Agglutination Tests basic skills

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Agglutination Tests basic skills


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Agglutination Tests Principles and Practice:

Agglutination Tests Principles and Practice Dr.T.V.Rao MD

Beginning of Serology:

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 . 1/3/2013 Dr.T.V.Rao MD 2

Karl Landsteiner (1868-1943):

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, 1/3/2013 Dr.T.V.Rao MD 3

Purpose of Serological Tests:

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. 1/3/2013 Dr.T.V.Rao MD 4


Serology The branch of laboratory medicine that studies blood serum for evidence of infection and other parameters by evaluating antigen-antibody reactions in vitro 1/3/2013 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 1/3/2013 Dr.T.V.Rao MD 6


Agglutination Agglutinins Antibodies that produce such reactions Involves two-step process: Sensitization or initial binding Lattice formation or formation of large aggregates 1/3/2013 Dr.T.V.Rao MD 7


Agglutination Types of particles that participate in such reactions: Erythrocytes Bacterial cells Inert carriers such as latex particles 1/3/2013 Dr.T.V.Rao MD 8

Agglutination tests:

9 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 1/3/2013 Dr.T.V.Rao MD


10 Examples Slide Agglutination Test Plate Agglutination Test Tube Agglutination Test Passive Agglutination Test Microscopic Agglutination Test Haemagglutination test (HAT) 1/3/2013 Dr.T.V.Rao MD

Steps in Agglutination:

Steps in Agglutination Primary phenomenon (SENSITIZATION) First reaction involving Ag-Ab combination Single antigenic determinant on the surface particle Initial reaction: rapid and reversible Cross link formation  visible aggregates (stabilization) 1/3/2013 Dr.T.V.Rao MD 11


SENSITIZATION 1/3/2013 Dr.T.V.Rao MD 12

PowerPoint Presentation:

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 1/3/2013 Dr.T.V.Rao MD 13

Secondary Phenomenon :

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, p recipitation 1/3/2013 Dr.T.V.Rao MD 14


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. 1/3/2013 Dr.T.V.Rao MD 15


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 groups 1/3/2013 Dr.T.V.Rao MD 16

PowerPoint Presentation:

1/3/2013 Dr.T.V.Rao MD 17


DIRECT AGGLUTINATION These reactions can be performed on slides (rapid tests) or on microliter plates or tubes for Antibody titration if required. 1/3/2013 Dr.T.V.Rao MD 18

Slide Agglutination Test:

19 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 agglutination 1/3/2013 Dr.T.V.Rao MD

PowerPoint Presentation:

1/3/2013 Dr.T.V.Rao MD 20

Slide Agglutination Test:

21 Slide Agglutination Test 1/3/2013 Dr.T.V.Rao MD

Tube Agglutination Test:

22 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 1/3/2013 Dr.T.V.Rao MD

PowerPoint Presentation:

23 Tube Agglutination Test 1/3/2013 Dr.T.V.Rao MD

Tube Agglutination Test:

No agglutination Agglutination 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:

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 1/3/2013 Dr.T.V.Rao MD 25

Passive Agglutination:

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 1/3/2013 Dr.T.V.Rao MD 26

Passive Agglutination Test:

27 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. 1/3/2013 Dr.T.V.Rao MD

PowerPoint Presentation:

The latex particles are coated with IgG and mixed with the patient's serum 1/3/2013 Dr.T.V.Rao MD 28

Quantitative Micro Hemagglutination Test (HA):

29 Quantitative Micro Hemagglutination Test (HA) Haemagglutination Test (HA) 1/3/2013 Dr.T.V.Rao MD


30 Haemagglutination 1/3/2013 Dr.T.V.Rao MD


31 Haemagglutination RBC 1/3/2013 Dr.T.V.Rao MD

Viral Haemagglutination:

32 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 1/3/2013 Dr.T.V.Rao MD

Viral Hemagglutination:

33 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 1/3/2013 Dr.T.V.Rao MD

Equivalence point: (suitable proportion between the virus particles and RBCs):

34 Equivalence point: (suitable proportion between the virus particles and RBCs) 1/3/2013 Dr.T.V.Rao MD

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

Negative control well (only RBCs+ buffer) (no Haemagglutinnins) Positive control well (contains haemagglutinin)

Readings The results :

36 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 2 8 (1:256 dilution contains (1 HA unit) (one haemagglutinating unit) 1/3/2013 Dr.T.V.Rao MD

Example of readings :

37 Example of readings 1/3/2013 Dr.T.V.Rao MD

PowerPoint Presentation:

Titer = 32 HA units/ml Hemagglutination test: method 1:8 1:2 1:2 1:2 1:2 1:2 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


39 WHAT DO WE NEED? 1/3/2013 Dr.T.V.Rao MD


40 PROCEDURE (CONTROLs) Always run four control rows: _ Positive : Contains antibodies against the specific virus _ Negative : Contains no antibodies against the specific virus _ Antigen _ RBCs 1/3/2013 Dr.T.V.Rao MD


41 WASHING RBCs 1/3/2013 Dr.T.V.Rao MD

Why do we have to wash RBCs?:

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 42 1/3/2013 Dr.T.V.Rao MD

Washing process :

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) 43 1/3/2013 Dr.T.V.Rao MD

Procedure :

44 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. 1/3/2013 Dr.T.V.Rao MD

In the absence of anti-virus antibodies:

45 In the absence of anti-virus antibodies Erythrocytes Virus Virus agglutination of erythrocytes 1/3/2013 Dr.T.V.Rao MD

In the presence of anti-virus antibodies:

46 In the presence of anti-virus antibodies Erythrocytes Virus Anti-virus antibodies Viruses unable to bind to the erythrocytes 1/3/2013 Dr.T.V.Rao MD

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47 1/3/2013 Dr.T.V.Rao MD

What is Antibody Titer:

48 What is Antibody Titer Is the lowest concentration of antibodies against a particular antigen. Figure 18.6 1/3/2013 Dr.T.V.Rao MD

PowerPoint Presentation:

49 1/3/2013 Dr.T.V.Rao MD

Readings :

50 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 (2 9 ). (the lowest concentration of Abs which inhibit HA caused by the virus ) 1/3/2013 Dr.T.V.Rao MD

Coombs Test an Agglutination Test:

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). 51 1/3/2013 Dr.T.V.Rao MD

Coombs (Antiglobulin)Tests :

Coombs (Antiglobulin)Tests Incomplete Ab Direct Coombs Test Detects antibodies on erythrocytes + ↔ Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Patient’s RBCs Coombs Reagent (Antiglobulin) 1/3/2013 Dr.T.V.Rao MD 52

Coombs Test Direct ant globulin test (also called the Coombs’ test, :

Coombs Test Direct ant globulin test (also called the Coombs’ test, 53 1/3/2013 Dr.T.V.Rao MD

Coombs (Antiglobulin)Tests :

Coombs (Antiglobulin)Tests Indirect Coombs Test Detects anti-erythrocyte antibodies in serum Y Y Y Y Y Patient’s Serum Target RBCs + ↔ Step 1 + ↔ Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Coombs Reagent (Ant globulin) Step 2 1/3/2013 Dr.T.V.Rao MD 54

Application of Coombs (Antiglobulin)Tests :

Application of Coombs (Antiglobulin)Tests Applications Detection of anti-Rh Ab Autoimmune hemolytic anemia 1/3/2013 Dr.T.V.Rao MD 55

REVERSE PASSIVE Agglutination Tests:

REVERSE PASSIVE Agglutination 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 1/3/2013 Dr.T.V.Rao MD 56

REVERSE PASSIVE Agglutination Tests:

REVERSE PASSIVE Agglutination Tests PRINCIPLE: latex particles coated with antibody are reacted with a patient sample containing suspected antigen 1/3/2013 Dr.T.V.Rao MD 57

Agglutination Inhibition:

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 1/3/2013 Dr.T.V.Rao MD 58

Agglutination Inhibition Tests:

Agglutination Inhibition Tests Pregnancy Testing - classic example of agglutination inhibition Human chorionic gonadotropin (hCG) Appears in serum and urine early in pregnancy 1/3/2013 Dr.T.V.Rao MD 59

Agglutination Inhibition:

Agglutination Inhibition Urine 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 hCG NOT PREGNANT NO AGGLUTINATION of carriers: Positive test for hCG PREGNANT 1/3/2013 Dr.T.V.Rao MD 60


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 1/3/2013 Dr.T.V.Rao MD 61


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 antibody 1/3/2013 Dr.T.V.Rao MD 62

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

Highly specific but not very sensitive in detecting small quantities of antigen 1/3/2013 Dr.T.V.Rao MD 63

Co agglutination Test:

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. 1/3/2013 Dr.T.V.Rao MD 64

Rickettsia and Serology:

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 1/3/2013 Dr.T.V.Rao MD 65

Weil and Felix contribute for testing:

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. 1/3/2013 Dr.T.V.Rao MD 66

Weil-Felix reaction – A Heterophile agglutination Test:

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. 1/3/2013 Dr.T.V.Rao MD 67

Weil-Felix a Heterophile agglutination test:

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 . 1/3/2013 Dr.T.V.Rao MD 68

Reading/Grading Agglutination Reactions :

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). 1/3/2013 Dr.T.V.Rao MD 69

Reading/Grading Agglutination Reactions :

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 1/3/2013 Dr.T.V.Rao MD 70

PowerPoint Presentation:

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 1/3/2013 Dr.T.V.Rao MD 71

PowerPoint Presentation:

2+ Medium-sized aggregates; clear supernatant 3+ Several large aggregates; clear supernatant 4+ One solid aggregate; clear supernatant 1/3/2013 Dr.T.V.Rao MD 72

Interpretations in Weil-Felix reaction :

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 infections Test may be negative in 50 percent scrub typhus 1/3/2013 Dr.T.V.Rao MD 73

Weil-Felix test indicated in when patients present with rashes:

Weil-Felix test indicated 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).. 1/3/2013 Dr.T.V.Rao MD 74

Latex Agglutination :

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) 1/3/2013 Dr.T.V.Rao MD 75

Coagulation and Liposome-enhanced testing :

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. 1/3/2013 Dr.T.V.Rao MD 76

Problems with Serology Other Health conditions interfere:

Problems with Serology Other 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 1/3/2013 Dr.T.V.Rao MD 77

Variables that affect the quality of results in Agglutination Tests:

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 1/3/2013 Dr.T.V.Rao MD 78

Errors in measurement:

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). 1/3/2013 Dr.T.V.Rao MD 79

Random Error:

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. 1/3/2013 Dr.T.V.Rao MD 80

Systematic Error:

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. 1/3/2013 Dr.T.V.Rao MD 81

Automations for Agglutination:

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. 1/3/2013 Dr.T.V.Rao MD 82

PowerPoint Presentation:

Programme Created By Dr.T.V.Rao MD for Medical Microbiologists in the Developing World Email 83 1/3/2013 Dr.T.V.Rao MD

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