BSMT-3 IMMUNOHEMATOLOGY Dr.
Andrea Villaruel, MD, DPSP
AGGLUTINATION
OUTLINE (CHAPTER 9)
I. Steps in Agglutination
a) Sensitization
b) Lattice Formation
c) Enhancement of Lattice Formation
II. Types of Agglutination Reactions
a) Direct Agglutination
b) Passive Agglutination
c) Reverse Passive Agglutination
d) Agglutination Inhibition
e) Coagglutination
III. Antiglobulin-Mediated Agglutination
a) Direct Antiglobulin Test
b) Indirect Antiglobulin Test
IV. Instrumentation
V. Quality Control And Quality Assurance
VI. Summary
INTRODUCTION
 Agglutination is the visible aggregation of
particles caused by combination with
specific antibody.
 Antibodies that produce such reactions are
often called agglutinins.
 Because this reaction takes place on the
surface of the particle, antigen must be
exposed and able to bind with antibody.
 Agglutination is actually a two-step process,
involving sensitization or initial binding
followed by lattice formation, or formation
of large aggregates.
 Types of particles participating in such
reactions include erythrocytes, bacterial
cells, and inert carriers such as latex
particles.
 Each particle must have multiple antigenic
or determinant sites, which are cross-linked
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to sites on other particles through the
formation of antibody bridges.
 In 1896, Gruber and Durham published the
first report about the ability of antibody to
clump cells, based on observations of
agglutination of bacterial cells by serum.
This finding gave rise to the use of serology
as a tool in the diagnosis of disease, and it
also led to the discovery of the ABO blood
groups.
 Widal and Sicard developed one of the
earliest diagnostic tests in 1896 for the
detection of antibodies occurring in
typhoid fever, brucellosis, and tularemia.
 Agglutination reactions can be classified
into several distinct categories: direct,
passive, reverse passive, agglutination
inhibition, and coagglutination.
STEPS IN AGGLUTINATION
SENSITIZATION
 Agglutination is a two-step process that
results in the formation of a stable lattice
network.
 The first reaction involves antigen–
antibody combination through single
antigenic determinants on the particle
surface and is often called the sensitization
step.
 This initial reaction follows the law of mass
action (see Chapter 8) and is rapid and
reversible.
 The second step is the formation of cross-
links that form the visible aggregates. This
represents the stabilization of antigen–
antibody complexes with the binding
together of multiple antigenic
determinants. Each stage of the process is
affected by different factors, and it is
important to understand these in order to
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manipulate and enhance end points for
such reactions.
 Sensitization is affected by the nature of
the antibody molecules themselves.
 The affinity and avidity of an individual
antibody determine how much antibody
remains attached.
 The class of immunoglobulin is also
important;
 IgM with a potential valence of 10 is over
700 times more efficient in agglutination
than is IgG with valence of 2.
 The nature of the antigen-bearing surface
is also a key factor in the initial
sensitization process. If epitopes are sparse
or if they are obscured by other surface
molecules, they are less likely to interact
with antibody.
LATTICE FORMATION
 The second stage, representing the sum of
interactions between antibody and
multiple antigenic determinants on a
particle, is dependent on environmental
conditions and the relative concentrations
of antigen and antibody.
 Bordet hypothesized that lattice formation
is governed by physicochemical factors such
as the milieu’s ionic strength, pH, and
temperature.
 Antibody must be able to bridge the gap
between cells in such a way that one
molecule can bind to a site on each of two
different cells.
 Erythrocytes and bacterial cells have a
slight negative surface charge, and because
like charges tend to repel one another, it is
difficult to bring such cells together into
lattice formation.
 In an ionic solution, red cells surround
themselves with cations to form an ionic
cloud, which keeps them about 25 nm
apart.
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 Antibodies of the IgG class often cannot
bridge the distance between particles,
because their small size and restricted
flexibility at the hinge region may prohibit
multivalent binding.
 IgM antibodies have a diameter of about
35nm, so they are strong agglutinins.
 Visible reactions with IgG often require the
use of enhancement techniques, which
vary physicochemical conditions.
ENHANCEMENT OF LATTICE FORMATION
 The surface charge must be controlled for
lattice formation, or a visible agglutination
reaction, to take place.
 One means of accomplishing this is by
decreasing the buffer’s ionic strength
through the use of low ionic strength
saline.
 The addition of albumin in concentrations
of 5 to 30 percent also helps to neutralize
the surface charge and allows red cells to
approach each other more closely.
 Other techniques that enhance
agglutination, especially that of red blood
cells, include increasing the viscosity, using
enzymes, agitating centrifuging, and
altering the temperature or the pH.
 Viscosity can be increased by adding agents
such as dextran or polyethylene glycol
(PEG). These agents reduce the water of
hydration around cells and allow them to
come into closer proximity for antibody to
join together.
 Bromelin, papain, trypsin, and ficin are the
enzymes most often used to enhance
agglutination, and these are thought to
work by reducing the surface charge on red
blood cells through cleaving of chemical
groups and decreasing hydration.
 Ficin cleaves sialoglycoproteins from the
red blood cell surface; in addition to
reducing the charge, this may change the
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external configuration of the membrane to
reveal more antigenic determinant sites.
 Agitation and centrifugation provide a
physical means to increase cell–cell contact
and thus heighten agglutination.
 All of these techniques plus the use of
antiglobulin reagents (discussed in the
section on antiglobulin testing) are used in
the blood bank to better detect antigen–
antibody reactions, especially in selecting
blood for transfusion.
 Temperature has an influence on the
secondary, or aggregation, phase.
 Antibodies belonging to the IgG class
agglutinate best at 30°C to 37°C
 IgM antibodies react best at temperatures
between 4°C and 27°C.
 Because naturally occurring antibodies
against the ABO blood groups belong to
the IgM class, these reactions are best run
at room temperature.
 These latter reactions are the most
important to consider in selecting
compatible blood for a transfusion, because
these are the ones that will actually occur in
the body.
 pH. Most reactions produce optimal
antigen–antibody combination when the
pH is between 6.5 and 7.5
 Human anti-M and antiP1, which react best
at a lower pH.
TYPES OF AGGLUTINATION REACTION
 Agglutination reactions are easy to carry
out, require no complicated equipment, and
can be performed as needed in the
laboratory without having to batch
specimens.
 They can be usedto identify either antigen
or antibody.
 Typically, most agglutination tests are
qualitative, simply indicating absence or
presence of antigen or antibody, but
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dilutions can be made to obtain
semiquantitative results.
DIRECT AGGLUTINATION
 Direct agglutination occurs when antigens
are found naturally on a particle.
 Best examples involve known bacterial
antigens used to test for the presence of
unknown antibodies in the patient.
 Typically, patient serum is diluted into a
series of tubes or wells on a slide and
reacted with bacterial antigens specific for
the suspected disease.
 Detection of antibodies is primarily used in
diagnosis of diseases for which the
bacterial agents are extremely difficult to
cultivate.
 Example. Widal test, a rapid screening test
to help determine the possibility of typhoid
fever.
 The antigens used in this procedure include
Salmonella O (somatic) and H (flagellar)
antigens.
 A significant finding is a fourfold increase in
antibody titer over time when paired
dilutions of serum samples are tested with
any of these antigens.
 If an agglutination reaction involves red
blood cells, then it is called
hemagglutination.
 Best example of this occurs in ABO blood
group typing of human red blood cells.
 Antisera of the IgM type can be used to
determine the presence or absence of the A
and B antigens, and this reaction is usually
performed at room temperature without
the need for any enhancement techniques.
This type of agglutination reaction is simple
to perform, is relatively sensitive, and is
easy to read. A titer that yields
semiquantitative results can be performed
in test tubes or microtiter plates by making
serial dilutions of the antibody. The
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reciprocal of the last dilution still exhibiting
a visible reaction is the titer, indicating the
antibody’s strength.
 Interpretation of the test is done on the
basis of the cell sedimentation pattern.
 If there is a dark red, smooth button at the
bottom of the microtiter well, the result is
negative.
 A positive result will have cells that are
spread across the well’s bottom, usually in
a jagged pattern with an irregular edge.
 Test tubes also can be centrifuged and then
shaken to see if the cell button can be
evenly resuspended. If it is resuspended
with no visible clumping, then the result is
negative.
 Positive reactions can be graded to indicate
the strength of the reaction.
PASSIVE AGGLUTINATION
 Passive, or indirect, agglutination employs
particles that are coated with antigens not
normally found on their surfaces.
 A variety of particles, including
erythrocytes, latex, gelatin, and silicates,
are used for this purpose.
 The use of synthetic beads or particles
provides the advantage of consistency,
uniformity, and stability.
 Reactions are easy to read visually and give
quick results. Particle sizes vary from 7 μm
for red blood cells down to 0.8 μm or less
for fine latex particles.
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 Many antigens, especially polysaccharides,
adsorb to red blood cells spontaneously, so
they are relatively easy to manipulate.
 Problems encountered with the use of
erythrocytes as carrier particles include the
possibility of cross-reactivity, especially with
heterophile antibody (see Chapter 3) if the
cells used are nonhuman.
 In 1955, Singer and Plotz found by
happenstance that IgG was naturally
adsorbed to the surface of polystyrene
latex particles.
 While other substances such as
polysaccharides and highly charged
proteins are not naturally adsorbed by
these particles, manipulation with certain
chemicals is usually successful in coating
latex particles with these substances.
 Latex particles are inexpensive, are
relatively stable, and are not subject to
cross-reactivity with other antibodies.
 A large number of antibody molecules can
be bound to the surface of latex particles,
so the number of antigen binding sites is
large.
 Additionally, the large particle size
facilitates reading of the test.
 Passive agglutination tests have been used
to detect rheumatoid factor; antinuclear
antibody occurring in the disease lupus
erythematosus; antibodies to group A
streptococcus antigens; antibodies to
Trichinella spiralis; antibodies to
Treponema pallidum; and antibodies to
viruses such as cytomegalovirus, rubella,
varicella-zoster, and HIV-1/HIV-2.
 Because many of these kits are designed to
detect IgM antibody, and there is always
the risk of nonspecific agglutination caused
by the presence of other IgM antibodies,
reactions must be carefully controlled and
interpreted.
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 Commercial tests are usually performed on
disposable plastic or cardboard cards or
glass slides.
 Kits contain positive and negative controls,
and if the controls do not give the expected
results, the test is not valid.
 Such tests are typically used as screening
tools to be followed by more extensive
testing if the results are positive.
REVERSE PASSIVE AGGLUTINATION
 In reverse passive agglutination, antibody
rather than antigen is attached to a carrier
particle.
 The antibody must still be reactive and is
joined in such a manner that the active
sites are facing outward.
 Adsorption may be spontaneous, or it may
require some of the same manipulation as is
used for antigen attachment.
 This type of testing is often used to detect
microbial antigens.
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 Numerous kits are available today for the
rapid identification of antigens from such
infectious agents as group B streptococcus,
Staphylococcus aureus, Neisseria
meningitidis, streptococcal groups A and B,
Haemophilus influenzae, rotavirus,
Cryptococcus neoformans, Vibrio cholera
01, and Leptospira.
 Rapid agglutination tests have found the
widest application in detecting soluble
antigens in urine, spinal fluid, and serum.
 The principle is the same for all these tests:
Latex particles coated with antibody are
reacted with a patient sample containing
the suspected antigen.
 In some cases, an extraction step is
necessary to isolate antigen before the
reagent latex particles are added.
 Organisms can be identified in a few
minutes with fairly high sensitivity and
specificity, although this varies for different
organisms.
 Use of monoclonal antibodies has greatly
cut down on cross-reactivity, but there is
still the possibility of interference or
nonspecific agglutination.
 Such tests are most often used for
organisms that are difficult to grow in the
laboratory or for instances when rapid
identification will allow treatment to be
initiated more promptly.
 For infections in which a large amount of
viral antigen is present, such as rotavirus
and enteric adenovirus in infants, latex
agglutination tests are extremely useful.
 Reverse passive agglutination testing has
also been used to measure levels of certain
therapeutic drugs, hormones, and plasma
proteins such as haptoglobin and C-reactive
protein.
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AGGLUTINATION INHIBITION
 Agglutination inhibition reactions are
based on competition between particulate
and soluble antigens for limited antibody-
combining sites, and a lack of agglutination
is an indicator of a positive reaction.
 This type of reaction involves haptens that
are complexed to proteins;
 The hapten–protein conjugate is then
attached to a carrier particle.
 The patient sample is first reacted with a
limited amount of reagent antibody that is
specific for the hapten being tested.
Indicator particles that contain the same
hapten one wishes to measure in the
patient are then added.
 If the patient sample has no free hapten,
the reagent antibody is able to combine
with the carrier particles and produce a
visible agglutination. In this case, however,
agglutination is a negative reaction,
indicating that the patient did not have
sufficient hapten to inhibit the secondary
reaction.
 Either antigen or antibody can be attached
to the particles.
 The sensitivity of the reaction is governed
by the avidity of the antibody itself.
 It can be a highly sensitive assay capable of
detecting small quantities of antigen.
 Detection of illicit drugs such as cocaine or
heroin are examples of very sensitive
agglutination inhibition tests.
 Hemagglutination inhibition reactions use
the same principle, except red blood cells
are the indicator particles.
 This type of testing has been used to detect
antibodies to certain viruses, such as
rubella, mumps, measles, influenza,
parainfluenza, HBV, herpesvirus,
respiratory syncytial virus, and adenovirus.
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 Red blood cells have naturally occurring
viral receptors.
 When virus is present, spontaneous
agglutination occurs, because the virus
particles link the red blood cells together.
 Presence of patient antibody inhibits the
agglutination reaction.
 To perform a hemagglutination inhibition
test, patient serum is first incubated with a
viral preparation. Then red blood cells that
the virus is known to agglutinate are added
to the mixture.
 If antibody is present, this will combine
with viral particles and prevent
agglutination, so a lack of or reduction in
agglutination indicates presence of patient
antibody.
 Controls are necessary, because there may
be a factor in the serum that causes
agglutination, or the virus may have lost its
ability to agglutinate.
COAGGLUTINATION
 Coagglutination is the name given to
systems using bacteria as the inert particles
to which antibody is attached.
 Staphylococcus aureus is most frequently
used, because it has a protein on its outer
surface, called protein A, which naturally
adsorbs the fragment crystallizable (FC)
portion of antibody molecules.
 The active sites face outward and are
capable of reacting with specific antigen
 These particles exhibit greater stability
than latex particles and are more refractory
to changes in ionic strength.
 However, because bacteria are not colored,
reactions are often difficult to read.
 Such testing is highly specific, but it may not
be as sensitive for detecting small quantities
of antigen, as is latex agglutination.
 Coagglutination reagents have been used in
identification of streptococci, Neisseria
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meningitidis, Neisseria gonorrhoeae, Vibrio
cholera 0139, and Haemophilus influenzae.
ANTIGLOBULIN-MEDIATED
AGGLUTINATION
 The antihuman globulin test, also known as
the Coombs’ test, is a technique that
detects non-agglutinating antibody by
means of coupling with a second antibody.
 The key component of the test is antibody
to human globulin that is made in animals
or by means of hybridoma techniques.
 Such antibody will react with the FC portion
of the human antibody attached to red
blood cells.
 Agglutination takes place because the
antihuman globulin is able to bridge the
distance between cells that IgG alone
cannot do.
 The strength of the reaction is proportional
to the amount of antibody coating the red
blood cells.
 The Coombs’ test can be divided into two
different types, direct and indirect, each of
which has a different purpose.
DIRECT ANTIGLOBULIN TEST
 The direct antiglobulin test is used to
demonstrate in vivo attachment of
antibody or complement to an individual’s
red blood cells.
 Serves as an indicator of autoimmune
hemolytic anemia, hemolytic disease of
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the newborn, sensitization of red blood
cells caused by the presence of drugs, or a
transfusion reaction.
 The test is called direct, because red blood
cells are tested directly as they come from
the body.
 A blood sample is obtained from the
patient, the red blood cells are washed to
remove any antibody that is not specifically
attached, and then cells are tested directly
with antibody to IgG or complement
components.
 If IgG or complement is present on the red
blood cells, the anti- human globulin
(Coombs’ reagent) is able to bridge the gap
between red blood cells and cause a visible
agglutination
 Polyspecific antiserum will react with IgG
and with complement component C3d.
 Antibodies to C3b, C4b, or C4d may also be
present.
 If such a reaction is positive, then
monospecific antibody, which will react
with only one component, is used.
 This will differentiate between IgG and
individual complement components on the
patient’s red blood cells.
 A positive test indicates that an immune
reaction is taking place in that individual.
INDIRECT ANTIGLOBULIN TEST
 The indirect antiglobulin test, or indirect
Coombs’ test, is used to determine the
presence of a particular antibody in a
patient
 It can be used to type patient red blood
cells for specific blood group antigens.
 This is a two-step process, in which washed
red blood cells and antibody are allowed to
combine at 37°C, and the cells are then
carefully washed again to remove any
unbound antibody. When antihuman
globulin is added, a visible reaction occurs
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where antibody has been specifically
bound.
 This test is most often used to check for the
presence of clinically significant
alloantibody in patient serum when
performing compatibility testing for a blood
transfusion.
 In this case, patient serum is used to
combine with reagent red blood cells of
known antigenicity.
 All reactions are run at 37°C to detect
clinically significant antibodies.
 Cells are then washed, and antihuman
globulin is added.
 Tubes are centrifuged and read for
agglutination.
 Possible sources of error in performing the
Coombs’ test include failure to wash cells,
improper centrifugation, failure to add test
serum or antihuman globulin, and use of
expired reagents or those that have not
been properly stored.
 An improper concentration of red cells may
alter the results.
 Too heavy a red cell concentration may
mask agglutination
 Too light a concentration will make the
reaction hard to read.
 It is important to use quality controls and
to interpret results carefully.
INSTRUMENTATION
 Several systems have been developed using
automation to increase sensitivity.
 TURBIDIMETRY. based on the principle that
as particles combine, light scatter
increases, and the absorbance of the
solution increases proportionally.
 NEPHELOMETRY. applied to the reading of
agglutination reactions, and the term
particle-enhanced immunoassay is used to
describe such reactions.
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 Nephelometry without the use of particles
is capable of detecting soluble antigen–
antibody complexes at a sensitivity of
between 1and 10 μg/mL.
 If particles are used, the sensitivity can be
increased to nanograms/mL.
 For this type of reaction, small latex
particles with a diameter of less than 1 μm
are used.
 One such type of instrumentation system is
called a PACIA.
 Particle-counting immunoassay (PACIA)
involves measuring the number of residual
non-agglutinating particles in a specimen.
 These are counted by means of a laser
beam in an optical particle counter similar
to one that is designed to count blood cells.
 Nephelometric methods are used to
measure forward light scatter.
 Latex particles are coated with whole
antibody molecules or with F(ab)2
fragments. Use of the latter reduces
interference and nonspecific agglutination.
 If antigen is present, complexes will form
and will be screened out by the counter
because of their large size.
 An inverse relationship exists between the
number of unagglutinated particles
counted and the amount of unknown in the
patient specimen.
 Measurements are made by looking at the
rate at which the number of unagglutinated
particles decrease, called a rate assay, or
the total number of unagglutinated
particles left at the end, known as an end-
point assay.
 PACIAs have been used to measure several
serum proteins, therapeutic drugs, tumor
markers, and viral antigens, such as those
associated with measles, herpes simplex,
and hepatitis B surface antigen.
 One disadvantage, however, is the need to
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invest in expensive equipment.
QUALITY CONTROL AND QUALITY
ASSURANCE
 Although agglutination reactions are simple
to perform, interpretation must be carefully
done.
 Techniques must be standardized as to
concentration of antigen, incubation time,
temperature, diluent, and the method of
reading.
 The possibility of cross-reactivity and
interfering antibody should always be
considered.
 Cross-reactivity is caused by the presence
of antigenic determinants that resemble
one another so closely that antibody
formed against one will react with the
other.
 Most cross-reactivity can be avoided
through the use of monoclonal antibody
directed against an antigenic determinant
that is unique to a particular antigen.
 Heterophile antibody and rheumatoid
factor are two interfering antibodies that
may produce a false-positive result.
 Heterophile antibodies are most often a
consideration when red blood cells are
used as the carrier particle.
 Patients may have an antibody that is
capable of reacting with an antigen on the
red blood cell other than the antigen that is
being tested for. Such cross-reactions can
be controlled by preadsorption of test
serum on red blood cells without the test
antigen or by treating red blood cells to
remove possible interfering antigens.
 Rheumatoid factor, as mentioned
previously, will react with any IgG present
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and is especially a problem in reverse
passive agglutination tests. If this is
suspected, samples can be treated with
pronase or the reducing agent 2-
mercaptoethanol to reduce false- positive
results due to the IgM rheumatoid factor.
 The use of positive and negative control
sera is essential in agglutination testing,
but it cannot rule out all false-positive
reactions.
 Other considerations include proper storage
of reagents and close attention to
expiration dates.
 Reagents should never be used beyond the
expiration date.
 Advantages of agglutination reactions
include rapidity; relative sensitivity; and
the fact that if the sample contains a
microorganism, it does not need to be
viable.
 In addition, most tests are simple to
perform and require no expensive
equipment.
 Test are conducted on cards, tubes, and
microtiter plates, all of which are extremely
portable.
 A wide variety of antigens and antibodies
can be tested.
 However, that agglutination tests are
screening tools only and that a negative
result does not rule out presence of the
disease or the antigen.
 Play an important role in the identification
of rare pathogens such as Francisella and
Brucella and more common organisms such
as rotavirus and Cryptococcus, for which
other testing is complex or unavailable.
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AGGLUTINATION September 14, 2020

REFERENCE
 Stevens, C. D. (2010). Clinical immunology & serology: A laboratory perspective.

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