TOPIC 14 - AGGLUTINATION Results can be:

-April 19-23,2021- & -May 11, 2021- Qualitative

-Dr. Ariana S. Fernando- ▪ Serves as a screening test for the presence or absence of the antigen
or antibody.
Blue Text are words from Dr. Arianna Semiquantitative
▪ Titration of serum to determine the titer of antibody present.
1.0 Agglutination ▪ A High titer of antibody from a single patient sample - only suggestive
• Agglutination in the secondary binding test is similar to precipitation of disease.
○ However, it differs from precipitation with that antibodies (Ab) are ▪ A four-fold rise or 2-tube increase in titer between paired sera - more
made to react with particulate Antigens (Ag) instead of antigens and meaningful or diagnostic.
antibodies being soluble in the solution. ▪ Paired sera - 2 serum samples taken form the same patient at
▪ Antigens are insoluble different stages of illness.
○ Precipitation reaction represent a phase change while agglutination • 1st sample from the acute phase.
manifest as clumping or sedimentation of antigen-antibody complexes • 2nd sample taken around 2-3 weeks after onset of disease -
▪ These particulate antigens in agglutination reactions can either be convalescent stage.
cells, such as bacteria, yeast cells, red blood cells
• They can also be inert particles such as platelets, charcoal
particles or even gelatin
▪ Participating antigen = “agglutinogen”
▪ Participating antibody = “agglutinin”
○ Agglutinin testing serves as a means of detecting antigen-antibody
reactions and diagnostic clinical tets for infectious and noninfectious
diseases.
• Secondary binding tests.
• Similar to precipitation except:
○ Antibodies (Ab) are made to react with particulate antigens (Ag).
○ Manifests as clumping of Ag-Ab complexes.
• Antibodies are made to react with: Factors that influence the reaction:
○ Cells (e.g., bacteria, yeast cells and RBC) 1. Concentration
○ Inert particles (e.g., latex, charcoal, gelatin) ▪ Optimum proportions must be met in order for reaction to occur.
• Participating antigen = Agglutinogen ▪ “Zone of Equivalence” - indicated by maximal agglutination.
• Participating antibody = Agglutinin ▪ Prozoning and Postoning reactions - false negative results.
▪ Prozone - excess antibodies.
Agglutination occurs in 2 phases. ▪ Postzone - excess antigens.
1. Sensitization ▪ Lattice formation/agglutination occur when antigens and antibodies
▪ Specific antigen-antibody binding. are in appropriate concentrations.
▪ No visible agglutination is seen. ▪ Prozone and postzone yield poor lattice formation thus false negative
▪ Antibody molecules attach to their corresponding antigenic site results.
(epitope) on the red blood cell membrane with no visible clumping 2. Temperature
seen. ▪ Some antibodies react best at warm temperatures (37C) - IgG
2. Lattice Formation ▪ Others favor colder temperature (4C) - IgM.
▪ Cross-linking between sensitized particles and antibodies. ▪ Any change in temperature may affect binding strength of antibody
▪ Visible agglutination is present. and the speed/rate of reaction.
▪ Antibody molecules crosslink RBCs forming a lattice that result in visible ▪ “Cold Agglutinins” - antibodies that exhibit maximal reactivity with
clumping or agglutination. antigens at 0-10C.
3. Motion
Performed on: ▪ Shaking, stirring and centrifugation will enhance the agglutination
SLIDE TESTS TUBE TESTS MICROTITER TESTS reaction.
• Rapid • Longer incubation • Adaptation of tube ▪ This is why most agglutination tests in clinical practice employ the use
• 2-3 minutes incubation • 15 minutes - 24 hours test. of movement, it may either be manual rotation or instruments such as
time. incubation time. • Require longer rotators or centrifuge.
• Rotation at room • 4 Celsius, 20 Celsius, or incubation time. 4. pH
temperature. 37 Celsius. • Less patient’s sample ▪ Most antibodies have optimal activity between pH 6.5-7.5
• Performed on glass • More sensitive than the and less reagent ▪ Any deviation from this pH would result in the change in the reaction.
slides but more tests rapid slide tests. needed (differs from ▪ Should be close to physiologic pH to resemble in vivo conditions.
make use of plastic • Performed on glass the tube test) 5. Time of Incubation
cards. test tubes but are • Important advantage: If prolonged:
replaced with plastic small sample and • Slide method: Ag-Ab complexes dry up (false positive)
alternatives when reagent needed. • Tube method: may cause dispersion/dissociation (false negative)
using automated
If shortened
systems.
• Reaction is weaker
6. Class of Antibody
Strength of Agglutination
IgM
APPEARANCE REPORT • Efficient agglutinator.
Many small aggregates with turbid solution. +1 • Pentamer molecule which allows more antigen to bind to the antibody
small-Medium Aggregates with a clear background +2 molecule.
Several Medium- Large Aggregates +3 • Made up of 5 structural units, large in size allows bridging despite the
One Solid Aggregate +4 presence of sialic acid on the RBC surface.
• Can reach antigenic sites up to 35nm.
Ruste, G; Momongan, MJ; 2021 //
IgG Abnormality found in antibody
• Efficient precipitator • Movement of the hinge region of the antibody is limited.
• Made up of a single structural unit. • Poor function of antibody → weak reactions or no reaction occurs.
• Can attach only up to 14nm. • A structural abnormality.
RBC Zeta Potential
• Net (-) charge naturally exerted 2.0 Major Categories of Agglutination Reaction
by cells, causing them to repel
each other. 1. Direct Immune Agglutination
• IgM - can span 35nm to overcome ○ Antibodies with antigens (Innate) naturally found on the surface of the
forces, causing agglutination. cells (RBC or bacteria).
• IgG - only spans about 14nm, so ○ E.g., ABO blood typing, Widal Test
cannot reach antigens on 2. Direct Non-Immune Agglutination
separate cells to cause ○ What causes agglutination is not antibodies.
agglutination. (“Non- 1. Lectins or Phytohaemagglutinin
Agglutinating Ab” or “Incomplete ▪ Derived from plants
Ab”) ▪ e.g., Anti-H lectin extracted from Ulex Europaeus - agglutinates
• IgG - no lattice formation Group O Cells
• IgM - cross-linking occurs. ▪ Lectin from Dolichos biflorus - agglutinates A1 cells
2. Hemagglutination caused by Viruses
Ways to Produce Lattice Formation with IgG ▪ Rubella, Mumps, Influenza, Measles, Dengue Viruses - agglutinates
1. Addition of Potentiating Media RBCs.
○ To reduce zeta potential.
3. Indirect or Passive Agglutination
1. Colloidal Diluents:
○ Presence of passive participant/carrier.
▪ Bovine albumin
○ Biologic Carriers - RBCs
▪ Polybrene
○ Inert Carriers - Latex, Gelatin, Charcoal
▪ Polyethylene glycol (PEG)
1. Passive Agglutination - antigens are attached or coated onto the
▪ Protamine, polyvinylpyrrolidone (PVP)
particulate carrier.
2. Proteolytic Enzymes
▪ Papain (from papaya)
▪ Bromelin (from pineapple)
▪ Ficin (from figs)
▪ Trypsin (from pig’s stomach)
2. Addition of AHG Reagent
2. Reverse Passive Agglutination - antibodies are attached to particulate
○ “Anti-human globulin” (used in the Coomb’s Test)
carriers.
○ To bridge the gap between the sensitized cells.
3. Centrifugation
○ Mostly done in blood banking.
○ Motion enhances lattice formation.
○ Overcomes the natural repulsive effect of RBC or bacteria to one
another.
○ Allows for an increased Ag-Ab bridging (lattice formation) 3. Coombs Test (Antiglobulin technique) - anti-human globulin is used to
○ Bring cells closer together to allow IgG to bridge the cells. bridge the gap between sensitized cells.
Note: DIRECT COOMBS TEST INDIRECT COOMBS TEST
○ IgM antibody agglutinating RBCs suspended in saline. Patient’s RBCs Patient’s Serum (Antibodies)
○ Cells with IgG attached; agglutination does not occur and can only be Diagnosis of HDN and Hemolytic Cross-Matching
obtained by: Anemia Detect the presence of Anti-Rh+
▪ Reducing the zeta potential or; antibodies in the serum of the Rh (-)
▪ Adding anti-human globulin serum. mother who has given birth to a Rh+
child.
Patient’s RBCs washed 3x with NSS Patient’s Serum → Washed Donor’s
Causes of Lack of Reactivity
→ Supernatant is removed → RBCs are added → incubated for 30
1. Prozone/Postzone effect
AHG/Coombs’ Serum is added minutes at 37C → AHG is added
▪ Poor lattice formation.
(+) Agglutination - cells were coated (+) Agglutination - antibodies in
▪ Produces false negative results.
in vivo with IgG patient’s serum reacted donor’s RBCs
▪ Antigens and antibodies need to exist in optimal concentrations to
achieve “Zone of Equivalence” for maximum agglutination.
2. Non-agglutinating/Incomplete Antibodies
▪ Generally IgG and IgA - do not agglutinate in saline medium even when
bound to antigen.
▪ Physiologic disadvantage not an abnormality of the antibodies that
prevents lattice formation.
3. Other Causes
Abnormality found in antigen
• Antigenic sites which lie very deep within the surface coat of the particle.
• Antigenic sites are not readily accessible.
• No cross-linking occurs.

Ruste, G; Momongan, MJ; 2021 //

 3.0 Direct Immune Agglutination • Associated with IM, Serum Sickness and Forssman Antibodies → IgM
Type
1. Hemagglutination Several Serologic Tests
○ RBCs are agglutinated. Rapid Plate Screening Test
A. Cold Hemagglutinin Test ○ 1 drop of patient serum + 1 drop SE onto a glass slide → (+)
▪ Detects the presence of cold agglutinins. ○ Agglutination - Heterophile antibody present in the patient serum.
▪ Measures the cold agglutinin ○ Screening test only, should be followed up by a confirmatory test.
titer (CAT) Presumptive Test/Paul-Bunnel Test
▪ “Cold Agglutinins” ○ Patient serum in 56C water bath for 30 minutes (Inactivation)
• Nonspecific antibodies. ○ doubling serial dilution of patient’s serum (1:7-1:7,168)
• Strongly agglutinate RBCs at ○ add 2% SE (sheep’s erythrocytes) to all tubes
4C, ○ incubated at 37C for 1 hour or room temperature for 2 hours.
• weakly at 34C ○ > 224 = suggestive
• very weakly or negatively at ○ 4-fold increase (paired sera) = diagnostic.
37C.
Differential Heterophile Test of Davidsohn
▪ “Auto Hemagglutinins”
3 antigens are employed
• Capable of agglutinating patient’s own RBCs.
1. 20% Guinea Pig Kidney and (2) 20% Beef (Bovine) Erythrocyte
2 Groups of Cold Agglutinins
(differential antigens)
HARMLESS (BENIGN) HARMFUL or PATHOLOGIC
3. Sheep Erythrocytes (detects unabsorbed heterophile antibodies)
Found in normal sera. Associated with disease Differential absorption technique
anti-I, anti-H, rarely anti-P anti-I, anti-i, rarely other specificities 1. Patient serum + GPK in 1 tube and patient serum + BE in another
Active between 10-15C Active in vitro up to 25-30C 2. Centrifuge, separated absorbed sera.
Diseases Associated with Harmful Cold Agglutinins 3. Doubling dilutions for absorbed sera.
Primary Atypical Pneumonia (PAP) 4. Add 3rd antigen (SE) to all tubes.
• “Walking pneumonia” 5. Incubate at 37C for 1 hour .
• Caused by Mycoplasma pneumoniae Read Titers
• 55% of patients produce IgM pathologic cold agglutinins Infectious GPK absorbed serum
Not absorbed by GPK
• Anti-I specificity - reacts with I antigens found on the surface of RBCs Mononucleosis produces high titer
• High titers produced during the 2nd-4th week of illness. Low titer or (-) result in
Serum Sickness Absorbed by GPK and BE
• Titers decline or will become (-) during the 4th-6th week. both absorbed sera.
• Titer of 32-64 is suggestive of PAP. High Titer with BE
Forssman Antibody Not Absorbed by BE
• A 2-tube/4-fold increase between paired sera is diagnostic. absorbed serum
Cold Hemagglutinin Syndrome
Rapid Slide Test
• Autoimmune disease.
• High levels of cold agglutinins → patient’s own RBCs are agglutinated ○ Modification of Davidsohn’s Differential Test
when exposed to cold temperature → blockage of blood vessels ○ 3 Antigens Employed: GPK, BE (differential antigens) and HE (Horse
(extremities or the face) Erythrocytes - 3-4x more sensitive than SE)
• Reversible - rewarming of exposed parts of the body → dissociation of Spot Test by Lee and Co.
cold agglutinins → dispersion of agglutinates. ▪ On a slide:
• Manifests as acrocyanosis of the hands or the feet and in complement 1. Mix patient serum + GPK (Left Spot) and Patient serum + BE (right
binding, may present as intravascular hemolysis. spot).
2. SE added onto both spots and mix.
Other Conditions that Produce Cold Agglutinins (IgM Type)
▪ Read the Results
• Liver Disorders
Stronger agglutination on Left Spot than on
• Chronic Sepsis Infectious Mononucleosis
the Right spot.
• AHA (Autoimmune Hemolytic Anemia)
Serum Sickness (-) results on both spots.
• Black Water Fever (Severe Complication of malignant tertian malaria -
Stronger agglutination on Right Spot than on
Plasmodium falciparum) Forssman Antibody
the left spot.
• Leishmaniasis •
Procedure for Cold Hemagglutinin Tests Modification for the Infectious Mononucleosis Heterophile Antibodies
1. prewarm , syringes, test tubes, centrifuge cups. ○ Differential Antigens (GPK and BE) are no longer employed.
2. Allow blood samples to clot inside the incubator at 37C. Examples
3. Serum is separated from clotted red cells and perform doubling serial ▪ Immuno-IM-Monocoll - utilizes dyed, color-enhanced HE.
dilution on serum (1:4-1:1,024) ▪ Cellognost Mononucleosis - utilizes acid-treated HE.
4. Add antigen (1-2% Group O cells in NSS) or Patient’s own RBCs ▪ Rapid Card Test - utilizes HE IM receptor glycoprotein incorporated into
5. Refrigerate at 4C overnight. lipid particles with charcoal to enhance visualization.
6. Immediately read the titer (reciprocal of highest dilution showing ▪ Monocol/Lex - latex particle with Bovine RBC membrane substrates.
agglutination)
▪ Confirmatory Tests: (+) tubes placed in a 37C water bath for 30 2. Bacterial Agglutination
minutes-2 hours. ○ Bacterial Cells are agglutinated.
▪ If agglutination will: 2 General Functions in Clinical Laboratory
• Disperse - cold agglutinins 1. Provides rapid ID of bacteria by identifying antigens present in isolated
• Persist - not caused by cold agglutinins. colonies with the use of known antisera.
B. Heterophile Agglutinin Test 2. Provides etiologic diagnosis based on a 4-fold increase in antibody
▪ Detects presence of Heterophile Antibodies that can be detected by the titers even when no bacteria can be isolated from the patient.
6th-10th day of illness.
▪ High titers observed during the 2nd-3rd week.
▪ Heterophile Antibodies - will react with sheep and horse erythrocytes.

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Bacterial Antigens ▪ 44% of patients produce heterophile antibodies - cross react with
Somatic (O) Antigen capsular polysaccharide of Streptococcus MG.
• Gram (-) - found in the polysaccharide side chains of LPS (Endotoxin; ▪ Antibodies are detectable 2-3 weeks after onset of illness.
Biovin Antigen) Procedure
• Gram (+) - found in the outer layer of teichoic acid. 1. Blood sample is allowed to clot at any temperature.
○ Made up of glycoproteins and polysaccharides. 2. Patient’s serum is serially diluted (1:10-1:640)
• Protoplast (cell wall-deficient bacteria) - proteins of cytoplasmic 3. Add antigen (NSS Suspension of Strep MG) to all tubes.
membrane (e.g., Mycoplasma and L-forms) 4. Shake and incubate overnight at room temperature
• The “O” Complex of Antigens determines the somatic serogroup to 5. Read titer (reciprocal of the highest dilution showing bacterial
which the bacterium belongs. agglutination)
• “O” Type Agglutination → compact clumps or granular masses of • Titer of > 20 = suggestive of PAP.
bacteria (not easily disrupted by vigorous shaking • 4-fold/2-tube increase between paired sera = Diagnostic.
Capsular (K) antigen or Vi antigen ▪ 55% of patients produce cold agglutinins and 44% will produce
• “Vi” antigen among the Salmonella sp. heterophile antibodies against Streptococcus MG - run both when PAP
• Polysaccharide (in nature) or complexes of polysaccharides and is suspected.
proteins. ▪ Negative Results in both tests does not rule out PAP.
• Prevents phagocytosis - associated with virulence. ▪ Best Diagnostic Tests - isolation of Mycoplasma pneumoniae.
• Interferes with agglutination between O antigen and O antibody ▪ Medium of Choice - Edward-Hayflick Agar (mulberry or fried egg
○ Remedy: boiling water bath for 10-30 minutes. appearance)
• Opsonization renders organisms susceptible to phagocytosis. C. Tests for Febrile Agglutinins
Flagellar (H) Antigen ▪ Widal Test and Weil-Felix Test
• Present in motile bacteria. ▪ Tests Commonly referred to tests for antibodies arising from individuals
• Made up of proteins called “Flagellin” from fever of unknown origin
• Strongly antigenic “Febrile Agglutinins”
• Important to serological classification (serotyping) of bacteria. • Antibodies in response to fever-producing bacteria.
• “H” type agglutination → large, fluffy or loose, filmy clumps. (easily • E.g., Salmonella typhi, Salmonella paratyphi A, C, Brucella 1, 2, 3,
disrupted by vigorous shaking) Francisella tularensis, Rickettsia.
2 Types of H Antigen Widal Test
1. Phase 1 Antigens • Measures the antibodies produced by patients with Typhoid Fever and
▪ Specific H antigen of a serotype. Paratyphoid Fever
▪ Designated by small letters. • Antigens: O antigen and H antigen or Vi antigen.
2. Phase 2 Antigens • Baseline Titers - Anti-O (50) and Anti-H (80)
▪ Group antigens or antigens common to several serotypes.
Weil-Felix Test
▪ Designated by numbers.
• Measures heterophile antibodies produced by patients with Rickettsial
2 Types of Motile Bacteria based on “H” Antigens
disease.
MONOPHASIC DIPHASIC • Antigens: P.vulgaris OX-19 and OX-2; P.mirabilis OX-K
Have H antigens of only 1 phase. Have both phase 1 and 2 H antigens. • Titers of 64-320 = suggestive
E.g., Salmonella typhimurium 1, 4, 5, 12: • 4-fold/2-tube increase = Diagnostic.
i: 1, 2
SLIDE Test TUBE Test
Can be phase 1 or 2 (1, 4, 5, 12 - O antigens)
Patient’s Serum (1:20-1:320) Patient’s Serum (1:10-1:320)
(i - phase 1 antigen)
(1, 2 - phase 2 antigens) Add antigen then rotate for not Add antigen then shake and incubate
E.g., Salmonella typhi 9, 12; Vi; d more than 3 minutes at room at 52C (18 Hours) or at 37C (24-48
(9 & 12 - O antigens) temperature. hours)
(Vi - capsular antigen) Read titer (reciprocal of highest Read titer (reciprocal of highest
(d - phase 1 H antigen) dilution showing +1 agglutination) dilution showing +1 agglutination)
some with +2 result.
A. Coagglutination Technique
Screening Test More sensitive Tet
▪ Enhances sensitivity of bacterial agglutination.
▪ Makes it possible to detect agglutination in weaker bacterial
suspensions. 3. Tests for Nonspecific Reagin
Coagglutination Reagents for Serotyping Streptococcus Groups A, B, C, D ○ Non-treponemal tests for diagnosis of Syphilis
and Gonococcus 2 Major Groups of Serologic Tests for Syphilis
• Each reagent is made up of dead 1. Treponemal Tests
staphylococcus aureus (whole cells) • Detect specific antibody against T.pallidum (IgG class)
coated with specific antibodies (IgG Class) 2. Non-treponemal Tests
• IgG attach protein A of S.aureus cells by • Detect the presence of nonspecific reagin.
their Fc portions. “Syphilitic Reagin”
• Fab portions are freed to bind the ▪ IgM antibody produced by patients with Treponemal Infections.
antigens on the isolated bacterium ▪ Detected in serum (1st and 2nd Stage) and CSF (3rd Stage)
producing large clumps. Common Non-Treponemal Tests
• Fc Portion - attaches to Protein A of ▪ Kolmer, Kline, Hinton, Kahn
S.aureus ▪ RPR*
• Fab Portion - bind to antigen of isolated ▪ VDRL*
bacterium. ▪ *most commonly performed in the lab.
• Not exactly passive/indirect agglutination - due to active participation ▪ Serve only as screening/presumptive tests for syphilis.
of S.aureus. ▪ Can be used to monitor efficiency of treatment.
B. Streptococcus MG Agglutination Test
▪ Alternative test to Cold Hemagglutinin Test for PAP.

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A. VDRL Tests (Venereal Disease Research Laboratory Test) • Uses plastic coated cards with 14-18mm circles.
▪ Serum or CSF sample depending on the stage of syphilis. • Procedure
▪ Sample should be drawn before meals. 1. 0.05mL patient’s serum + 1 drop RPR antigen using gauge 18 needle
▪ Sample should not be hemolyzed. (1/60mL)
▪ Alcohol diminishes the reaction. 2. Rotate at 100 rpm for 8 minutes.
▪ Serum should be inactivated (heated at 56C for 30 minutes) • Reading
▪ No inactivation necessary for CSF. ○ Reactive = any clumping
○ Non-reactive = slight roughness or no clumping.
VDRL Reagent
• Cardiolipin
4.0 Quantitative Agglutination Reactions
○ Beef heart extract.
○ Serves as antigen. • Inert particles (instead of RBCs) are used.
• Lecithin and Cholesterol • Immunoassays in which agglutination is the end product.
○ To enhance the sensitivity of reagent. • Assays may allow for simultaneous detection of more than 1 antigen.
○ Lecithin - Neutralizes the anticomplementary property of cardiolipin. • Higher sensitivity
○ Cholesterol - enhances the complement fixing activity of cardiolipin • Quantitative rather than just qualitative or semiquantitative.
with reagin.
Qualitative vs. Semiquantitative Tests 2 Major Categories
Qualitative Test 1. Those in which agglutination can be detected using the naked eye.
2. Those that allow instruments to be used to measure low levels of
• Uses a concave slide
agglutination.
• Procedure
1. 0.05mL of patient’s serum + 1 drop VDRL Antigen (1/60mL; using
syringe with gauge 18 needle with bevel cut off) A. Sol Particle Immunoassay
2. Rotate for 4 minutes at 180 rpm (manual: 120rpm for 4 minutes) ○ Immunoassays that use antibodies (or antigens) bound to colloidal
3. Read result under LPO (take not of clumps and free antigens) particles.
• Reading ○ Colloidal Particles (Sols) - fine particles that disperse instead of settling
out of solution.
○ Gold Particles - one of the most popular inorganic colloidal particles
used.; has special optical properties.
○ Antibodies or antigens absorbed onto the surface of gold particles
forming sensitized gold particles.
Advantages of Using Gold Particles
Medium to large clumps formed with few or no free antigens ▪ Dispersed gold particles in buffer solution are purple red (or magenta)
Reactive
(Patient’s serum has reagin) in color - not altered when antibody or antigen is affixed to the particle.
Weakly Small clumps with a few to moderate free antigens (Reagin is ▪ When they aggregate, magenta color is either significantly reduced or
Reactive present but in small amounts) disappears completely (color can be detected by the naked eye).
Non- ▪ If used in quantitative tests, a standard curve is constructed.
No Clumps; free antigens are well distributed (Reagin is Absent)
Reactive
Semiquantitative Test
• Uses a ceramic ringed slide.
• NSS - added using a gauge 23 without bevel (1/100mL drop)
• Antigen - added using a gauge 19 without bevel (1/75mL drop)
• Procedure
1. Patient’s serum - dilution (1:1-1:32) on ceramic ringed slide.
2. Add 1 drop of antigen to 0.04mL dilutions.
▪ (Figure) Sol-particle Immunoassay: a scheme of conjugate
3. Rotate for 4 minutes at 180 rpm.
aggregation caused by binding to target molecules (a) and
4. Read under LPO (titer = reciprocal of highest dilution with +2
corresponding changes in sol color and absorption spectra (b).
agglutination)
• Serial Dilution
B. Disperse Dye Immunoassay
1:1 1:2 1:4
○ Immunoassay in which colloidal dye is used as particles.
Undiluted Patient’s Serum 0.04mL 0.02mL 0.01mL
○ Aggregated antigen/antibody particles can be detected by the naked
NSS (O.01mL/drop - 2 drops 3 drops eye (or quantitated using photometric techniques)
VDRL Antigen (1/75mL/drop) 1 drop 1 drop 1 drop ○ Organic Colloidal Dye Particles - not as popular as gold. Do not
○ change color when aggregated.
1:8 1:16 1:32 ○ Dye Particles - useful as indicator labels on antibody molecules.
Diluted 1:8 patient’s serum 0.04mL 0.02mL 0.01mL
NSS (O.01mL/drop - 2 drops 3 drops C. Immunoassay by Particle Counting (IMPACT)
VDRL Antigen (1/75mL/drop) 1 drop 1 drop 1 drop ○ Employs instrumentation to achieve a higher sensitivity.
• Reading ○ Antigen or antibody coated latex particles are incubated with biologic
○ A titer > 8 = indicates active Syphilis. sample in an automated instrument (includes incubator or agitator and
○ < 8 = suggests latent or dormant Syphilis, previous treatment or late particle counter linked to a computer processor and data analyzer)
Syphilis. ○ Particle counting component operates on the same principle as Flow
B. RPR (Rapid Plasma Reagin) Test Cytometer.
• Uses VDRL Cardiolipin antigen incorporated with choline chloride and ○ Following incubation, the number of unagglutinated particles are
charcoal particles. determined.
• Choline Chloride - inactivates nonspecific inhibitors. ○ Absent antibody/antigen → number of non agglutinated particles will
• Charcoal - enhances visualization of reaction. be high.
• Serum or plasma (Heparin or EDTA) ○ Agglutination will only occur if antigen/antibody is present in serum.
• Not suitable for CSF. ○ Computer transforms unagglutinated particle counting data into a
Ruste, G; Momongan, MJ; 2021 //
 standard curve from which the concentration of antigen/antibody can
be determined.
Main Advantage
▪ Automation
▪ Short incubation period
▪ High sensitivity.

▪ (Figure) Principle of immuno aggregation assay, which can be readily

coupled with optical microscopes or particle counters for quantitative
and qualitative detection of biomacromolecules.

IMMUNOASSAY TECHNIQUE INERT PARTICLE DETECTION

Quali - color change
SPIA Indirect or reverse agglutination Gold - inorganic colloidal particles
Quanti - colorimetric analysis
Quali - color visualized.
DIA Indirect or reverse agglutination Dye - organic colloidal particle
Quanti - color measured optically (photometric analysis)
IMPACT Indirect or Reverse Agglutination Latex Particles Quanti - automated particle counter.

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