and they represent
the normal immune response.
The proliferation of a single lymphocyte clone
producing antibody of identical specificity and
having identical heavy and light chains results in a
monoclonal antibody. This situation is abnormal and
often pathologic, as in cold hemagglutination disease,
in which the monoclonal antibody is IgM and may be
anti-I or anti-i.
In 1975, it was shown that mouse myeloma cells
grown in culture could be fused with lymphocytes
from immunized animals to produce a cell, called a
hybridoma, that would grow continuously and secrete
antibody specific for the immunizing antigen. The
advantages of this new technology were at once clear.
The synthesized antibodies made from the single-
cell clone are structurally identical to each other and
therefore are monospecific, capable of binding a single
epitope. Further, cell lines can be maintained continu-
ously in a culture and may be frozen and recovered.
Hybridomas provide a highly reproducible, well-
defined, and replenishable supply of homogeneous
antibody. The application of monoclonals has had a
profound effect in immunology. They have made
diagnostic testing more sensitive and specific. This
is true for the monoclonal reagents used in blood
banking.
ANTIGEN–ANTIBODY REACTIONS
The structural framework comprising the typical
mammalian cell membrane is a bilayer of lipid and
phospholipid molecules, approximately 4.5 nm thick,
arranged with the hydrophilic heads forming the
outer and inner membrane surfaces. The hydrophobic
tails meet at the center of the membrane. Many other
molecules are also present in this membrane structure,
including, on RBCs, the carbohydrate molecules com-
prising the A, B, and H antigens, the lipoprotein struc-
tures of the Rh system antigens, and other essential
membrane molecules, such as cholesterol and N-
acetylneuraminic acid (also called sialic acid) and
other blood group system molecules. In transfusion,
these molecules may act as antigens.
The Zeta Potential
The microenvironment of the red cell and the ions that
may be present in human plasma or serum can
dramatically influence whether an antibody is able to
attach to RBCs, or whether the red cells themselves
spontaneously aggregate. In 1965, the work of Pollack3–5
and others showed that the red cell carries a net nega-
tive charge that results from the ionization of carboxyl
groups of the essential membrane constituent,
N-acetylneuraminic acid. The negatively charged RBC
attracts positively charged cations in an ionic environ-
ment such as human serum. The red cell then travels
in a cloud of positive cations, the density of which de-
creases as the distance from the cell increases. The
zone separating the most dense layer surrounding the
cell from the remainder of the cationic environment is
referred to as the slipping plane of plane of shear. The
zeta potential is the force expressed at this boundary
that results from the difference in electrostatic poten-
tial at the red cell surface and the boundary. Any
increase in the ionic strength of the microenvironment
82043_ch05.qxd 11/11/09 5:51 PM Page 67
(or suspending medium) results in an increase in the
charge density and a decrease in the thickness of the
cation cloud and the zeta potential.
Hydration and Surface Tension
Zeta potential theory is not the entire story concerning
the physics of red cell–antibody binding. Steane6 have
also investigated the ability of IgG molecules to cause
agglutination. They concluded that the degree of
hydration at the red cell surface also contributes to the
agglutination phenomenon. These investigators found
that the hydrophilic heads of phospholipids comprising
the red cell membrane attract and orient water molecules.
Phospholipid structures in the membrane are not static,
but move more or less freely in a dynamic shifting and
reorienting of water, creating a surface tension effect at
the water–lipid interface. Many other factors, including
the number, size, and distribution of antigen sites and
van der Waals forces, also contribute. Artificial alter-
ation of the normal ionic environment through the use
of intravenous solutions in patients who have lost
blood and as in vitro suspending media, such as
low–ionic-strength saline (LISS), causes complex prob-
lems for the blood banker when determining the pres-
ence and compatibility of IgG alloantibody.
IMMUNE RESPONSE TO BLOOD
PRODUCTS AND SUBSEQUENT
HEMAGGLUTINATION
The immune response has already been described in
some detail, with specific reference to antigen presen-
tation and the formation of antibodies. The chapter
has described the concepts of the primary and
secondary immune responses and the regulation of
these responses.
The blood banker may be primarily concerned
with the B-cell antibody product made in response to
antigenic material such as allogeneic (“foreign”: from
a donor other than the transfusion recipient) red cells
and sometimes white blood cells, platelets, and drugs.
Immunization, or sensitization (exposure to foreign
antigens resulting in immune response) to these sub-
stances, occurs through transfusion or pregnancy.
Cellular elements from the donor or fetus contain
antigens recognized by the immune system of the re-
cipient as nonself. When presented to the recipient,
these antigens are processed by the recipient’s im-
mune system and may result in the formation of de-
tectable antibody. This occurs in 30% to 70% of all
people transfused with blood products that contain
leukocytes (nonleukoreduced). The antibodies made
in response to foreign blood products may be of the
CHAPTER 5 Basic Immunologic Principles 67
IgG or IgM subclasses. IgM antibodies are usually the
result of the primary immune response, are of a rela-
tively low concentration, and are detectable within 3
to 4 weeks. On reexposure to a nonself antigen, a
secondary response may occur, with typical IgG anti-
body production in 1 to 2 days and in much larger
amounts than the IgM response. This secondary re-
sponse is known as the anamnestic response.
Many factors, known and unknown, affect the
primary and secondary responses. Among the most
important are the immunogenicity of the antigen, its
survival in the circulation, and the strength of the
immune system of the recipient. The immune
response of the recipient depends on factors such as
age, nutritional status, and prior exposure.
Mechanisms of Agglutination
Antigen–antibody reactions follow the law of mass
action7 in a simple combination reaction (Ab + Ag Δ
AbAg complex) that is followed by secondary and ter-
tiary reactions. The reactions are reversible and
depend on many factors, the most important of which
are goodness of fit of antibody-binding site and anti-
gen, complementarity of charge, the concentration
of antigen and antibody, the pH of the suspending
medium, temperature, and ionic strength.
Antibody Binding to Red Blood Cells
For red cell–antibody binding to form a visible agglu-
tination reaction, a minimum number of antibody
molecules must be bound to antigen. It follows that
the larger the number of antibodies bound on each red
cell, the stronger the observed reaction will be. Also,
increasing the serum-to-cell ratio has a desirable effect
regarding the appearance of observable agglutination;
the test sensitivity is increased. Conversely, increas-
ing the antigen concentration by increasing the
strength of the red cell suspension in a test system
results in lower sensitivity, and fewer antibody
molecules are bound per red cell.
The law of mass action also allows for a description
of binding in several stages. The primary reaction may
be viewed simply as one of recognition in which an
antigen and its specific antibody possess complemen-
tary structures that enable them to come into very close
apposition to each other. They then are held together by
weak intermolecular noncovalent bonds. The weak-
ness of these intermolecular bonds may or may not be
sufficient to hold the complex together. Several types of
noncovalent bonds have been described. The simplest
ionic bonds arise from electrostatic attraction of posi-
tive and negative charges, which are dramatically influ-
enced by the distance between the charges.
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68 UNIT 2 Genetic and Immunologic Principles

Weak hydrogen bonds result from the sharing of
hydrogen atoms between protons and contribute to
bonding. van der Waals forces are normally present
from the shifting in the ionic environment of various
positive cations and the resultant attraction of nega-
tive ions. (Remember that electrons are in constant
motion around the atomic nucleus.) Hydrophobic
bonds are extremely weak and result from water
molecule exclusion from the antigen–antibody com-
plex formation. Figure 5-14 describes the binding of
an antibody to its antigen.
Hemagglutination
The second stage of a red cell antigen–antibody reaction
involves agglutination, or, more precisely, hemaggluti-
nation. Hemagglutination as an observable reaction
may or may not occur as a consequence of a patient
becoming immunized to allogeneic red cell antigens,
and depends on many variables, including the amount
and type of antibody present; the size, number, and
location of available antigen sites; and the pH, tempera-
ture, and ionic strength of the test system.
Although much progress has been made in the bio-
chemical characterization of blood group antigens, their
location over the surface of the red cell membrane, and
their approximate numbers, many problems must be
resolved before a more complete picture emerges.
Essentially, however, the membrane surface should be
thought of as a fluid moiety of phospholipids and
glycolipids with antigen molecules having the ability to
move around and reorient themselves. Furthermore, the
antigens protrude from the membrane bilipid layer. The
reaction of specific antibodies largely depends on the pH
of the medium in which they are suspended. Most
blood group antibodies react within a relatively narrow
pH range, 6.5 to 7.5, but there are many exceptions to
this range. For example, pH-dependent forms of anti-M
and anti-Pr (Sp1) can be encountered.
Blood group antibodies, depending on their class,
may react in the range of 4°C to 37°C. The rate at which
antibody complexes with antigen increases with
temperatures up to 37°C, and the rate of dissociation
increases as the temperature is increased above that
temperature. This principle is used in all antigen–
antibody tests, from the compatibility test to the anti-
body elution procedure that uses heat to remove the
antibody from the red cell surface. The clinical signifi-
cance of antibodies reacting throughout this range is
discussed later, but as an example of the influence of
temperature, it has been demonstrated that the binding
of anti-D is 20 times more efficient at 37°C than at 4°C.
The important findings of Hughes-Jones7 and
Hughes-Jones et al.8 revealed that reducing the ionic
strength of the suspending medium results in an in-
crease in the antigen–antibody association rate. In other

Antigen

VL VH

◆ Interaction between the three dimensional structure of

the Ag and the cleft between the heavy and light chains.

◆ Non-covalent interaction. Weak van der Waals forces, H

bonding forces. Strong electrostatic and hydrophobic
forces.

Fab site of an
◆ The closer the 3D fit, the stronger the interaction, the
higher the affinity. Competition reactions. immunoglobin
molecule

FIGURE 5-14 Binding of antigen to its corresponding antibody.