CHAPTER

64
Antigen–Antibody Reactions in the
Laboratory
CHAPTER CONTENTS
Introduction
Types Of Diagnostic Tests
Agglutination
Precipitation (Precipitin)
Radioimmunoassay (RIA)
Enzyme-Linked Immunosorbent Assay (ELISA)
Immunofluorescence (Fluorescent Antibody)
Complement Fixation
Neutralization Tests
Immune Complexes
Hemagglutination Tests
Antiglobulin (Coombs) Test
Western Blot (Immunoblot)
Fluorescence-Activated Cell Sorting (Flow Cytometry)
Antigen–Antibody Reactions Involving Red Blood Cell Antigens
The ABO Blood Groups & Transfusion Reactions
Rh Blood Type & Hemolytic Disease of the Newborn
Self-Assessment Questions
Practice Questions: USMLE & Course Examinations

INTRODUCTION
Reactions of antigens and antibodies are highly specific. An antibody will react only
with the antigen that induced it or with a closely related antigen. Because of the great
specificity, reactions between antigens and antibodies are suitable for identifying

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one by using the other. This is the basis of serologic reactions. However, cross-
reactions between related antigens can occur, and these can limit the usefulness of
the test.
The results of many immunologic tests are expressed as a titer, which is defined
as the highest dilution of the specimen (e.g., serum) that gives a positive reaction in
the test. Note that a patient’s serum with an antibody titer of, for example, 1/64
contains more antibodies (i.e., is a higher titer) than a serum with a titer of, for
example, 1/4.
Table 64–1 describes the medical importance of serologic (antibody-based)
tests. Their major uses are in the diagnosis of infectious diseases, in the diagnosis of
autoimmune diseases, and in the typing of blood and tissues prior to transplantation.

Table 64–1 Major Uses of Serologic (Antibody-Based) Tests

Microorganisms and other cells possess a variety of antigens and thus induce
antisera containing many different antibodies (i.e., the antisera are polyclonal).
Monoclonal antibodies excel in the identification of antigens because cross-reacting
antibodies are absent (i.e., monoclonal antibodies are highly specific).

TYPES OF DIAGNOSTIC TESTS

Many types of diagnostic tests are performed in the immunology laboratory. Most of
these tests can be designed to determine the presence of either antigen or antibody.

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To do this, one of the components, either antigen or antibody, is known and the other
is unknown. For example, with a known antigen such as influenza virus, a test can
determine whether antibody to the virus is present in the patient’s serum.
Alternatively, with a known antibody, such as antibody to herpes simplex virus, a
test can determine whether viral antigens are present in cells taken from the patient’s
lesions.

Agglutination
In this test, the antigen is particulate (e.g., bacteria and red blood cells)1 or is an
inert particle (latex beads) coated with an antigen. Antibody, because it is divalent
or multivalent, cross-links the antigenically multivalent particles and forms a
latticework, and clumping (agglutination) can be seen. This reaction can be done in a
small cup or tube or with a drop on a slide. One very commonly used agglutination
test is the test that determines a person’s ABO blood group (Figure 64–1; see the
section on blood groups at the end of this chapter).

FIGURE 64–1 Agglutination test to determine ABO blood type. On the slide at
the bottom of the figure, a drop of the patient’s blood was mixed with antiserum
against either type A (left) or type B (right) blood cells. Agglutination (clumping)
has occurred in the drop on the left containing the type A antiserum but not in the
drop containing the type B antiserum, indicating that the patient is type A (i.e., has A
antigen on the red cells). The slide at the top shows that the red cells (circles) are
cross-linked by the antibodies (“Y” shapes) in the drop on the left but not in the drop
on the right. If agglutination had occurred in the right side as well, it would indicate
that the patient was producing B antigen as well as A and was type AB.

Precipitation (Precipitin)
In this test, the antigen is in solution. The antibody cross-links antigen molecules in

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variable proportions, and aggregates (precipitates) form. In the zone of
equivalence, optimal proportions of antigen and antibody combine; the maximal
amount of precipitates forms, and the supernatant contains neither an excess of
antibody nor an excess of antigen (Figure 64–2). In the zone of antibody excess,
there is too much antibody for efficient lattice formation, and precipitation is less
than maximal.2 In the zone of antigen excess, all antibody has combined, but
precipitation is reduced because many antigen–antibody complexes are too small to
precipitate (i.e., they are “soluble”).

FIGURE 64–2 Precipitin curve. In the presence of a constant amount of

antibody, the amount of immune precipitate formed is plotted as a function of
increasing amounts of antigen. In the top part of the figure, the binding of antigen ( )
and antibody (Y) in the three zones is depicted. In the zones of antibody excess and
antigen excess, a lattice is not formed and precipitation does not occur, whereas in
the equivalence zone, a lattice forms and precipitation is maximal. (Modified and
reproduced with permission from Stites D, Terr A, Parslow T, eds. Basic &
Clinical Immunology. 9th ed. Originally published by Appleton & Lange. Copyright
1997 McGraw-Hill.)

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 Precipitin tests can be done in solution or in semisolid medium (agar).
Precipitation in Solution
The concept of precipitation in solution is used clinically to measure the amount of
immunoglobulins (IgM, IgG, etc.) in the blood plasma. The lab test used is called
nephelometry, in which the amount of precipitate formed is measured by optical
density of the precipitate. In the test, antibody specific for IgM, IgG, IgA, or IgE is
reacted with the patient’s serum and the optical density measured. This value is then
compared with a standard curve, which displays the optical density caused by
known amounts of the immunoglobulins.
Precipitation in Agar
This is done as either single or double diffusion. It can also be done in the presence
of an electric field.
Single Diffusion— In single diffusion, antibody is incorporated into agar and
antigen is placed into a well. As the antigen diffuses with time, precipitation rings
form depending on the antigen concentration. The greater the amount of antigen in the
well, the farther the ring will be from the well. By calibrating the method, such
radial immunodiffusion is used to measure IgG, IgM, complement components, and
other substances in serum. (IgE cannot be measured because its concentration is too
low.)
Double Diffusion—In double diffusion, antigen and antibody are placed in different
wells in agar and allowed to diffuse and form concentration gradients. Where
optimal proportions (see zone of equivalence, above) occur, lines of precipitate
form (Figure 64–3). This method (Ouchterlony) indicates whether antigens are
identical, related but not identical, or not related (Figure 64–4).

FIGURE 64–3 Double diffusion in agar. Antigen is placed in the well on the

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left, and antibody is placed in the well on the right. The antigen and antibody diffuse
through the agar and form a precipitate in the zone of equivalence. Close to the
antigen-containing well is the zone of antigen excess, and close to the antibody-
containing well is the zone of antibody excess. No precipitate forms in the zones of
antigen and antibody excess.

FIGURE 64–4 Double-diffusion (Ouchterlony) precipitin reactions. In these

Ouchterlony reactions, wells are cut into an agar plate and various antigens and
antisera are placed in the wells. The antigens and antibodies diffuse toward each
other within the agar, and a line of precipitate forms in the zone of equivalence.
Close to the antigen-containing well, a zone of antigen excess exists and no
precipitate forms; close to the antibody-containing well, a zone of antibody excess
exists and no precipitate forms. A and B are unrelated antigens (i.e., they have no
epitopes in common). B and C are related antigens (i.e., they have some epitopes in
common but some that are different). For example, chicken lysozyme (well B) and
duck lysozyme (well C) share some epitopes because they are both lysozymes but
have unique epitopes as well because they are from different species. The line of
identity between B and C is caused by the reaction of the anti-B antibody with the
shared epitopes on antigens B and C. The spur pointing toward well 4 is caused by
the reaction of some of the anti-B antibody with the unique epitopes on antigen B in
well 3. These lines of partial identity occur because antibody to B (chicken
lysozyme) is polyclonal and has some immunoglobulins that react with the epitopes
common to chicken and duck lysozyme and other immunoglobulins that react only
with the epitopes unique to chicken lysozyme. (Modified and reproduced with
permission from Brooks GF et al. Medical Microbiology. 19th ed. Originally
published by Appleton & Lange. Copyright 1991 McGraw-Hill.)

Precipitation in Agar with an Electric Field

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Immunoelectrophoresis—A serum sample is placed in a well in agar on a glass
slide (Figure 64–5). A current is passed through the agar, and the proteins move in
the electric field according to their charge and size. Then a trough is cut into the agar
and filled with antibody. As the antigen and antibody diffuse toward each other, they
form a series of arcs of precipitate. This permits the serum proteins to be
characterized in terms of their presence, absence, or unusual pattern (e.g., human
myeloma protein).

FIGURE 64–5 Immunoelectrophoresis. A: Human serum placed in the central

well is electrophoresed, and the proteins migrate to different regions (orange
ellipses). Antiserum to human serum is then placed in the elongated trough (gray
areas). B: Human serum proteins and antibodies diffuse into agar. C: Precipitate
arcs (orange lines) form in the agar. (Modified and reproduced with permission
from Stites D, Terr A, Parslow T, eds. Basic & Clinical Immunology. 9th ed.
Originally published by Appleton & Lange. Copyright 1997 McGraw-Hill.)

Counter-Immunoelectrophoresis— This method relies on movement of antigen

toward the cathode and of antibody toward the anode during the passage of electric
current through agar. The meeting of the antigen and antibody is greatly accelerated
by this method and is made visible in 30 to 60 minutes. This has been applied to the

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detection of bacterial and fungal polysaccharide antigens in cerebrospinal fluid.

Radioimmunoassay (RIA)
This method is used for the quantitation of antigens or haptens that can be
radioactively labeled. It is based on the competition for specific antibody between
the labeled (known) and the unlabeled (unknown) concentration of material. The
complexes that form between the antigen and antibody can then be separated and the
amount of radioactivity measured. The more unlabeled antigen is present, the less
radioactivity there is in the complex. The concentration of the unknown (unlabeled)
antigen or hapten is determined by comparison with the effect of standards. RIA is a
highly sensitive method and is commonly used to assay hormones or drugs in serum.
The radioallergosorbent test (RAST) is a specialized RIA that is used to measure the
amount of serum IgE antibody that reacts with a known allergen (antigen).

Enzyme-Linked Immunosorbent Assay (ELISA)

This method can be used for the quantitation of either antigens or antibodies in
patient specimens. It is based on covalently linking an enzyme to a known antigen or
antibody, reacting the enzyme-linked material with the patient’s specimen, and then
assaying for enzyme activity by adding the substrate of the enzyme. The method is
nearly as sensitive as RIA yet requires no special equipment or radioactive labels
(Figure 64–6).

FIGURE 64–6 Enzyme-linked immunosorbent assay (ELISA). The term

enzyme-linked refers to the covalent binding (linking) of an enzyme to antibody to
human IgG. If the patient has antibodies to the microbial or viral antigen, those
antibodies will bind to the microbial or viral antigens. The antibody to human IgG
linked to the enzyme will then bind to the patient’s antibodies. Then when the

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substrate of the enzyme is added, the substrate changes color, indicating that the
patient’s serum contained antibodies.

For measurement of antibody, known antigens are fixed to a surface (e.g., the
bottom of small wells on a plastic plate), incubated with dilutions of the patient’s
serum, washed, and then reincubated with antibody to human IgG labeled with an
enzyme (e.g., horseradish peroxidase). Enzyme activity is measured by adding the
substrate for the enzyme and estimating the color reaction in a spectrophotometer.
The amount of antibody bound is proportional to the enzyme activity. The titer of
antibody in the patient’s serum is the highest dilution of serum that gives a positive
color reaction.

Immunofluorescence (Fluorescent Antibody)

Fluorescent dyes (e.g., fluorescein and rhodamine) can be covalently attached to
antibody molecules and made visible by ultraviolet (UV) light in the fluorescence
microscope. Such “labeled” antibody can be used to identify antigens (e.g., on the
surface of bacteria such as streptococci and treponemes, in cells in histologic
section, or in other specimens) (Figure 64–7). The immunofluorescence reaction is
direct when known labeled antibody interacts directly with unknown antigen and
indirect when a two-stage process is used. For example, known antigen is attached
to a slide, the patient’s serum (unlabeled) is added, and the preparation is washed; if
the patient’s serum contains antibody against the antigen, it will remain fixed to it on
the slide and can be detected on addition of a fluorescent dye–labeled antibody to
human IgG and examination by UV microscopy. The indirect test is often more
sensitive than direct immunofluorescence, because more labeled antibody adheres
per antigenic site. Furthermore, the labeled antiglobulin becomes a “universal
reagent” (i.e., it is independent of the nature of the antigen used because the antibody
to IgG is reactive with all human IgG).

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FIGURE 64–7 Fluorescent antibody test. A: In the direct fluorescent antibody
test, the fluorescent dye is attached directly to the antibody that is interacting with the
antigen (dark triangles) on the surface of the cell. B: In the indirect fluorescent
antibody test, the fluorescent dye is attached to antibody made against human IgG.

Complement Fixation
The complement system consists of 20 or more plasma proteins that interact with one
another and with cell membranes. Each protein component must be activated
sequentially under appropriate conditions for the reaction to progress. Antigen–
antibody complexes are among the activators, and the complement fixation test can
be used to identify one of them if the other is known.
The reaction consists of the following two steps (Figure 64–8): (1) Antigen and
antibody (one known and the other unknown; e.g., use a known antigen and determine
whether a patient’s serum contains antibodies to that antigen) are mixed, and a
measured amount of complement (usually from guinea pig) is added. If the antigen
and antibody match, they will combine and use up (“fix”) the complement. (2) An
indicator system, consisting of “sensitized” red blood cells (i.e., red blood cells
plus anti–red blood cell antibody), is added. If the antibody matched the antigen in
the first step, complement was fixed and less (or none) is available to attach to the
sensitized red blood cells. The red blood cells remain unhemolyzed (i.e., the test is
positive) because the patient’s serum had antibodies to that antigen. If the antibody
did not match the antigen in the first step, complement is free to attach to the
sensitized red blood cells and they are lysed (i.e., the test is negative).

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FIGURE 64–8 Complement fixation. Left: Positive reaction (i.e., the patient’s
serum contains antibody). If a known antigen is mixed with the patient’s serum
containing antibody against that antigen, then complement (solid circles) will be
fixed. Because no complement is left over, the sensitized red cells are not lysed.
Right: Negative reaction. If a known antigen is mixed with the patient’s serum that
does not contain antibody against that antigen, complement (solid circles) is not
fixed. Complement is left over and the sensitized red cells are lysed. Ab, antibody;
Ag, antigen.

Complement must be carefully standardized, and the patient’s serum must be

heated to 56°C for 30 minutes to inactivate any human complement activity. The
antigen must be quantitated. The result is expressed as the highest dilution of serum
that gives positive results. Controls to determine whether antigen or antibody alone
fixes complement are needed to make the test results valid. If antigen or antibody
alone fixes complement, it is said to be anticomplementary.

Neutralization Tests
These use the ability of antibodies to block the effect of toxins or the infectivity of
viruses. They can be used in cell culture (e.g., inhibition of cytopathic effect and

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plaque-reduction assays) or in host animals (e.g., mouse protection tests).

Immune Complexes
Immune complexes in tissue can be stained with fluorescent complement. Immune
complexes in serum can be detected by binding to C1q or by attachment to certain
(e.g., Raji lymphoblastoid) cells in culture.

Hemagglutination Tests
Many viruses clump red blood cells from one species or another (active
hemagglutination). This can be inhibited by antibody specifically directed against the
virus (hemagglutination inhibition) and can be used to measure the titer of such
antibody. Red blood cells also can absorb many antigens and, when mixed with
matching antibodies, will clump (this is known as passive hemagglutination,
because the red cells are passive carriers of the antigen).

Antiglobulin (Coombs) Test

Some patients with certain diseases (e.g., hemolytic disease of the newborn [Rh
incompatibility] and drug-related hemolytic anemias) become sensitized but do not
exhibit symptoms of disease. In these patients, antibodies against the red cells are
formed and bind to the red cell surface but do not cause hemolysis. These cell-bound
antibodies can be detected by the direct antiglobulin (Coombs) test, in which
antiserum against human immunoglobulin is used to agglutinate the patient’s red
cells. In some cases, antibody against the red cells is not bound to the red cells but is
in the serum and the indirect antiglobulin test for antibodies in the patient’s serum
should be performed. In the indirect Coombs test, the patient’s serum is mixed with
normal red cells, and antiserum to human immunoglobulins is added. If antibodies
are present in the patient’s serum, agglutination occurs.

Western Blot (Immunoblot)

This test is typically used to determine whether a positive result in a screening
immunologic test is a true-positive or a false-positive result. For example, patients
who are positive in the screening ELISA for human immunodeficiency virus (HIV)
infection or for Lyme disease should have a Western blot test performed. Figure 64–
9 illustrates a Western blot test for the presence of HIV antibodies in the patient’s
serum. In this test, HIV proteins are separated electrophoretically in a gel, resulting
in discrete bands of viral protein. These proteins are then transferred from the gel
(i.e., blotted) onto filter paper, and the person’s serum is added. If antibodies are
present, they bind to the viral proteins (primarily gp41 and p24) and can be detected

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by adding antibody to human IgG labeled with either radioactivity or an enzyme such
as horseradish peroxidase, which produces a visible color change when the enzyme
substrate is added.

FIGURE 64–9 Western blot (immunoblot test). In this test, microbial or viral
proteins are separated on an acrylamide gel and then transferred (blotted) onto
paper. The patient’s serum then interacts with the separated proteins. If antibodies
are present in the patient’s serum, they bind to the proteins. The patient’s antibodies
are then detected by using labeled antibody to human IgG.

Fluorescence-Activated Cell Sorting (Flow Cytometry)

This test is commonly used to measure the number of the various types of
immunologically active blood cells (Figure 64–10). For example, it is used in HIV-
infected patients to determine the number of CD4-positive T cells. In this test, the
patient’s cells are labeled with monoclonal antibody to the protein specific to the
cell of interest (e.g., CD4 protein if the number of helper T cells is to be
determined). The monoclonal antibody is tagged with a fluorescent dye, such as
fluorescein or rhodamine. Single cells are passed through a laser light beam, and the
number of cells that fluoresce is counted by use of a machine called a fluorescence-
activated cell sorter (FACS).

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FIGURE 64–10 Flow cytometry. At the top of the figure, a cell has interacted
with monoclonal antibody labeled with a fluorescent dye. As the cell passes down
the tube, ultraviolet light causes the dye to fluoresce and a sensor counts the cell.
Farther down the tube, an electrical charge can be put on the cell, which allows it to
be deflected into a test tube and subjected to additional analysis.

ANTIGEN–ANTIBODY REACTIONS INVOLVING

RED BLOOD CELL ANTIGENS
Many different blood group systems exist in humans. Each system consists of a gene
locus specifying antigens on the erythrocyte surface. The two most important blood
groupings, ABO and Rh, are described next.

The ABO Blood Groups & Transfusion Reactions

All human erythrocytes contain alloantigens (i.e., antigens that vary among
individual members of a species) of the ABO group. A person’s ABO blood group
is a very important determinant of the success of both blood transfusions and organ
transplants.
The A and B genes encode enzymes that add specific sugars to the end of a

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polysaccharide chain on the surface of many cells, including red cells (Figure 64–
11). People who inherit neither gene are type O. The genes are codominant, so
people who inherit both genes are type AB. People who are either homozygous AA
or heterozygous AO are type A, and, similarly, people who are either homozygous
BB or heterozygous BO are type B.

FIGURE 64–11 ABO blood groups. Structures of the terminal sugars that
determine ABO blood groups are shown. Blood group O cells have H antigen on
their surface; blood group A cells have N-acetylgalactosamine added to the end of
the H antigen; and blood group B cells have galactosamine added to the end of the H
antigen. (Reproduced with permission from Stites DP, Stobo JD, Wells JV, eds.
Basic & Clinical Immunology. 6th ed. Originally published by Appleton & Lange.
Copyright 1987 McGraw-Hill.)

The A and B antigens are carbohydrates that differ by a single sugar. Despite this
small difference, A and B antigens do not cross-react. Erythrocytes have three
terminal sugars in common on their surface: N-acetylglucosamine, galactose, and
fucose. These three sugars form the H antigen (Figure 64–11). People who are blood
group O have only the H antigen on the surface of their red cells. People who are
blood group A have N-acetylgalactosamine added to the galactose of the H antigen,
whereas people who are blood group B have galactose added to the galactose of the

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H antigen.
There are four combinations of the A and B antigens, called A, B, AB, and O
(Table 64–2). A person’s blood group is determined by mixing the person’s blood
with antiserum against A antigen on one area on a slide and with antiserum against B
antigen on another area (Figure 64–1). If agglutination occurs only with A antiserum,
the blood group is A; if it occurs only with B antiserum, the blood group is B; if it
occurs with both A and B antisera, the blood group is AB; and if it occurs with
neither A nor B antisera, the blood group is O.

Table 64–2 ABO Blood Groups

The plasma contains antibody against the absent antigens (i.e., people with blood
group A have antibodies to B in their plasma). These antibodies are formed against
cross-reacting bacterial or food antigens, are first detectable at 3 to 6 months of age,
and are of the IgM class. Individuals are tolerant to their own blood group antigens,
and therefore a person with blood group A does not form antibodies to A antigen.
The end result is that antigen and corresponding antibody do not coexist in the same
person’s blood. Transfusion reactions occur when incompatible donor red blood
cells are transfused (e.g., if group A blood were transfused into a group B person
[because anti-A antibody is present]). The red cell–antibody complex activates
complement, and a reaction consisting of shock caused by large amounts of C3a and
C5a (anaphylatoxins) and hemolysis caused by C5, C6, C7, C8, and C9 (membrane
attack complex) occurs (Figure 64–12).

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FIGURE 64–12 Transfusion reaction. Top panel: Red blood cells bearing A
antigen are transfused into a person who is type B and therefore has antibodies to A
antigen. Middle panel: Anti-A antibodies bind to A antigen on the red cells causing
agglutination of red cells that can block movement of blood through capillaries
causing anoxia to tissue. Bottom panel: Complement is activated by the antigen–
antibody complexes and the membrane attack complex lyses the red cells, causing
hemolysis and anemia. (Reproduced with permission from Cowan MK, Talaro KP,
eds. Microbiology: A Systems Approach. New York: McGraw-Hill; 2009.)

To avoid antigen–antibody reactions that would result in transfusion reactions, all

blood for transfusions must be carefully matched (i.e., erythrocytes are typed for
their surface antigens by specific sera). As shown in Table 64–2, persons with group
O blood have no A or B antigens on their red cells and so are universal donors (i.e.,
they can give blood to people in all four groups) (Table 64–3). Note that type O
blood has A and B antibodies. Therefore when type O blood is given to a person
with type A, B, or AB blood, you might expect a reaction to occur. A clinically

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detectable reaction does not occur because the donor antibody is rapidly diluted
below a significant level. Persons with group AB blood have neither A nor B
antibody and thus are universal recipients.

Table 64–3 Compatibility of Blood Transfusions Between ABO Blood Groups 1

In addition to red blood cells, the A and B antigens appear on the cells of many
tissues. Furthermore, these antigens can be secreted in saliva and other body fluids.
Secretion is controlled by a secretor gene. Approximately 85% of people carry the
dominant form of the gene, which allows secretion to occur.
ABO blood group differences can lead to neonatal jaundice and anemia, but the
effects on the fetus are usually less severe than those seen in Rh incompatibility (see
next section). For example, mothers with blood group O have antibodies against both
A and B antigens. These IgG antibodies can pass the placenta and, if the fetus is
blood group A or B, cause lysis of fetal red cells. Mothers with either blood group
A or B have a lower risk of having a neonate with jaundice because these mothers
produce antibodies to either B or A antigens, respectively, that are primarily IgM,
and IgM does not pass the placenta.

Rh Blood Type & Hemolytic Disease of the Newborn

About 85% of humans have erythrocytes that express the Rh(D) antigen [i.e., are
Rh(D)+]. When an Rh(D)– person is transfused with Rh(D)+ blood or when an
Rh(D)– woman has an Rh(D)+ fetus (the D gene being inherited from the father), the
Rh(D) antigen will stimulate the development of antibodies (Table 64–4). This
occurs most often when the Rh(D)+ erythrocytes of the fetus leak into the maternal
circulation during delivery of the first Rh(D)+ child (Figure 64–13).

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Table 64–4 Rh Status and Hemolytic Disease of the Newborn

FIGURE 64–13 Hemolytic disease of the newborn (erythroblastosis fetalis).

Left panel: Fetal red cells (RBCs) bearing the Rh antigen enter the mother’s blood
when the placenta separates during the birth of the first Rh-positive child. IgG

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antibodies to Rh antigen are then produced by the mother. Center panel: During a
second pregnancy with an Rh-positive fetus, IgG antibodies pass from the mother
into the fetus via the placenta. The antibodies bind to the fetal red cells, complement
is activated, and the membrane attack complex lyses the fetal red cells. Right panel:
Anemia and jaundice occur in the fetus/newborn. As a result of the anemia, large
numbers of erythroblasts are produced by the bone marrow and are seen in the blood
of the newborn. (Reproduced with permission from Cowan MK, Talaro KP, eds.
Microbiology: A Systems Approach. New York: McGraw-Hill; 2009.)

Subsequent Rh(D)+ pregnancies are likely to be affected by the mother’s anti-D

antibody, and hemolytic disease of the newborn (erythroblastosis fetalis) often
results. This disease results from the passage of maternal IgG anti-Rh(D) antibodies
through the placenta to the fetus, with subsequent lysis of the fetal erythrocytes. The
direct antiglobulin (Coombs) test is typically positive (see earlier description of the
Coombs test).
The problem can be prevented by administration of high-titer Rh(D) immune
globulins (Rho-Gam) to an Rh(D)– mother at 28 weeks of gestation and immediately
upon the delivery of an Rh(D)+ child. These antibodies promptly attach to Rh(D)+
erythrocytes and prevent their acting as sensitizing antigen. This prophylaxis is
widely practiced and effective.

SELF-ASSESSMENT QUESTIONS
1. Which one of the following laboratory tests would be the best to determine the
number of CD4-positive cells in the blood of a patient infected with HIV?
(A) Agglutination
(B) Complement fixation
(C) Enzyme-linked immunosorbent assay (ELISA)
(D) Flow cytometry
(E) Immunoelectrophoresis
2. You have just received a lab report that says your patient is positive for IgM
antibody to Borrelia burgdorferi in an enzyme-linked immunosorbent assay
(ELISA). This supports your clinical impression that the patient has Lyme
disease. Which one of the following best describes how the ELISA was
performed? (For brevity, the wash steps have been left out.)
(A) The patient’s serum was reacted with antibody to human mu heavy chain.

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