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IMMUNOHAEMATOLOGY AND HUMAN BLOOD TRANSFUSION.
IMMUNOHEMATOLOGY;
Section I. THE IMMUNE RESPONSE AND THE INTERACTION OF ANTIGENS,
ANTIBODIES, AND COMPLEMENT IN IMMUNOHEMATOLOGY
1-1. BACKGROUND
Immunology, a field once dominated by bacteriologists, has become important to
scientists in many other areas. The field of immunohematology came into being when
Landsteiner discovered that the blood antigens (ABO) present on RBCs (RBCs) would
react with their respective antibodies present in plasma, and that this reaction had great
clinical significance. Since that time, many discoveries in this field have added to the
understanding of immune mechanisms operative in health and disease. It is important
that scientists working in areas associated with blood transfusion understand basic
immunology and try to be familiar with the recent advances in this field that might relate
directly to their work.
1-2. THE IMMUME RESPONSE
According to Roitt, the basic of immunology is memory, specificity, and the
recognition of "nonself". The original basis for this was the protection (immunity)
afforded by exposure to infectious illness. The first contact with an infectious organism
imprints some information (for example, memory) so that the body will recognize and
attack that organism when it encounters it in the future. The protection is usually
specific (for example, only against the original infecting organism). The body also has
to recognize that organism as being foreign (that is., "nonself"). The substance initially
responsible for an immune response is known as an antigen or more specifically an
immunogen.
ANTIGENS;
a. Antigens are substances that can induce a specific immunologic response orcan interact with specific antibody or immune cells "in vivo" or "in vitro". The immune response can be either humoral or cellular (paragraph 1-4). Blood group serology is mainly concerned with the humoral response that leads to the production of free antibody in the plasma. The antibodies, under appropriate conditions of reaction (temperature, pH, ionic strength, and so forth.), will react specifically with the antigen in some observable way (for example., agglutination, hemolysis).
b. An antigen [1]contains structural chemical groups in a specific three-dimensional arrangement, known as antigenic determinants (epitopes), which are lacking or foreign to the immunized animal. Each antigen can contain many of these epitopes. The specific three-dimensional shape of these antigenic determinants, or chemical groupings, is what determines the specificity of its reaction with a particular antibody molecule.
c. An Important factor affecting the immunogenicity of an antigen is its molecular size. Immunogenic molecules are rarely less than 4,000 daltons. Much smaller molecules (for example, drugs such as penicillin) can be immunogenic if coupled to a
protein “carrier” of larger molecular weight. Such a molecule is termed a hapten and can be defined as a small molecule that, by itself, cannot stimulate antibody synthesis
but will combine with antibody once formed. Indeed, most of our basic understanding of
antigen specificity came from work by Landsteiner using haptens.
d. Blood group antigens are chemical groupings present on the RBC membrane.
We are only just beginning to learn the exact nature of these determinants. The ABH
antigens have been the most thoroughly studied and when present on RBCs are
predominantly glycolipids. A and B antigens are composed of the same fatty acids and
sugars, the difference in specificity being caused by the terminal sugar in the chain of
sugars joined to the fatty acid backbone. The specificity is a result not only of the
particular sugar but also the configuration of the end grouping it forms. As the sugars
responsible for A or B specificity (N-acetylgalactosamine and galactose, respectively)
are structurally identical except for the substitution of an hydroxyl group for an N-
acetylamino group at carbon atom number two, they serve as a good example of the
remarkable specificity of antigen-antibody reactions.
e. Proteins are direct gene products, whereas carbohydrates, such as the A and
B antigens, are indirect products of genes (for example, A or B genes). The direct
(protein) products of the A and B genes are enzymes that recognize and then transfer
specific sugars from their nucleotide carriers to specific acceptor molecules. Thus, the
A gene product is an N-acetyl-D-galactose-aminyltransferase enzyme and the B gene product in
a D-galactosyltransferase.
f. The biologic role of blood group antigens, if any, is at present unknown. The
ABH antigens are widely distributed throughout the body, being present on many types
of cells, organs, and body fluids. Some antigens such as Rh and Kell (K) appear to play
a part in cell membrane integrity. Rare individuals lacking Rh antigens (Rhnull) on their
RBCs often have an associated hemolytic anemia (“Rh-null syndrome”), whereas, in
contrast, rare individuals lacking A, B, and H antigens (Bombay phenotype) do not. It
has been suggested that this is because the ABH antigens are glycolipids projecting
above the cell membrane, whereas Rh appears to be lipoprotein, an integral part of the
RBC membrane. An association between a rare inherited defect of neutrophil
bactericidal function (chronic granulomatous disease) and the Kell blood group system
has recently been described. Another report suggests a possible relationship between
the Duffy blood group antigens and resistance to malaria. There are many other
associations of blood groups with disease, particularly malignancy; many of them are
purely statistical and their causes unknown.
An Important factor affecting the immunogenicity of an antigen is its molecular
size. Immunogenic molecules are rarely less than 4,000 daltons. Much smaller
molecules (for example, drugs such as penicillin) can be immunogenic if coupled to a
protein “carrier” of larger molecular weight. Such a molecule is termed a hapten and
can be defined as a small molecule that, by itself, cannot stimulate antibody synthesis
but will combine with antibody once formed. Indeed, most of our basic understanding of
antigen specificity came from work by Landsteiner using haptens.
d. Blood group antigens are chemical groupings present on the RBC membrane.
We are only just beginning to learn the exact nature of these determinants. The ABH
antigens have been the most thoroughly studied and when present on RBCs are
predominantly glycolipids. A and B antigens are composed of the same fatty acids and
sugars, the difference in specificity being caused by the terminal sugar in the chain of
sugars joined to the fatty acid backbone. The specificity is a result not only of the
particular sugar but also the configuration of the end grouping it forms. As the sugars
responsible for A or B specificity (N-acetylgalactosamine and galactose, respectively)
are structurally identical except for the substitution of an hydroxyl group for an N�
acetylamino group at carbon atom number two, they serve as a good example of the
remarkable specificity of antigen-antibody reactions.
e. Proteins are direct gene products, whereas carbohydrates, such as the A and
B antigens, are indirect products of genes (for example, A or B genes). The direct
(protein) products of the A and B genes are enzymes that recognize and then transfer
specific sugars from their nucleotide carriers to specific acceptor molecules. Thus, the
A gene product is an N-acetyl-D-galactose-aminyltransferase enzyme and the B gene product in
a D-galactosyltransferase.
f. The biologic role of blood group antigens, if any, is at present unknown. The
ABH antigens are widely distributed throughout the body, being present on many types
of cells, organs, and body fluids. Some antigens such as Rh and Kell (K) appear to play
a part in cell membrane integrity. Rare individuals lacking Rh antigens (Rhnull) on their
RBCs often have an associated hemolytic anemia (“Rh-null syndrome”), whereas, in
contrast, rare individuals lacking A, B, and H antigens (Bombay phenotype) do not. It
has been suggested that this is because the ABH antigens are glycolipids projecting
above the cell membrane, whereas Rh appears to be lipoprotein, an integral part of the
RBC membrane. An association between a rare inherited defect of neutrophil
bactericidal function (chronic granulomatous disease) and the Kell blood group system
has recently been described. Another report suggests a possible relationship between
the Duffy blood group antigens and resistance to malaria. There are many other
associations of blood groups with disease, particularly malignancy; many of them are
purely statistical and their causes unknown.
ANTIBODY SYNTHESIS
a. The Process of Antibody Synthesis. When an antigen enters the body, it
may evoke a humoral response, in which antibodies are synthesized by plasma cells
and released into the body fluids (for example, plasma), and/or a cellular response, in
which lymphocytes participate in cell-mediated immunity (for example, rejection of
transplanted tissue and delayed hypersensitivity). That two different responses were
present was originally shown by Chase and Landsteiner in the early 1940s when they
demonstrated that some kinds of immune reaction could be transferred from one animal
to another by the exchange of living cells, whereas others could be transferred by blood
serum. The cells required for the former experiment were lymphocytes. It was not until
the early 1960s that involvement of the lymphocyte was proven.
b. Lymphocyte PopulatioNS. Stem cells from the bone marrow are thought to
differentiate to form two distinct lymphocyte populations. The cells that pass through
the thymus become known as T-lymphocytes (T-cells) and the others that are
independent of the thymus B-lymphocytes (B-cells). Although these lymphocytes look
similar by conventional light or electron microscopy, they do look very different by
scanning electron microscopy, and also they can be differentiated by a variety of
surface markers. Their functions are of course different, but there is mounting evidence
for the possibility of cooperation between the two systems.
c. T-Lymphocytes. Once leaving the thymus, where they are known as
thymocytes, the T-lymphocytes are immunocompetent, that is to say, capable of
participating in an immune response. This is the basis of cellular immunity.
T-lymphocytes constitute the greater part of the recirculating pool of small lymphocytes
and have a relatively long half-life. When they encounter an antigen (which may have
to be first processed by a macrophage), they transform to lymphoblasts (See
figure 1-1). These T-lymphoblasts, which have no demonstrable intracellular
immunoglobulin, have several functions:
(1) They divide further into primed antigen-sensitive cells, which provide
immunologic memory because of their long life span.
(2) They release a number of soluble factors (lymphokines) which mediate
delayed-type hypersensitivity.
(3) They are “killer” cells, which are cytotoxic for cells bearing the
histo-compatibility antigens of a graft or tumor cells.
(4) They may cooperate with the humoral system by triggering
B-lymphocytes.
B-Lymphocytes. The B-lymphocyte gets its name from the Bursa of
Fabricius, a lymphoid organ present in birds, which controls the production of
lymphocytes responsible for making humoral antibody. The equivalent organ in man has
yet to be found. Thymus-independent, or B-lymphocytes synthesize and excrete
specific antibodies (surface immunoglobulins) that serve as receptors for antigens.
When triggered by antigen, the B-lymphocytes change to plasma cells, which are
responsible for the excretion of free antibody into the body fluids (for example, humoral
antibody), see figure 1-1. There is much evidence to suggest that macrophages are
required to process antigens for appropriate presentation to lymphocytes before the
humoral response occurs. In addition, many antigens appear to require the cooperation
of both B- and T-lymphocytes. The mechanisms by which T- and B-lymphocytes
interact are complex and far from clear at present. As mentioned previously, it is
humoral antibodies that are dealt with routinely in blood transfusion science, but
possibly cellular reactions will increase in importance in the future.
e. Differentiation of T- and B-Lymphocytes. Approximately 25 percent of
human blood lymphocytes are B cells, 70 percent T cells, and 5 percent have neither T
nor B markers (they are called “null cells”). Immunoglobulins are readily demonstrable
on B, but not T-lymphocytes by immunofluorescence. T- but not B-lymphocytes will
form "spontaneous” rosettes with unsensitized sheep erythrocytes.
PRIMARY AND SECONDARY IMMUNE RESPONSES
a. Following a first exposure to a foreign antigen, specific antibodies can appear
after about five days, rise slowly to a modest level, remain for a variable period, then
gradually decline, eventually becoming undetectable, until further stimulation occurs.
The first antibodies produced in this primary response are usually lgM, but eventually
other immunoglobulins [2](for example, lgG) may appear. The type of antigen and the
route of administration will influence the pattern observed.
b. After the primary response, a second dose of the same antigen, given days or
even years later, will usually elicit an intense and accelerated secondary (memory)
response. The serum antibody usually begins to rise within two or three days, reaching
a peak in about 10 days. In this secondary response, lgM antibody is often transiently
produced, following a similar pattern to the primary response, but the predominant
antibody produced is lgG, which rises to a much greater concentration than the lgM, and
remains in the plasma much longer. The secondary response is sometimes called an
anamnestic response.
IMMUNIZATION TO BLOOD-GROUP ANTIGENS
a. Within a few months after birth, an infant makes anti-A and/or anti-B, if lacking
those antigens on its RBCs. Such antibodies are termed naturally occurring since they
have no apparent antigenic stimulus. Experiments in chicks have shown that these
antibodies probably develop as a result of exposure to bacterial antigens, closely
related chemically to blood group antigens (for example, Escherichia coli has an antigen
on its membrane closely resembling human B antigen). Naturally occurring antibodies
to antigens other than ABO are also often encountered, particularly in the I, Lewis, P,
and MN systems. These antibodies are usually lgM and react better at lower
temperatures.
b. Immune antibodies to blood group antigens usually develop as a result of
pregnancy, transfusion, or immunization (intential sensitization). Following
immunization, lgM antibodies are often seen first, followed by lgG antibodies, which
often predominate. These antibodies usually react better at 37ºC.
c. Antibodies other than anti-A or anti-B are usually called “irregular", "atypical",
or "unexpected” antibodies. The preferred term is unexpected.
d. There is extensive evidence in animals, such as mice, that the immune
response is genetically controlled (by the so-called Ir genes). It has been suggested
that this may apply in man also. Studies in man on the immune response to the Rh (D)
antigen indicate that approximately 30 percent of the Rho(D)-negative individuals
appear to be incapable of forming anti-Rho(D) even after repeated and/or large
transfusions of Rho(D)-positive blood. The antibody response in individuals who do
make antibody will depend on many factors, including the relative potency of the
antigen, the route of immunization, and the amount of blood given.
ANTIBODY STRUCTURE, FUNCTION, AND PROPERTIES
a. Plasma proteins with antibody activity are called immunoglobulins (lg).
During the last ten years, great advances have been made in defining their structure,
physiochemical properties, antigenic characteristics, serologic behavior, and biological
properties.
b. Each immunoglobulin molecule consists of basic units, each composed of
four polypeptide chains, two light chains (L) and two heavy chains (H), held together by
covalent disulfide bonds (S-S), and noncovalent interactions (see figure 1-2).
c. Five classes of immunoglobulins have been recognized on the basis of
antigenic differences in the heavy chain: lgG, lgA, lgM, lgD, and lgE (see figure 1-3). No
blood groups antibodies have yet been found to be lgD or lgE. There are two types of
light chains (kappa chain and lambda chain), which are common to, and found in, all
five immunoglobulin classes, but each individual immunoglobulin molecule has only one
type of light chain. Approximately 66 percent of the molecules of each class have
kappa light chains, and 33 percent have lambda light chains.
ANTIGEN-ANTIBODY REACTIONS IN BLOOD GROUP SEROLOGY
a. Antibodies may react with their specific antigens in a number of ways. The
following reactions have all been used to demonstrate “in vitro” antigen-antibody
reactions in blood transfusion science:
(1) Agglutination.
(2) Hemolysis.
(3) Inhibition.
(4) Absorption and elution.
(5) Precipitation.
(6) Complement-fixation.
(7) Radioimmunoassay.
(8) Fluorescence.
b. The first two methods are the most commonly used in blood group serology
and will be discussed in more detail. Inhibition and absorption/elution techniques,
although not used every day in the routine blood bank, are used regularly in the forensic
laboratory (for example, blood grouping of blood stains) and in reference laboratories.
Absorption techniques lead to a decrease in antibody activity following treatment of a
serum with RBCs having the appropriate antigens; elution refers to the technique used
to dissociate or remove antibody bound to sensitized RBCs. Precipitation, complement�
fixation, and radioimmunoassay have been utilized more in blood banks in the last few
years, particularly for the detection of hepatitis virus. Fluorescence has been used to
demonstrate blood group antigens (for example, ABH) in tissues.
AGGLUTINATION
a. Background. It is convenient to consider antibody-mediated agglutination of
RBCs as involving two distinct stages. First, there is physical attachment of antibody to
the antigenic determinant on the RBC surface. This stage, representing the specific
immunochemical reaction, is referred to as sensitization. It may go on to involve the
binding or fixing of complement components. The second stage involves agglutination
of the sensitized cells. Agglutination results from collision of sensitized cells, allowing
cross-linking of cells to occur by the formation of antibody bridges. As the aim of blood
group serology is to obtain maximum sensitivity without loss of specificity, it is important
to understand and recognize the factors that influence the complex agglutination
phenomenon.
b. Factors Affecting the First Stage (Sensitization). Red blood cell
sensitization with antibody obeys the law of mass action. Thus, the reaction between
antigen on the RBC surface and antibody is reversible and the quantity of
cell-bound antibody at equilibrium will vary depending on the reaction conditions and the
equilibrium constant of the antibody. The reaction conditions should be designed to
maximize the quantity of cell-bound antibody at equilibrium in order to facilitate
detection of either blood group antigen or antibody. Some of these reaction conditions
are described below:
(1) Temperature. Most blood group antibodies show their greatest reactivity
over a restricted temperature range, some reacting optimally at 4ºC, others at 37ºC.
Antibodies reacting optimally at 37ºC have been described as “warm” antibodies, and
those reacting optimally at lower temperatures as “cold” antibodies. Agglutinins
(antibodies) having maximum reactivity at one temperature may have sufficient thermal
amplitude to be active at others. Antibody activity is usually tested at room temperature
and at 37ºC. Antibodies active at 37ºC are the most clinically significant, although “cold”
antibodies cannot be ignored if they have a wide thermal amplitude (for example, above
30ºC). Antibodies only reacting at lower temperatures may be of importance in patients
subjected to hypothermia.
(2) pH. The pH optima for antibody reactivity in most blood-group systems
have not been investigated. For anti-RhO(D), the optimum pH lies between 6.5 and 7.
Antibodies of other blood-group specificities may have different pH optima (for example,
some examples of anti-M react best at pH 5.5).
(3) Incubation time. Time is required for the antibody RBC reaction to reach
equilibrium. The amount of time required to reach this state will depend upon other
variables. The rate of antibody binding is greatest initially, so incubation times for
routine laboratory procedures may be relatively short (for example, 15 to 30 minutes).
(4) Ionic strength.
(a) The ionic strength of the reaction medium is one of the
physiochemical conditions that play an important role in the binding of antibody to RBC
antigens. Ionic strength is a measure of intensity of the electrical field resulting from
ions in solution. Electro-static forces (interaction of positive and negative charges) play
an important role in antibody reaction involving RBCs. Red blood cells carry a large
electronegative charge, which serves to keep them from spontaneously aggregating.
This enables them to function efficiently in oxygen transport by maintaining a maximum
surface area available for gas diffusion. When RBCs are suspended in an electrolyte
solution (0.85 percent NaCl), the cations (positive) are attracted to the negatively
charged RBCs, and the RBC becomes surrounded by a diffuse double layer (“ionic
cloud”), that travels with the RBC as if it were part of it. The outer edge of this layer is
called the surface of shear or the
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1-13 slipping plane. The effective charge (potential) of the RBC, called the zeta potential, is
determined at this plane and is responsible for the electrostatic repulsion between one
RBC and another.
(b) In the first stage of agglutination, reducing the ionic strength of the
medium decreases the electropositive clouds of cations surrounding the RBCs and
facilitates the interaction of electropositive lgG with the negatively charged RBC. This
absorption of antibody to the RBC reduces the electronegative charge of the RBC and
reduces the zeta potential, thereby accelerating the second stage. Experiments have
shown that the initial rate of association of
anti-RhO(D) with RhO(D)-positive RBCs is increased 1,000-fold by a reduction of ionic
strength from 0.17 to 0.03 (for example, instead of using 0.9 percent NaCI, 0.2 percent
NaCI in 7 percent glucose or 0.3M glycine is used as a RBC diluent).
(5) Antigen-antibody ratio. The rate at which antibody is bound to the cell,
and the quantity of antibody bound, depend on the concentration of cells and of
antibody. In general, an increase in sensitivity is obtained by increasing the amount of
antibody in relation to antigen. This is often achieved in the blood bank by using less
antigen in the form of weaker cell suspensions (for example, it is a more sensitive
technique to add one volume of two percent RBCs to two volumes of serum, than to add
one volume of ten percent RBCs to two volumes of serum). Some agglutination
reactions are weakened or even become negative in the presence of an excess of
antibody, the prozone reactions phenomenon. The optimal proportion of antigen to
commercial antiserum is usually determined by the manufacturer; the directions issued
with each antiserum should be followed.
c. The Second Stage (Agglutination).
Blood group antibodies were characterized empirically before the immunoglobulin(1) Once RBCs are sensitized, they may or may not directly agglutinate.
classes were recognized. Those antibodies that could produce agglutination in a saline
medium were called “complete” antibodies or ‘bivalent” antibodies, and those that did
not were called "incomplete" antibodies or “univalent” antibodies. Current evidence
indicates that all antibodies are at least bivalent; that is, each molecule has at least two
antigen-combining sites. The term incomplete antibody is used to denote an antibody
that reacts with, but fails to cause visible agglutination of a saline suspension of RBCs
possessing the corresponding antigenic determinant; such antibodies tend to be of
class lgG.
(2) The failure of “incomplete" antibodies to produce agglutination in a saline
environment may be a result, in part, of location, number, and mobility of antigenic
determinants on the RBC surface, of the size and configuration of the antibody
molecule, and of the electrostatic forces involved.
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1-14 (3) It has been suggested that the zeta potential, mentioned previously, is
the most important factor In explaining why most lgG antibodies do not directly
agglutinate RBCs, the span of the lgG molecules not being sufficient to bridge adjacent
RBCs under the conditions created by the electrostatic forces keeping the RBCs apart.
The same workers suggested that lgG antibodies agglutinated RBCs in the presence of
albumin because albumin raises the dielectric constant (charge dissipating power) of
the suspending medium, thus lowering the zeta potential, allowing RBCs to come close
enough together for agglutination to occur. They also suggested that proteolytic
enzymes (for example, papain, ficin, bromelin, and trypsin) produce the same final
effect by cleaving sialic acid from the RBC membrane, thus reducing the zeta potential.
It should be noted that lgG antibodies (for example, lgG anti-A and -B) do sometimes
directly agglutinate saline-suspended RBCs; this may be a result of the large number of
antigenic sites present, the orientation of these antigens above the surface of the RBC
membrane, and/or the clustering of these antigens during the antigen-antibody
interaction. Recently some workers have argued that zeta potential may not be the
most important factor involved in these reactions.
HEMOLYSIS
Some blood group antibodies can activate the complement cascade (see
paragraph 1-11), leading to Iysis of RBCs possessing the appropriate antigens.
Antibodies showing this characteristic are termed hemolysins and usually will
agglutinate or sensitize RBCs in the absence of complement. Examples of blood-group
antibodies that can sometimes act as hemolysins are anti-A, -B, -A, B, -I, -i, -Lea , -Leb , -
Lex , -Jka , -Jkb , -PP1Pk (TJa ), -Vel. Some of these antibodies and others may sensitize
the RBCs with complement, but not hemolyze them. This complement sensitization can
be detected by the antiglobulin test.
THE CLASSIC COMPLEMENT PATHWAY
a. Activation of the classic pathway can be initiated by a number of substances,
the best known of these, and probably the most important, being the immunoglobulin
molecule. Only one molecule of lgM on the cell membrane is necessary to activate the
complement system (two subunits of the lgM molecule combining with adjacent
antigens on the membrane). In contrast, it is thought that lgG needs to form a "doublet”;
that is to say, two separate lgG molecules have to combine with adjacent antigens on
the cell membrane as close together as 250 to 400 A, before they are able to activate
C1. Only certain subclasses of lgG are able to activate complement through this
pathway. IgG3 is the most efficient, followed by IgG1; IgG2 is the least efficient. IgG4
does not activate the complement; neither does IgA.
b. Appropriate interaction of antibody with antigen leads to sequential activation
of the complement system, often ending in cytolysis. This involves a series of
protein-protein interactions resulting in the generation of a series of cellular
intermediates bearing successively bound complement components. An
antibody-sensitized erythrocyte is designated EA, and successive complement
components are designated by numbers, for example, EACI, EAC1, 4. The protein
components of complement circulate in the plasma in an inactive state, and once
activated are designated by a bar over the component number, for example, C1. The
activation process usually is achieved by cleavage of the next complement molecule
into fragments, which are designated by lower case letters, for example, C3a, C3b. The
activated products usually have enzymatic properties; thus the whole pathway is an
enzymatic cascade similar to the coagulation cascade (see figure 1-4). The system is
held in check by the instability of the complexes formed and the naturally occurring
inhibitors and inactivators present in normal plasma (for example, C3b INA).
c. The pathway consists of three operationally defined functional units, the
recognition unit (C1), the activation unit (C4, C2, C3), and the membrane attack unit
(C5, C6, C7, C8, C9).
(1) Recognition unit. C1 is a complex of three proteins held together by
calcium. C1q is a collagen-like protein with binding sites for lgG and gM; C1r is the
activating enzyme of the critical catalytic site of the C1 complex, C1s is a proenzyme,
activated by C1r. When C1 collides with an antigen-antibody complex (EA), it is bound
to the Fc fragment of the immunoglobulin molecule through the C1q subunit. This
activates C1r and subsequently C1s by cleavage of a single polypeptide chain.
(2) Activation unit. This unit is assembled in two stages. Activated C1(C1s)
acts on native C4 by cleaving the molecule into C4a and C4b. The major fragment C4b
attaches to the cell membrane. A shower of fragments is produced by a single CIs
enzyme, so that many C4b molecules may cluster around the EAC1 site on the cell.
C1s also cleaves native C2 into two fragments; the major C2 fragment, C2a, combines
with C4b on the cell membrane to form an active complex C4b2a (C3 convertase), that
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1-16 has enzymatic activity directed against C3. Magnesium ions are necessary for the
formation of the C4b2a complex. C3 is cleaved by the C4b2a complex into two
molecules, C3a and C3b. The smaller C3a (MW 10,000) does not bind to the cell
membrane, but is released into the fluid phase as a mediator of inflammation
(anaphylatoxin I). The C3b molecule (MW 175,000) binds to the cell membrane and
can also bind to its own activation enzyme. As the C4b2a complex is an enzyme, it can
react more than once, and produce a shower of C3b fragments each time. Only the
C3b fragments that become bound adjacent to the C4b2a enzyme, however, are
believed to participate in the next reaction, in which C5 is cleaved.
(3) Membrane attack unit. Some of the C3b molecules combine with C4b2a
to form C4b, 2a, and 3b, which will cleave the C5 molecule into C5a (anaphylatoxin II)
and C5b. This is the last enzymatic reaction in the pathway. C5b appears to bind C6
and C7 by absorption. The resulting trimolecular complex attaches to the cell
membrane and binds C8 and C9. Fully assembled, the membrane attack complex
consists of one molecule of C5b, C6, C7, C8, and up to six molecules of C9. It has a
molecular weight of about one million. The end result of the pathway is lysis of the cell
(see figure 1-4).
d. Electron microscopy shows that lesions start appearing in the cell membrane
after C8 is absorbed, although the cell does not Iyse until C9 is complexed. It is not
understood how these lesions are made. In most instances, the lesions are not large
enough to allow the hemoglobin molecule to escape directly through the lesion, so it is
thought that cell lysis is caused by an osmotic effect. When cells are attacked by
complement, they swell until the cell membrane is ruptured. The cause of the swelling
is salt and water entering the cell. Mayer has postulated a theory he calls his
“doughnut” hypothesis: a stable hole is produced by the assembly of a rigid,
doughnut-shaped structure in the lipid bilayer of the cell membrane. The hole forms a
channel connecting the inside of the cell with the extracellular fluid. The outside of the
doughnut could be composed of nonpolar polypeptides, that is, protein chains that were
hydrophobic; the interior would need polar peptides so that it could be hydrophilic. He
suggests that C5b, C6, C7, C8, and C9 may be the proteins that form the doughnut or
funnel shape, penetrating the lipid bilayer of the membrane.
THE ALTERNATIVE COMPLEMENT PATHWAY
a. The alternative pathway bypasses C1, C2, C4, and enters the classic
pathway at the C3 stage. It can be activated by several substances including
aggregated immunoglobulins and endotoxin. Thus, an antigen-antibody reaction is not
necessary to initiate the cascade.
b. This pathway is not fully understood at present, but several factors necessary
for its reaction have been isolated. Once C3 is activated in this pathway, the molecular
consequences are identical to the classic pathway. The alternative pathway has not
been incriminated in many immunohaematologic problems yet, but it is of interest to note
that the red -blood cells of patients suffering with paroxysmal nocturnal hemoglobinuria
(PNH) have been shown to hemolyse through this pathway.
STABILITY OF COMPLEMENT WITH PARTICULAR REFERENCE TO THE
DETECTION OF BLOOD-GROUP ANTIBODIES
a. In order to demonstrate hemolysis or RBC bound complement by the
antiglobulin test, one must sensitize RBCs with complement-fixing antibodies in the
presence of complement, or in a two-stage technique, in which cells are first incubated
with antibody, washed, and then incubated with a source of complement. The most
practical source of complement is human serum, but in order to utilize this source, one
must have information about the stability of complement under varying conditions of
storage. A study by Garratty was specifically designed to determine the effect of
storage on the activity of complement with a reference to the detection of blood-group
antibodies. In this study, normal serums were stored at -90ºC, -55ºC,
-20ºC, 22ºC, and 37ºC from 24 hours to 3 months. Complement activity was assessed
by a standard hemolytic assay and also by antiglobulin test assay, which measured the
ability of the stored serums to serve as a source of complement in the detection of
blood-group antibodies by the antiglobulin test. Hemolytic assays closely paralleled
antiglobulin assay. At levels below 60 percent complement activity, there was a danger
of missing weak complement-binding antibodies. An average of these assays showed:
at 37ºC, activity fell to 30 percent In 24 hours; at room temperature, activity was 40
percent at 48 hours, 8O percent at 24 hours, zero at 72 hours; at 4ºC, as 90 percent at
72 hours and 60 percent at two weeks. At -20ºC, activity was more than 60 percent for
2 months, and at -55ºC and -90ºC, activity was retained at three months.
b. It should be stressed that these studies were carried out on normal serums,
and that serums from hospital patients may be deficient in certain complement
components before storage, or they may develop anticomplementary properties faster
than normal serums. The Standards of the American Association of Blood Banks states
that tests for compatibility should be performed on nonactivated, refrigerator-stored
serum, collected within 72 hours of performance test.
Section II. THE ANTIGLOBULIN TEST
PRINCIPLES OF THE ANTIGLOBULIN TEST
a. In 1945, Coombs, Mourant, and Race described a test that detected
non-agglutinating (coating) Rh antibodies in serum, and later used the same test to
demonstrate “in vivo” coating of RBCs with antibodies. In 1957, Dacie et al. showed
that complement components attached to the RBC could also be detected by the test.
This test, now known as the antiglobulin test, depends on the following simple
principles:
NOTE: “In vivo” means something is measured, seen, or tested inside a living
organism. “In vitro” means something is measured, seen, or tested outside a
living organism after it has been removed from that organism.
(1) Antibody molecules and complements components are globulins.
(2) If an animal (for example, rabbit or goat) is injected with human globulin
(either purified or in whole human serum), the animal will make antibodies to the foreign
protein (for example, antihuman globulin, AHG).
(3) This antiglobulin serum, after suitable treatment, will react specifically
with human globulin. If this globulin (for example, antibody or complement) is attached
to the RBC membrane, the antiglobulin serum will combine with globulin on adjacent
RBCs, and cause agglutination of the sensitized RBCs. Non-sensitized RBCs will not
react .
b. As mentioned in Section I, most blood group antibodies are lgM or lgG. Most
lgM antibodies can be detected by direct agglutination; thus, the principal purpose of the
antiglobulin test is to detect lgG (sensitizing) antibodies. In certain
circumstances, it may be advantageous to detect RBC-bound lgA, lgM and/or
complement components by this test.
c. The antiglobulin test can be used to detect “in vivo” or “in vitro" RBC
sensitization.
NOTE: Below you are given the procedure for the direct antiglobulin test (testing for
"in vivo" coating of red cells). For variations of the indirect antiglobulin test
(testing for “in vitro” coating of red cells), see the test for DU in paragraph
1-17, and the two crossmatch procedures on pages found in Section 1-47.
a. The direct antiglobulin test is used for the detection of “in vivo” coating of
RBCs with globlins. Washed RBCs from the patient or donor are directly tested with the
antiglobulin reagents.
b. The direct antiglobulin test is useful for:
(1) Diagnosis of hemolytic disease of the newborn.
(2) Diagnosis of autoimmune hemolytic anemia.
(3) Investigation of RBC sensitization caused by drugs.
(4) Investigation of transfusion reactions.
c. The procedure for the direct antiglobulin test is presented below.
(1) STEP 1. Place one drop of a 2 to 5 percent saline suspension of cells to
tested in a labeled 10-x 75-mm tube. Wash 3 or 4 times with saline. After last wash,
decant completely. Add one or two drops of antiglobulin serum: mix.
(2) STEP 2. Centrifuge and examine for agglutination with an optical aid;
grade and record results. (The manner in which the RBCs are dislodged from the
bottom of the tube is critical. The tube should be held at an angle and shaken gently
until all cells are dislodged. Then it should be tilted gently back end forth until an even
suspension of cells or agglutinates is observed.)
(3) STEP 3. To control for inadvertent contamination of the antiglobulin
serum, add one drop of lgG-sensitized RBCs to any tubes that have been recorded as
negative and recentrlfuge. If the patient’s cells were washed adequately in the first
stage of the test, the control cells should be agglutinated, and the negative result on the
patient is valid.
NOTE: If monospecific anticomplement [3](-C3, -C4 or nongamma) reagents are used
(para 1-21), complement-sensitized cells should be substituted for the
lgG-sensitized cells in step 3.
INDIRECT ANT IGLOBULIN TEST
a. The indirect antiglobulin test is used to demonstrate antibodies that may
cause RBC sensitization “in vitro”. The antibody-containing serum is incubated with
specific RBCs, which, following washing, are reacted with antiglobulin serum to see
whether RBC sensitization has occurred.
b. The indirect antiglobulin test is useful for:
(1) Detection and identification of unexpected antibodies.
(2) Crossmatching.
(3) Detecting RBC antigens not demonstrable by other techniques.
(4) Special studies, (for example, leukocyte and platelet antibody tests).
c. The technique of the indirect antiglobulin test is included in other sections of
this manual where this technique is utilized, (for example, Lesson I, Sections Ill and IV).
ROLE OF COMPLEMENT IN THE ANTIGLOBULIN REACTION
Red blood cells can become sensitized with complement components both
“in vivo” or “in vitro.” One of two main mechanisms is usually involved: (1) complement
components may sensitize the RBCs through the action of complement-binding blood
group antibody(ies), or (two) immune complexes may activate the complement cascade,
causing complement components to attach to RBCs nonspecifically. Whichever of the
mechanisms is operative, the end product is the same, in that the RBC becomes
sensitized with complement components and may or may not continue to hemolysis. If
the RBC does not hemolyse, complement components may still be detected on the
RBC membrane by the antiglobulin test. C3 and C4 are the most readily detected, and
C3 is the most clinically significant. C3 and C4 are beta globulins (B1O and B1E,
respectively); thus, if a RBC is sensitized with C3 or C4, the cell is, in chemical terms,
sensitized with beta globulin. When antiglobulin serum is made by injecting a rabbit with
human globulin (either fractionated or in human serum), the rabbit will make antibodies
to gamma globulin, and also to the “nongamma” globulins, (for example, beta globulin).
This anti-non-gamma fraction of the antiglobulin serum containing antibeta globulin will
react with the RBC-bound complement (C3 or C4), causing agglutination, a positive
antiglobulin test. It is also possible to prepare purified C3 and C4 as immuunogens, so
that specific anti-C3 and anti-C4 can be produced.
MECHANISMS FOR SENSITIZATION OF RED BLOOD CELLS BY
COMPLEMENT
a. Activation of Complement by Blood-Group Antibodies.
(1) Some blood group antibodies bind complement to the RBC membrane.
Some of them do this very efficiently and commonly cause lysis of normal red cells (for
example, anti-A, -B, -Vel, -PP1Pk (Tja ); others may sensitize the RBC with complement
components, not causing lysis of normal RBCs, unless they are enzyme-treated (for
example, anti-Lewis and -Kidd); still, other examples of anti-Lewis, -Kidd, -KeIl, -Duffy, -
S, -s, -I, -i, -H, and -P1, may sensitize RBCs with complement components without
causing lysis of untreated cells or enzyme-treated cells. It is not understood why some
antibodies sensitize RBCs with enough C3 to give strongly positive antiglobulin tests,
yet do not proceed to lysis. Some of these antibodies are lgM and cause agglutination
as well as complement sensitization of the RBCs (for example, anti-A, -B, -I, -i, and
some anti-Lewis). Other, rarer, examples may be lgM and do not cause agglutination
under normal conditions (for example, some anti-Lewis, -KeII, -Duffy, and -Kidd); but
may sensitize RBCs with complement, detectable by the antiglobulin test. Still others
are lgG (for example, some anti-Kell, -Duffy, anti-Kidd); in most cases, the lgG
sensitization is readily detected by anti- globulin serum in addition to the complement
sensitization, but in some cases (for example, some anti-Kidd), the lgG sensitization is
very weak, and the complement sensitization strong
Activation of Complement by Immune Complexes.
(1) Red blood cells can become sensitized with complement because of
activation of the complement cascade by immune complexes. Sometimes these
immune complexes are attached to the RBC membrane; at other times, they are remote
from the cell. A good example of this is the formation of immune complexes involving
certain drugs, for example, phenacetin or quinidine. The drug-antidrug complex can
attach nonspecifically to RBCs, and cause activation of complement with subsequent
attachment of complement compounds to the RBC membrane.
(2) It is important that anticomplement (in particular anti-C3d) activity be
present in antiglobulin serums used for direct antiglobulin tests in the diagnosis of
autoimmune hemolytic anemia; however, as alloantibodies detectable only by their
ability to bind complement are so rare, the Importance of anticomplement activity in
reagents used for compatibility tests is open to debate.
STANDARDIZATION OF ANTIGLOBULIN SERUMS
a. Background.
(1) The most important function of antiglobulin serum is to detect RBC�
bound lgG. The present standards used by the Bureau of Biologics (BoB) are in terms
of activity against Rh antibodies only (for example, lgG). Most reagents on the market
today are prepared and standardized to detect lgG, not only in terms of Rh, but are
tested against a wide selection of lgG blood group antibodies. In addition, most of them
are prepared and standardized to detect cell-bound complement (in particular C3 and
sometimes C4). The presence of other antibodies in the reagents (for example, anti�
lgM, -lgA, -albumin, and so forth) is variable, and is usually coincidental, rather than
deliberate.
(2) The reagent can be prepared by injecting rabbits with highly purified
immunogens, or by harvesting from immuno-secreting whole human serum or protein
fractions. Injection of whole serum has several disadvantages, one being that the
animal will respond much better to single antigens, than to the many present in whole
serum; another being that antibodies to certain proteins (for example, human lgW) show
a prozone effect, whereas others (for example, antihuman beta globulin), usually do not.
Therefore, it is difficult to select one dilution that detects both lgG and complement
optimally. Because of these problems, it is more usual for the animals to be injected
with fractions of human serum, either purified proteins (for example, lgG or complement
components) or cruder fractions such as gamma and "nongamma” globulin. The
commercial houses prepare careful blends from antibodies to the different fractions, and
the antlglobulin serum is tested as below. Finally, after being licensed by the FDA, it is
distributed as antihuman globulin.
b. Specificity Testing. Following a course of immunization, the rabbits are
bled, and their serum screened for reactivity against normal non-sensitized RBCs, lgG,
and complement-sensitized RBCs. Most of the rabbit serums will contain antispecies,
which will have to be removed from the final product, by either fractionation, dilution, or
absorption with nonsensitized human RBCs, before it is sold to the consumer.
c. Antl-IgG Standardization. Antiglobulln serums are tested against
Rh-sensitized RBCs by the so-called checkerboard titration. Rh-positive RBCs are
incubated with a series of dilutions of anti-RhO(D) to yield RBCs sensitized with IgG,
varying from strongly positive to barely positive. Each sample of sensitized cells is
tested against each of a range of dilutions prepared from the rabbit anti-globulin serum.
In hyperimmunized animals, a prozone is often observed (for example, the more diluted
rabbit serum may react better than the less diluted reagent). This means that the
weakest sensitized RBCs may react with a 1:200 dilution of the antiglobulin serum, yet
not react with lower dilutions (or indeed higher dilutions). Thus, 1:200 would be the
optimal dilution of this particular reagent for the detection of Rh (lgG) antibodies. Most
commercial houses test their antiglobulin serums against many other lgG antibodies,
unfortunately, the optimal dilution for detecting lgG anti-Rh may not be the same for the optimal detection of IgG anti-Fya or Jka . Generally speaking, though, the optimal
dilutions are similar for most lgG antibodies.
. d. Anticomplement Standardization.
(1) The FDA requires that reagents marketed as polyspecific AHG contain
anti-C3d activity at a level that equals or exceeds the FDA’s anti-C3d reference serum.
(2) When the C3 (B10) molecule is acted on by C3 convertase (C142) in an
immune reaction, the molecule is cleaved into two fragments. The smaller C3a
fragment does not attach to the RBC membrane, but the larger C3b fragment does.
(3) (Thus, if RBCs are sensitized with complement “in vitro” by a
complement binding blood group antibody, they will be sensitized with C3b. They will
give a positive antiglobulin test with antiglobulin serums containing antibodies to
determinants on the C3b molecule (for example, anti-C3b, anti-C3c [(1A or anti-C3d2D)]).
(4) When RBCs are sensitized with complement “in vivo” (for example,
autoimmune hemolytic anemia or transfusion reactions), or following prolonged
incubation “in vitro”, the C3b is acted on by the C3 inactivator, which is present in all
normal plasma, and the molecule is cleaved into C3c and C3d. The C3c (B1A) fragment
is lost from the RBC, and the C3d (2D) fragment remains on the cell membrane. (see
figure 3-6). Thus, in order to detect C3 bound to the RBC “in vivo”, the antiglobulin
serum must contain activity against C3d.
(5) Recent published data indicate that C4 is also fragmented in a similar
fashion, possibly by the same inactivator. Thus, RBCs sensitized “in vivo” with C4 have
only C4d present on their surface.
(6) Generally, the commercial houses standardize their antiglobulin serums
against RBCs sensitized with C3, C4, and C3d. The cells are usually sensitized with
complement by complement-binding blood group antibodies (for example, anti-Lewis or
anti-I) and/or low ionic strength methods. The C3d-sensitized cells can be from patients
with autoimmune hemolytic anemia or cells prepared “in vitro”, by treating C3b�
sensitized red blood celIs with C3 inactivator (for example, in normal serum) or trypsin. POLYSPECIFIC ANTIGLOBULIN REAGENTS
POLYSPECIFIC ANTIGLOBULIN REAGENTS
a. Polyspecific antiglobulin reagents are used for routine compatibility tests,
alloantibody detection, and the DAT. They contain antibody to human IgG and to the
C3d component of human complement. Other anticomplement antibodies may be
present, including anti-C3d, anti-C4b, and anti-C4d.
b. Since most clinically significant antibodies are lgG, the most important
function of polyspecific antiglobulin is to detect the presence of IgG.
MONOSPECIFIC ANTIGLOBULIN SERUMS
a. As discussed previously, the antiglobulin serum, used routinely in the blood
bank for procedures such as compatibility testing, usually reacts with several plasma
proteins (particularly IgG and complement). It is possible to prepare monospecific
antiglobulin serums by injecting animals with highly purified proteins, such as lgG, IgA,
lgM, C3, or C4; or by absorbing unwanted antibodies from the antiglobulin serum, the
first method being preferable (See Table 1-2).
b. The main use of monospecific antiglobulin serums is to extend the direct
antiglobulin test by evaluating which protein is responsible for the positive direct
antiglobulin test obtained with the broad-spectrum reagent (see Lesson 2, Section I).
On occasion, monospecific reagents such as anti-lgG and anti-C3 may be of value in
the indirect antiglobulin test to show different patterns of reactivity when mixtures of lgG
noncomplement-binding and complement-binding antibodies are present
(for example, anti-hr" (e) + -Leb ). The technique used for testing RBCs with these
reagents is exactly the same as that for broad-spectrum reagents.
CAUTION: Never use anti-IgA, -lgM, -C3, -C4 alone for compatibility testing or for
antibody detection.
Reagents used
Polyspecific:Contains anti-lgG and anti-C3d; may
contain other anticomplement and other and anti-immunoglobulin antibodies.
Contains rabbit antihuman lgG and murine monoclonal anti-C3b and -C3d.
Anti-lgG; Contains anti-lgG with no complement
activity.
Anti-lgG; (heavy chain)Contains only antibodies reactive againstand human gamma chains.
Anti-C3d and Anti-C3b, Anti-C3d;Contains only antibodies reactive against the designated complement component(s),with no anti-immunoglobulin activity.
Anti-C3d (murine monoclonal);
Anti-C3b, -C3d (monoclonal);Contains only antibodies reactive against the designated complement component,with no anti-immunoglobulin activity.
As defined by the FDA: Code of Federal Regulations 21 CFR 660.
FACTORS AFFECTING THE ANTIGLOBULIN TEST
a. Sensitization Phase ("In Vitro" Only).
(1) Temperature. Incubation is normally at 37ºC, as most clinically
significant lgG antibodies react optimally at 37ºC. Complement sensitization also
occurs optimally at 37ºC.
(2) Medium. The suspending medium for the RBCs may be saline, albumin,
or low-ionic-strength saline (LlSS) serum. There is evidence that a shorter incubation
period can be used if albumin is present in the incubation mixture, and also that
uncommon antibodies are detected in the presence of albumin that fail to react when
RBCs are suspended in saline. Antibody association is considerably enhanced if RBCs
are suspended in a simple low-ionic-strength medium.
3. Proportions of serum to cells. The same general principles apply here
as discussed in paragraph 1-9. Increasing the proportion of antibody to antigen will
increase the degree of antibody coating. Two drops of serum to one drop of two
percent to five percent RBCs, a proportion of 100:1 to 40:1 in terms of packed RBCs is
commonly used. Mollison has shown that by increasing this proportion (for example, to
1000:1), sometimes weak antibodies can be detected that are not detectable in our
routine incubation mixtures. In special investigations, such as hemolytic transfusion
reaction with no antibody detectable by routine procedures, it might be useful to try
increasing the proportion of serum to cells.
(4) Incubation time. A period of 15 to 30 minutes of incubation (especially in
the presence of albumin) at 37ºC permits detection of most clinically significant
antibodies. Extension of the incubation period to 30 to 60 minutes may detect a few
weaker examples of antibodies that are undetected after 15 minutes of incubation.
b. Washing Phase.
(1) Washing must be rapid and uninterrupted to minimize loss of cell-bound
antibody by elution.
(2) Decant the saline as completely as possible between each washing.
Shake to loosen and resuspend the cells completely. Add the saline in a forceful
stream. An automatic washer achieves these objectives more efficiently than washing
by hand.
(3) Do not cover the mouth of the test tube with the finger, or the palm of the
hand, when mixing. Serum remaining on the fingers after handling the specimen can
inactivate the antiglobulin reagent.
NOTE: As little as one drop of a 1:4,000 dilution of human serum can neutralize one
drop of antiglobulin serum.
(4) Use adequate volumes of saline. When 10- X 75-mm or 12- X 75-mm
test tubes are filled at least three-quarters full of saline, three or four washings are
usually adequate.
(5) After the final wash, discard the saline as completely as possible.
Resuspend the cells and add the appropriate amount of antiglobulin serum. Mix well,
and centrifuge. It is important that the antiglobulin serum be added immediately,
following completion of washing.
SOURCES OF ERROR
a. False Negative Results.
(1) Inadequate washing of cells will result in neutralization of the antiglobulin
serum by trace amounts of residual globulin. A final concentration of only two mg of
lgG/ml can cause neutralization of the antiglobulin serum. Thus, the RBCs have to be
washed free of unbound IgG, until it is below this figure.
(2) Contamination with human serum will neutralize the reagent. If the
reagent dropper is contaminated with serum and replaced in the vial, the entire contents
of the vial may be neutralized. If the tube is inverted over the thumb or finger in the
washing process, serum contaminating the skin may result in neutralization.
(3) Elution of antibody from the RBCs may take place if the test procedure is
interrupted or delayed, particularly during the washing phase.
(4) The optimum temperature for reactivity of the antibody must be
maintained during incubation to achieve maximal coating of the cells.
(5) A cell suspension that is too heavy will not permit optimum coating with
the antibody; if too weak, reading agglutination may be difficult. A 2 to 5 percent
suspension of RBCs is preferred.
(6) Test cells, test serum, and antiglobulin serum lose reactivity if improperly
stored.
(7) Some antibodies may be detected only in the presence of active
complement. Anticoagulants such as ACD, CPD, or EDTA will chelate calcium,
preventing activation of complement. Thus, the use of plasma rather than serum may
lead to a false negative reaction. Old or improperly stored serum will also have
impaired complement activity.
(8) A prozone reaction should not be a problem with licensed products.
Standardization is done by the manufacturer; directions for the test must be followed.
(9) Antiglobulin serum may have been omitted.
(10) Undercentrifugation or overcentrifugation (the latter because of the
excessive force needed to resuspend cells).
(11) Insufficient incubation time.
NOTE: The lack of agglutination of presensitized RBCs, added following
completion of a negative antiglobulin test, will demonstrate a false-negative
determination caused by 1, 2, 6, or 9 above.
False Positive Results.
(1) Cells having a positive direct antiglobulin test cannot be used with
reagent antiserums that require an antiglobulin phase because all such cells will be
agglutinated by the antiglobulin serum.
(2) Bacterial contamination of test cells or septicemia in a patient may result
in a positive antiglobulin test. If the RBCs are T-activated, they may react as some
antiglobuLin sera contain anti-T.
(3) Extreme reticulocytosis has been reported to give a positive result
because of transferrin bound to reticulocytes reacting with antitransferrin in the
antiglobulin reagents. Most antiglobulin reagents today have little antitransferrin activity.
(4) Saline stored in glass bottles may contain coilcical silica leached from
the container; this has been reported to cause false positive reactions.
(5) Saline stored in metal containers, or used in equipment with metal parts,
may contain metallic ions that may bring about nonspecific protein-coating of the RBCs.
(6) Improperly prepared antiglobulin serum may contain traces of
species-specific antibodies (This should not be a problem if tests are performed with
licensed AHG reagents).
(7) When all the antiglobulin tests are weakly positive, the cause may be
improperly cleaned glassware or other form of contamination.
(8) Overcentrifugation may give false-positive results (aggregation vs.
agglutination).
(9) Patients’ or donors’ serums can contain a naturally occurring cold
autoantibody (normal incomplete cold antibody) that can sensitize their own or other
cells with complement. Usually this only occurs at 4ºC, but it may occur up to room
temperature. If antiglobulin sera contain potent anticomplement, positive reactions may
occur with RBCs from refrigerated clots. These positive reactions have been found to
be largely a result of C4 sensitization, and can be avoided if the RBCs from
anticoagulanted blood is used. For cross matching, RBCs from ACD or CPD segments
can be used for direct antiglobulin tests; however, EDTA is preferable. These
anticoagulants will chelate Ca++ and Mg++, thus preventing any “in vitro” complement
uptake, without interfering with the complement already bound to the RBC “in vivo”.
(10) Red blood cells may be autoagglutinated, before they are washed, and
this agglutination may persist through washing, leading to a false-positive reaction when
antiglobulin serum is added.
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