IMMUNOLOGY AND SEROLOGY
(Midterm)
AGGLUTINATION METHODS
 Precipitation and agglutination: visible
expression of the aggregation of
antigens and antibodies.
 Precipitation: aggregation of soluble
test antigens
 soluble antigen + soluble antibody
= visible insoluble complex
 Agglutination: whereby specific
antigens (e.g., red blood cells)
aggregate to form larger visible clumps
when the corresponding specific
antibody is present in the serum
 Artificial carrier particles may be
needed to indicate visibly that an
antigen-antibody reaction has taken
place (latex particles and colloidal
charcoal)
 easy to perform; most sensitive
tests currently available
 1960s: first agglutination tests were
developed
1. LATEX AGGLUTINATION
 Antibody molecules: bound to the
surface of latex beads (C-reactive
protein)
 forming visible cross-linked
aggregates of latex beads and
antigen
 Antigen: coated the latex particles
(pregnancy testing, rubella antibody
testing)
 agglutinate into large visible
clumps
A. Coagglutination
 Uses antibodies bound to a particle
to enhance the visibility of
agglutination
 highly specific method
 not as sensitive as latex
agglutination
B. Liposome-enhanced testing
 detect minute amounts of human
chorionic gonadotropin (hCG)
 a glycoprotein hormone
secreted by the trophoblast of
the developing embryo
 first 6 to 8 weeks after
conception: helps maintain the
corpus luteum and stimulate the
 production of progesterone
 α unit: identical to luteinizing
hormone (LH), follicle-
stimulating hormone (FSH), and
thyroid-stimulating hormone
(TSH)
 β subunit
 many pregnancy test kits contain
monoclonal antibody (MAb)
directed against the β subunit to
increase the specificity of the
reaction
 level of hCG should double every
2 to 3 days
Agglutination Inhibition: incubation of the
patient’s specimen with anti-hCG, then
addition of latex particles coated with hCG.
 (+) rxn: no agglutination (neutralizes
the antibody)
 (-) rxn: agglutination
 Pregnancy Latex Slide Agglutination
 rapid, direct, uses monoclonal
Antibodies
 (+) rxn: agglutination within 2 mins
 (-) rxn: no agglutination
 agglutination should be read within 3
minutes to avoid erroneous results
caused by evaporation.
False-Positive Results
 hCG injection (Pregnyl): can remain
in the patient’s system for as long as
10 days
 Chorioepithelioma
 hydatidiform mole
IMMUNOLOGY AND SEROLOGY
(Midterm)
 excessive ingestion of aspirin
 testicular tumor
False-Negative
 Testing before reaching detectable
levels of hCG
2. FLOCCULATION TESTS
 for antibody detection
 soluble antigen + antibody =
formation of a precipitate of fine
particles
 used in syphilis serologic testing
 Venereal Disease Research
Laboratories (VDRL) test
o antibody-like protein, reagin,
binds to the test antigen,
cardiolipin-lecithin–coated
cholesterol particles,
o produces the particles that
flocculate
 rapid plasma reagin (RPR) test
o antigen, cardiolipin-lecithin–
coated cholesterol with
choline chloride, also
contains charcoal particles
that allow for
macroscopically visible
flocculation
3. DIRECT BACTERIAL
AGGLUTINATION
 In a thick suspension of the bacteria,
the binding of specific antibodies to
surface antigens of the bacteria
causes the bacteria to clump
together in visible aggregates
 use of sterile physiologic saline:
enhances the aggregation of
bacteria
 most bacterial surfaces exhibit a
negative charge that causes
them to repel each other
 tube testing is considered more
sensitive than slide testing
 tube testing: allows more time
for the antigen-antibody
reaction
4. HEMAGGLUTINATION
 detects antibodies to erythrocyte
antigens
 Specimen: contains Ab
Indirect hemagglutination or passive
hemagglutination (PHA)
 detect antibodies to antigens other
than those present on the cells
 used to cross-link antigens to the
cells: chromic chloride, tannic acid,
and glutaraldehyde
 Agglutination is the clumping of
particles that have antigens on their
surface
1. Sensitization
 First phase
 physical attachment of antibody
molecules to antigens on the
erythrocyte membrane
 combination of antigen and antibody
is a reversible chemical reaction
 Elution: when physical conditions
are purposely manipulated to break
the antigen-antibody complex, with
subsequent release of the antibody
into the surrounding medium
 zeta potential: net negative surface
charge
IMMUNOLOGY AND SEROLOGY
(Midterm)
 Lattice hypothesis: each antibody
molecule must have at least 2
binding sites and that an antigen
must be multivalent
 Zone of equivalence=detectable
rxn
 prozone phenomenon:
overcome by serially diluting the
antibody-containing serum until
optimum amounts of antigen
and antibody are present in the
test system
 postzone phenomenon: small
aggregates (clumps) are
surrounded by excess antigen
o Repeat blood specimen
should be collected 1 or
more weeks later
 placement and number of antigenic
determinants both affect
agglutination
 Steric hindrance: If dissimilar
antibodies with the same binding
constant are directed against
antigenic determinants located close
to each other, antibodies will
compete for space in reaching their
specific receptor sites
 mutual blocking, antibody
competition
 pH of the medium used for testing:
optimum pH of 6.5 to 7.5
 IgM antibodies: cold-reacting
(thermal range, 4° C to 22° C)
 IgG antibodies are warm-
reacting, with an optimum
temperature of reaction at 37° C
2. Lattice Formation
 establishment of cross-links between
sensitized particles (RBCs) and
 antibodies, resulting in aggregation
 slower process
 Methods of Enhancing Agglutination
A. Centrifugation
 physically forces the cells
together
 overcome the problem of
distance by subjecting sensitized
cells to a high gravitational force
B. Treatment with proteolytic
enzymes
 alters the zeta potential or
dielectric constant
C. Use of colloids
D. AHG testing
 antigens may be so deeply
embedded in the membrane
surface
 Graded Agglutination Reactions
 Observation of agglutination: gently
shaking the test tube containing the
serum and cells and viewing the
lower portion, the button
 Avoid hard shaking
 Pseudoagglutination
 False appearance of clumping
 rarely occur because of rouleaux
formation
 patients with high or abnormal
types of globulins in their blood
(multiple myeloma or after
 receiving dextran as a plasma
expander
 To disperse:few drops of
physiologic NaCl(saline) can be
added to the reaction tube,
remixed, and reexamined.
IMMUNOLOGY AND SEROLOGY
(Midterm)
Microplate Agglutination Reactions
 allows for the performance of a large
number of tests on a single plate
(hemagglutination and solid-phase
adherence assays)
 microplate is a compact plate of rigid
or flexible plastic with multiple wells
 U-shaped well has been used
most often in immunohematology
 volume capacity of each well:
approx 0.2 mL
 Samples and reagents are
dispensed with small-bore Pasteur
pipettes
 mixed by gentle agitation of the
plates then centrifuged
 After centrifugation, the cell buttons
are resuspended by gently tapping
the microplate or by using a flat-
topped mechanical shaker
 shaker provides a more
consistent and standard
resuspension of the cells
 (+) rxn: particles settle in a diffuse
uneven button
 (-) rxn: smooth compact button

Reading red blood cell agglutination

reactions