Chapter 9: Agglutination
1. Erythrocytes
 Agglutination
- Visible aggregation of particles caused by combination with
specific antibody
 Agglutinins
- Antibodies that produce agglutination reactions
 Agglutinogen
- Antigen that produce agglutination reactions
 Gruber & Durham (1896)
- Published first report about ability of antibody to clump cells
- Agglutination of bacterial cells by serum
- Gave rise to the use of Serology as a tool in the diagnosis of
disease.
- Led to the discovery of ABO blood groups
 Widal Sicard (1896)
- Developed diagnostic tests for detection of antibodies
occurring in: Typhoid fever, Brucellosis, Tularemia
Steps in Agglutination
1. Sensitization
- Initial reaction follows the law of mass action
- Rapid and reversible
2. Lattice formation
- Stabilization of antigen-antibody complexes
- Represents sum of interactions between
antibody and multiple antigenic determinants
 Bordet
- Hypothesized that lattice formation is governed
by physicochemical factors:
o Milieu’s ionic strength, pH, Temperature
 Enhancement of Lattice Formation:
- The surface charge must be controlled for
lattice formation by means of:
 Low ionic strength saline
- Decreases the buffer’s ionic strength to
control the surface charge for lattice
formation
 Albumin (5-30%)
- Neutralize surface charge
- Allow red cells to approach each other
closely
 Types of particles participating in Agglutination
2. Bacterial cells
3. Inert carriers – ex. Latex particles
 Each particle must have multiple antigenic or determinant
sites (cross-linked to sites on other particles through the
formation of antibody bridges)
 Types of Agglutination Reactions
 Batching specimens
- done if a test is expensive or complicated
- large number of specimens run at one time, which may result
in a time delay
 Kits
- available for standard testing (reagent preparation is minimal)
- can be used to identify either antigen or antibody
 Most agglutination tests are qualitative
- Indicates absence or presence of antigen or antibody
- Dilutions can be made to obtain semiquantitative results
 Antigen-antibody combination through simple antigenic determinants on the particle surface
 Factors that affect Sensitization
1. Nature of antibody molecule
2. Affinity and avidity of antibody
3. Class of immunoglobulin
 IgM (Valence: 10) is 700 times more efficient
in agglutination than is IgG (Valence: 2)
4. Nature of antigen-bearing surface
 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.
 Formation of cross-links that form visible aggregates
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:
o have a slight negative surface charge (like charges tend to repel one another).
 Ionic solution:
o red cells surround themselves with cations to form an ionic cloud
(keeps them about 25 nm apart).
 Nature of the antibody: where the ability to link cells together depends .
IgG antibodies:
- cannot bridge the distance between particles (small size and restricted flexibility at the
hinge region prohibit multivalent binding).
- Visible reactions require the use of enhancement techniques, which vary
physicochemical conditions.
IgM antibodies:
- have a diameter of about 35nmso (strong agglutinins).
  Enhancement of Agglutination 3. Altering temperature/pH
1. Increase viscosity
 using Enzymes: Bromelin, Papain, Trypsin, Ficin Temperature
o Ficin  influence the secondary or aggregation phase
- cleaves sialoglycoproteins from RBC surface
- may change the external configuration of the membrane  IgG agglutinates: 30-37°C
to reveal more antigenic determinant sites.  Contain: antibodies to other human blood groups
 These enzymes work by reducing the surface charge on red
blood cells through cleaving of chemical groups and  IgM agglutinates: 4-27°C
decreasing hydration.  Contain: naturally occurring antibodies against the ABO blood
 using Adding agents : Dextran, Polyethylene glycol (PEG) groups
 These agents reduce the water of hydration around cells and
allow them to come closer for antibody joining. pH
2. Agitating centrifuging:  Most reactions: 6.5-7.5 (optimal pH)
 provide a physical means to increase cell–cell contact  Exception: Anti-M and antiP1 – lower pH
and thus heighten agglutination.

Classification/Categories of Agglutination

1. Direct Agglutination  Occurs when antigens are found naturally

on a particle

Example: Widal’s Test Hemagglutination

1. Bacterial antigens:
o used to test for the presence of unknown
antibodies in the patient
 Patient serum is diluted into a series of tubes
on a slide and reacted with bacterial antigens
specific for the suspected disease.
- Rapid screening test to determine
possibility of typhoid fever by
serotyping of Salmonella spp.
 Antigens used:
- Salmonella O Antigen (Somatic)
o found on the bacterial cell wall
o compact globular agglutination
pattern
- Salmonella H Antigen (Flagellar)
o found on flagellar
o snowflake-like agglutination pattern
 Significant finding:
- fourfold increase in antibody titer over
time in paired dilutions of serum samples
- If agglutination reaction involves red blood
cells
- Simple to perform but relatively sensitive
- ABO blood group typing of human RBCs is
one of the most frequently used
immunoassays.
Direct/Forward Typing: Ag is detected.
Indirect/Reverse Typing: Ab is detected.
** Tube Method (gold standard), Slide Method
 IgM Antisera
– determines A and B antigens at 37°C
Titer
- the reciprocal of the last dilution that still
exhibit a visible reaction (antibody’s
strength).

2. Passive/ Indirect Agglutination  Employs particles that are coated with

antigens not normally found on surfaces

Example of particles: Reactions  Passive Agglutination tests detects:

a) Erythrocytes o Sizes:
o might cause crossreactivity, especially 7 μm for RBC down to  Rheumatoid factor
with heterophile antibody if the cells < 0.8 μm for fine latex
used are nonhuman.  Antinuclear antibody
b) Latex Many antigens (polysaccharides) adsorb to RBC (Lupus erythematosus)
o Inexpensive, relatively table; not subject spontaneously.
to cross-reactivity  Antibodies
c) Gelatin Singer and Plotz (1955) - Group A streptococcus antigens
d) Silicates o Fount that IgG was naturally adsorbed to - Trichinella spiralis
the surface of polystyrene latex particles. - Treponema pallidum
Synthetic beads or particles - Virus: Cytomegalovirus, Rubella,
o provides the advantage of consistency, Varicella-zoster, HIV-1/HIV-2
uniformity, and stability.
3. Reverse Passive Agglutination
Principle: Latex particles coated with antibody are
reacted with a patient sample containing the
suspected antigen
 Infectious agents that has kits for Rapid
Identification of Antigens:
- Group A & B strep, Staphylococcus aureus,
Neisseria meningitidis, Haemophilus
influenzae, Rotavirus, Cryptococcus
neoformans, Vibrio cholera 01, Leptospira
Rapid agglutination tests detect soluble antigen:
In urine, spinal fluid, and serum.
4. Agglutination Inhibition
POSITIVE REACTION:
- Lack of agglutination reaction
- Involves haptens that are complexed to
proteins.
o Hapten–protein conjugate is then
attached to a carrier particle.
NEGATIVE REACTION:
- Visible agglutination due to the patient
sample has no free hapten.
- Either antigen or antibody can be
attached to the particles.
o No Hapten that can inhibit the
secondary reaction
5. Coagglutination
Inert particle: Staphylococcus aureus
 most frequently used because of the
presence of protein A on its outer
surface.
Hemagglutination Interpretations :
 Cell sedimentation pattern:
Negative:
- dark red
- smooth button (bottom)
Positive:
- jagged pattern with irregular edge
- cells are spread (across the bottom)
 Antibody (than antigen) is attached to a o Often used to detect microbial antigens
carrier particle.
Monoclonal antibodies Used to measure levels of:
o greatly cut down on cross-reactivity - Therapeutic drugs
o most often used for organisms that are - Hormones
difficult to grow in the laboratory - Plasma proteins
- Haptoglobin
Latex agglutination tests - C-reactive protein
o for infections in which a large amount of - Rheumatoid factor - will cause FALSE
viral antigen is present. POSITIVE, as it reacts with ANY IgG
o Example: Rotavirus and enteric antibody
adenovirus in infants.
 Based on competition between
particulate and soluble antigens for
limited antibody-combining sites
Sensitivity of reaction Hemagluttination inhibition
- is govern by the avidity of the antibody. - Use the same principle; except red blood
- capable of detecting small quantities of cells are the indicator particles
antigen.  RBC has naturally occurring viral
- can be used for detection of illicit drugs: receptors
 Cocaine  When virus is present spontaneous
 Heroin agglutination occurs .
 The patient antibody inhibits the
agglutination reaction.
Used to detect antibodies to certain viruses
- Mumps
- Measles
- HBV
- Herpes virus
- Respiratory syncytial virus
- Rubella
- Adenovirus
- Parainfluenza
- Influenza
 Systems using bacteria as the inert
particles to which antibody is attached
Protein A Identifies:
- Naturally adsorbs fragment - Neisseria meningitides
crystallizable (Fc) portion of antibody - Neisseria gonorrhoeae
molecules - Vibrio cholerae 0139
- Exhibit greater stability than Latex - Haemophilus influenzae
particles - Streptococci
- More refractory to change in ionic
strength  highly specific, but it may not be as sensitive
for detecting small quantities of antigen.
Test tubes can be centrifuged:
 shaken to see if the cell button can be resuspended.
Negative:
- resuspended
- with no visible clumping
Positive:
- clumping
- graded to indicate the strength of the reaction
GRADING OF AGGLUTINATION REACTIONS
4+ = solid clump
3+ = several large clumps
2+ = small to medium sized clumps; clear background
1+ = small clumps; cloudy background
+w = tiny aggregates; cloudy background
+ micro = positive upon microscopic examination only
+ MF = Mixed field:
Small clumps amidst many unagglutinated cells.
Can be confused with 1+
hem = hemolyzed (a positive reaction)
neg = negative, no agglutination
(Never use – for negative; either write neg or 0)

Antiglobulin-Mediated Agglutination

Coomb’s Test (Antihuman globulin test)
 Detects nonagglutinating antibody by means  Possible Source of Errors in Coomb’s Test
of coupling with second antibody 1. Failure to wash cells
2. Improper centrifugation
3. Failure to add test serum or antihuman
globulin
4. Use of expired reagents
5. Improper concentration of RBC
 Too heavy concentration
– mask agglutination
 Too light concentration
– hard to read

- Most widely used procedures in Blood 1. Direct Antiglobiun Test 2. Indirect Antiglobulin Test
banking - Used to demonstrate in vivo the attachment
of antibody Used to:
- Key component: a) determine presence of particular
 Antibody to human globulin - Indicator of: antibody in a patient
(made from Animals or Hybridoma a) Autoimmune hemolytic anemia b) type patient RBC for specific blood
technique) b) Hemolytic disease of the newborn group antigens
c) Sensitization of RBC due to drugs
Antibody will react with the FC portion of the d) Transfusion reaction Two-step process:
human antibody attached to RBC. 1. RBC and antibody is combined at 37°C
Polyspecific Antiserum 2. Antihuman globulin is added, visible
 Agglutination takes place because the - will react to IgG and Complement reaction occurs
antihuman globulin is able to bridge the component C3d.
distance between cells that IgG alone o Otheres: C3b, C4b, or C4d Most often:
cannot do. - check for the presence of clinically
alloantibody in patient serum
Strength of reaction is proportional to amount of Positive result: - for Compatibility testing for blood
antibody coating the RBC  indicates immune reaction is taking place transfusion

Instrumentation

Several systems have been developed using automation to increase sensitivity:

Turbidimetry Principle: as particles combine, light scatter increases, and the absorbance
of the solution increases proportionally.

Nephelometry Decribed by particle-enhanced immunoassay:

o used to measure forward light scatter. - without particles: detect soluble antigen–antibody complexes at
a sensitivity of between 1 and 10 μg/mL
- with particles: sensitivity can be increased to nanograms/mL;
small latex particles with a diameter of less than 1 μm are used
Particle-counting Immunoassay (PACIA) Latex particles: coated with whole antibody molecules or with F(ab)2
fragments that reduces interference and nonspecific agglutination.
Used to measure:
- the number of residual nonagglutinating particles in a specimen Inverse relationship: between the number of unagglutinated particles
- several serum proteins counted and the amount of unknown in the patient specimen.
- therapeutic drugs
- tumor markers Measurements are made by looking at:
- viral antigens (measles, herpes simplex, and hepatitis B sAg) 1. Rate assay:
o the rate at which the number of unagglutinated particles
Three orders of magnitude more sensitive than standard manual decrease.
agglutination method. 2. End-point assay:
o the total number of unagglutinated particles left at the end.
Counted by : means of a laser beam in an optical particle count
Disadvantage: Expensive

Quality Control and Quality Assurance

- Techniques must be standardized:  Cross-reactivity

 concentration of antigen - Caused by presence of antigenic determinants that resemble
 incubation time one another so closely that antibody formed against one will
 temperature react with the other.
 diluent - Can be AVOIDED by the use of monoclonal antibody.
 method of reading

Interfering bodies  May produce false positive results

1. Heterophile Antibodies - Most often consideration when RBC are used as the carrier
particle.

Can be CONTROLLED by:

- Preadsorption of test serum on RBC without the test antigen

2. Rheumatoid factor - Will react with any IgG present

- Problem in reverse passive agglutination tests

Can be AVOIDED by:

- Treating samples with:
o Pronase
o 2-mercaptoethanol:
Reduce false – positive results due to the IgM
rheumatoid factor

Causes of False Reactions in Agglutination Testing

False-Positive Reactions False-Negative Reactions

 Overcentrifugation  Under centrifugation
 Contaminated glassware, slides, or reagents  Inadequate washing of cells
 Autoagglutination  Reagents not active
 Saline stored in glass bottles  Delays in testing procedures
 cross-reactivity  Incorrect incubation temperature
 rheumatoid factor  Insufficient incubation time
 heterophile antibody  Prozone phenomenon
 Delay in reading a slide test  Failure to add antiglobulin reagent

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