Agglutination TestsPrinciples and Practice

Dr.T.V.Rao MD

Beginning of Serology• Serology as a science began in 1901.

Austrian American immunologist Karl Landsteiner (1868-1943) identified groups of red blood cells as A, B, and O. From that discovery came the recognition that cells of all types, including blood cells, cells of the body, and microorganisms carry proteins and other molecules on their surface that are recognized by cells of the immune system.

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Karl Landsteiner (1868-1943)

• An Austrian physician by training, Landsteiner played an integral part in the identification of blood groups. He demonstrated the catastrophic effect of transfusing with the wrong type of blood,

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Purpose of Serological Tests

• Serological tests may be performed for diagnostic purposes when an infection is suspected, in rheumatic illnesses, and in many other situations, such as checking an individual's blood type. Serology blood tests help to diagnose patients with certain immune deficiencies associated with the lack of antibodies, such as X-linked agammaglobulinemia.

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Serology• The branch of

laboratory medicine that studies blood serum for evidence of infection and other parameters by evaluating antigen-antibody reactions in vitro 04/11/2023 Dr.T.V.Rao MD 5

Agglutination

• Is the aggregation of particulate matter caused by the combination with specific antibody

• 1896: First observed by Gruber and Durham when serum antibody was found to react with bacterial cells

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Agglutination• Agglutinins

– Antibodies that produce such reactions

• Involves two-step process:– Sensitization or initial

binding– Lattice formation or

formation of large aggregates

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Agglutination

• Types of particles that participate in such reactions:–Erythrocytes–Bacterial cells–Inert carriers such as latex

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Agglutination tests• Antibodies can agglutinate multivalent

particulate antigens, such as Red Blood Cells (RBCs) or bacteria

• Some viruses also have the ability to agglutinate with RBCs.

• This behavior is called agglutination.• Serological tests based on agglutination

are usually more sensitive than those based on precipitation

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Examples• Slide Agglutination Test• Plate Agglutination Test• Tube Agglutination Test• Passive Agglutination Test• Microscopic Agglutination Test• Haemagglutination test (HAT)04/11/2023 Dr.T.V.Rao MD

Steps in Agglutination•Primary phenomenon

(SENSITIZATION) First reaction involving Ag-Ab combination Single antigenic determinant on the surface

particle1) Initial reaction: rapid and reversible2) Cross link formation visible aggregates

(stabilization)

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SENSITIZATION

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Secondary phenomenon:LATTICE FORMATION

– Ab + multivalent Ag stable network (visible reaction)

– conc. of Ag and Ab– Governed by physiochemical factors:

• Ionic strength of milieu• pH• temperature

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Secondary Phenomenon • Lattice Formation• The Fab portion of the Ig molecule attaches to

antigens on 2 adjacent cells-visible results in agglutination

• If both antigen and antibody are SOLUBLE reaction will become visible over time, ie, precipitation

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DIRECT AGGLUTINATION- Test patient serum against large,

cellular antigens to screen for the presence of antibodies.

• Antigen is naturally present on the surface of the cells.

• In this case, the Ag-Ab reaction forms an agglutination, which is directly visible.

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DIRECT AGGLUTINATION• The particle antigen may be a

bacterium.

e.g.: Serotyping of E. coli, Salmonella using a specific antiserum

• The particle antigen may be a parasite.

e.g.: Serodiagnosis of Toxoplasmosis• The particle antigen may be a red blood

cell.

e.g.: Determination of blood groups04/11/2023 Dr.T.V.Rao MD 16

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DIRECT AGGLUTINATION

• These reactions can be performed on slides (rapid tests) or on microliter plates or tubes for Antibody titration if required.04/11/2023 Dr.T.V.Rao MD 18

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Slide Agglutination Test• Used for serotyping (e.g. Salmonella)• Antigen: isolated Salmonella in

suspension• Antibody: specific antisera against

Salmonella• Place test Salmonella in a drop of saline

on a slide • Add a drop of antiserum, mix and rock

slide for approx. 1 minute• Examine for agglutination04/11/2023 Dr.T.V.Rao MD

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Slide Agglutination Test

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Tube Agglutination Test• Also known as the standard agglutination test or serum

agglutination test (SAT)• Test serum is diluted in a series of tubes (doubling

dilutions)• Constant defined amount of antigen is then added to

each tube and tubes incubated for ~20h @37°C• Particular antigen clumps at the bottom of the test tube• Test is read at 50% agglutination• Quantitative• Confirmatory test for ELISA reactors• Example: Brucellosis screening , Widal Testing

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Tube Agglutination Test

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No agglutinationAgglutination

1/10 1/20 1/40 1/80 1/160 1/320 Neg. ctrl

In this case, the titre is 1/40

Tube Agglutination Test

Passive Agglutination• An agglutination reaction that employs

particles that are coated with antigens not normally found in the cell surfaces

• Particle carriers include:– Red blood cells– Polystyrene latex– Bentonite– charcoal

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Passive Agglutination

• Passive agglutination has been used in the detection of :–Rheumatoid factor–Antinuclear antibody in LE–Ab to group A streptococcus

antigens–Ab to Trichinella spiralis

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Passive Agglutination Test• Converting a precipitating test to an

agglutinating test• Chemically link soluble antigen to inert

particles such as LATEX or RBC• Addition of specific antibody will cause the

particles to agglutinate• Reverse PAT: antibody linked to LATEX

e.g. Lancefield grouping in Streptococci.

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The latex particles are coated with IgG and mixed with the patient's serum

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Quantitative Micro Hemagglutination Test (HA)

Haemagglutination Test (HA)

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Haemagglutination

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Haemagglutination

RBC

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Viral Haemagglutination• Some viruses and microbes contain proteins

which bind to erythrocytes (red blood cells) causing them to clump together

• NDV• Adenovirus III• AIV• IBV• Mycoplasma

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Viral Hemagglutination

• the attachment of viral particles by their receptor sites to more than 1 cell.

• As more and more cells become attached in this manner agglutination becomes visible

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Equivalence point: (suitable proportion between the virus particles

and RBCs)

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Negative control well (only RBCs+ buffer) (no Haemagglutinnins)

Positive control well (contains haemagglutinin)

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Readings The results • Titer: The maximum dilution that gives

visible agglutination.• The end point: is the well with the lowest

concentration of the virus where there is haemagglutination

2 4 8 16 32 64 128 256 512 1024 2048 4096

The HA titer of this virus in this row is 256 or 28

(1:256 dilution contains (1 HA unit) (one haemagglutinating unit)

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Example of readings

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Titer = 32 HA units/ml

Hemagglutination test: method

1:8

1:2 1:21:21:21:2

8 16 32 64 128 256

virus

serial dilution

mix with red blood cells

side view

top view

One HA unit :minimum amount of virus that causes complete agglutination of RBCs

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WHAT DO WE NEED?

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PROCEDURE(CONTROLs)

• Always run four control rows:

_ Positive: Contains antibodies against the specific virus

_ Negative: Contains no antibodies against the specific virus

_ Antigen

_ RBCs04/11/2023 Dr.T.V.Rao MD

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WASHING RBCs

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Why do we have to wash RBCs?

•To obtain pure RBCs and to get rid from any other blood components such as WBCs, immune complexes, and Abs

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Washing process • Take place 4-5 time .• Until get clear solution

above the RBCs after centrifugation .

• Using PBS or normal saline .

Note :(avoid using water to wash RBCs because it will definitely lead to RBCs lyses) 4304/11/2023 Dr.T.V.Rao MD

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Procedure • Obtain blood samples in tubes, spin at 1500

RPM for 5 minutes.• Draw off the supernatant using Pasteur pipette.• Add 2ml PBS to each tube and move to a clean

test tube.• Centrifuge again. Each time draw off the

washing solution and add 10 ml PBS until the solution above the RBCs layer becomes clear.

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In the absence of anti-virus antibodies

Erythrocytes

Virus

Virus agglutination of erythrocytes

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In the presence of anti-virus antibodies

Erythrocytes

Virus Anti-virus antibodies

Viruses unable to bind to

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What is Antibody Titer• Is the lowest

concentration of antibodies against a particular antigen.

Figure 18.604/11/2023 Dr.T.V.Rao MD

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Readings • The end point is the well with the lowest concentration of the serum where a clear

button is seen. 2 4 8 16 32 64 128 256 512 1024 2048 4096

The antibody titer in this row will be 512 (29).(the lowest concentration of Abs which inhibit HA caused

by the virus )

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Coombs Test an Agglutination Test

• The Coombs test is actually two separate tests: the "direct" and "indirect" Coombs tests. Both aim to identify autoimmune haemolysis of red blood cells (erythrocytes).

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Coombs (Antiglobulin)Tests

• Incomplete Ab• Direct Coombs Test

– Detects antibodies on erythrocytes

+ ↔

Patient’s RBCs Coombs Reagent(Antiglobulin)

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Coombs Test Direct ant globulin test (also called the

Coombs’ test,

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Coombs (Antiglobulin)Tests • Indirect Coombs Test

– Detects anti-erythrocyte antibodies in serum

Patient’s Serum

TargetRBCs

+ ↔Step 1

+ ↔

Coombs Reagent(Ant globulin)

Step 2

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Application of Coombs (Antiglobulin)Tests

• Applications–Detection of

anti-Rh Ab–Autoimmune

hemolytic anemia

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REVERSE PASSIVEAgglutination Tests

• Antibody rather than antigen is attached to a carrier particle

• For the detection of microbial antigens such as:▫ Group A and B streptococcus▫ Staphylococcus aureus▫ Neisseria meningitides▫ Haemophilus influenza▫ Rotavirus▫ Cryptococcus neoformans▫ Mycoplasma pneumoniae▫ Candida albicans

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REVERSE PASSIVE Agglutination Tests

• PRINCIPLE: latex particles coated with antibody are reacted with a patient sample containing suspected antigen04/11/2023 Dr.T.V.Rao MD 57

Agglutination Inhibition• Based on the competition between

particulate and soluble antigens for limited antibody combining site

• Lack of agglutination is indicator of a positive reaction

• Usually involves haptens complexed with proteins

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Agglutination InhibitionTests

• Pregnancy Testing-classic example of

agglutination inhibition

– Human chorionic gonadotropin (hCG)• Appears in serum

and urine early in pregnancy

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Agglutination InhibitionUrine Antiserum

No hCG in urine:

Anti-hCG free

hCG in urine:Anti-hCG neutralized

Carriers coated with hCG added Carriers coated with hCG added

AGGLUTINATION of carriers:Negative test for hCGNOT PREGNANT

NO AGGLUTINATION of carriers:

Positive test for hCGPREGNANT

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Co-agglutination• Co agglutination is similar to the latex

agglutination technique for detecting antigen (described above). Protein A, a uniformly distributed cell wall component of Staphylococcus aureus, is able to bind to the Fc region of most IgG isotype antibodies leaving the Fab region free to interact with antigens present in the applied specimens. The visible agglutination of the S. Aureus particles indicates the antigen-antibody reactions

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Coagglutination• Name given to

systems using inert bacteria as the inert particles to which the antibody is attached

• S.aureus: most frequently used because it has protein A in its outer surface that naturally adsorbs the Fc portion of the antibody04/11/2023 Dr.T.V.Rao MD 62

Highly specific but not very sensitive in detecting small quantities of antigen

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Co agglutination Test Agglutination test in

which inert particles (latex beads or heat-killed S aureus Cowan 1 strain with protein A) are coated with antibody to any of a variety of antigens and then used to detect the antigen in specimens or in isolated bacteria.

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Rickettsia and Serology• Rickettsia is a genus of motile, Gram-negative,

non-spore forming, highly pleomorphic bacteria that can present as Cocci (0.1 μm in diameter), rods (1–4 μm long) or thread-like (10 μm long). Obligate intracellular parasites

• Because of this, Rickettsia cannot live in artificial nutrient environments and are grown either in tissue or embryo cultures (typically, chicken embryos are used).

• Still we have to dependent on Weil Felix test

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Weil and Felix contribute for testing

• In 1915, Weil and Felix showed that serum of patients infected with any member of the typhus group of diseases contains agglutinins for one or more strains of O X Proteus. In cases of typhus fever the reaction usually appears before the sixth day and reaches its height in the second week.

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Weil-Felix reaction – A Heterophile agglutination Test

• A Weil-Felix reaction is a type of agglutination test in which patients serum is tested for agglutinins to O antigen of certain non-motile Proteus and Rickettsial strains(OX19, OX2, OXk)

• OX19, OX2 are strains of Proteus vulgaris.OXk is the strain of Proteus mirabilis. 04/11/2023 Dr.T.V.Rao MD 67

Weil-Felix a Heterophile agglutination test

• The agglutination reactions, based on antigens common to both organisms, determine the presence and type of rickettsial infection

• Because Rickettsiae are both fastidious and hazardous, few laboratories undertake their isolation and diagnostic identification

• Weil-Felix test that is based on the cross-reactive antigens of OX-19 and OX-2 strains of Proteus vulgaris.

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Reading/Grading Agglutination Reactions

• Done by gently shaking the tubes containing the serum and cells, and observing the cell button as it is dispersed

• Hard shaking must be avoided because this may yield to false result

• Attention should also be given to whether discoloration of the supernatant is present (Hemolysis).

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Reading/Grading Agglutination Reactions

• Pseudo agglutination or the Rouleaux Formation also occurs– Red blood cells appear as stacks of coins.

• Addition of physiologic Nacl will disperse pseudo agglutination

• Saline Replacement is done after pseudo agglutination is observed so that it may not give false negative result due to the dilution effect of the saline

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GRADING AGGLUTINATION REACTIONS

GRADE DESCRIPTION Appearance

Negative (-) No aggregates

Weak (+/-) Tiny aggregates that are barely visible macroscopically; turbid and reddish supernatant

1+ A few small aggregates just visible macroscopically; turbid and reddish supernatant

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2+ Medium-sized aggregates; clear supernatant

3+ Several large aggregates; clear supernatant

4+ One solid aggregate; clear supernatant

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Interpretations in Weil-Felix reaction

• Sera from endemic typhus agglutinate OX19, OX2.Tick borne spotted fever agglutinate OX19, OX2.

• Scrub Typhus agglutinate OXk strain • Test is negative in rickettsia pox, trench fever

and Q-fever.False positive reaction may occur in urinary or other Proteus infectionsTest may be negative in 50 percent scrub typhus

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Weil-Felix testindicated in when patients present

with rashes• Test for diagnosis of

typhus and certain other Rickettsial diseases. The blood serum of a patient with suspected Rickettsial disease is tested against certain strains of (OX-2, OX-19, OX-K)..

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Latex Agglutination

•Antibody molecules can be bound to each latex beads

• It will increase the potential number of exposed antigen-binding sites.

•When an antigen is present in test specimen, it may bind to the latex bead thus forming visible cross-linked aggregates.

•Latex particles can be coated with antigen (pregnancy testing, rubella antibody testing)

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Coagulation and Liposome-enhanced testing

• Are variations of latex agglutination

• uses antibodies bound to a particle to enhance the visibility of agglutination

• is a highly specific method but may not be sensitive.

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Problems with SerologyOther Health conditions interfere• Immunocompromised patients often give a

reduced or absent Humoral immune response.

• Patients with infectious mononucleosis and those with connective tissue diseases such as SLE may react non-specifically giving a false positive result.

• Patients given blood or blood products may give a false positive result due to the transfer of antibody

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Variables that affect the quality of results in Agglutination Tests

•The educational background and training of the laboratory personnel

•The condition of the specimens•The controls used in the test runs•Reagents•Equipment•The interpretation of the results•The transcription of results•The reporting of results

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Errors in measurement• True value - this is an ideal concept which

cannot be achieved.

• Accepted true value - the value approximating the true value, the difference between the two values is negligible.

• Error - the discrepancy between the result of a measurement and the true (or accepted true value).

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Random Error• An error which varies in an unpredictable manner, in

magnitude and sign, when a large number of measurements of the same quantity are made under effectively identical conditions.

• Random errors create a characteristic spread of results for any test method and cannot be accounted for by applying corrections. Random errors are difficult to eliminate but repetition reduces the influences of random errors.

• Examples of random errors include errors in pipetting and changes in incubation period. Random errors can be minimized by training, supervision and adherence to standard operating procedures.

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Systematic Error• An error which, in the course of a number of

measurements of the same value of a given quantity, remains constant when measurements are made under the same conditions, or varies according to a definite law when conditions change.

• Systematic errors create a characteristic bias in the test results and can be accounted for by applying a correction.

• Systematic errors may be induced by factors such as variations in incubation temperature, blockage of plate washer, change in the reagent batch or modifications in testing method.

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Automations for Agglutination

• One of the first successful attempts to automate antibody tests was made by Weitz (1967) at the Lister Institute, London. The apparatus developed by Weitz allowed the performance of up to 12 titrations in a single operation, with even less manipulation than that required for a single test done by a more conventional technique.

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• Programme Created By Dr.T.V.Rao MD for Medical Microbiologists in the

Developing World• Email

• doctortvrao@gmail.com

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