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Agglutination basic skills


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Agglutination Tests basic skills

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  • so basically the titre is the Maximum dilution of serum that gives visible agglutination? please answer, i have an exam tomorrow!
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Agglutination basic skills

  1. 1. Agglutination Tests Principles and Practice Dr.T.V.Rao MD
  2. 2. 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
  3. 3. 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
  4. 4. 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 individuals 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
  5. 5. Serology• The branch of laboratory medicine that studies blood serum for evidence of infection and other parameters by evaluating antigen-antibody reactions in vitro Dr.T.V.Rao MD1/3/2013 5
  6. 6. 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 cells1/3/2013 Dr.T.V.Rao MD 6
  7. 7. Agglutination• Agglutinins – Antibodies that produce such reactions• Involves two-step process: – Sensitization or initial binding – Lattice formation or formation of large1/3/2013 aggregates Dr.T.V.Rao MD 7
  8. 8. Agglutination• Types of particles that participate in such reactions: –Erythrocytes –Bacterial cells –Inert carriers such as latex particles1/3/2013 Dr.T.V.Rao MD 8
  9. 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 precipitation1/3/2013 Dr.T.V.Rao MD 9
  10. 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 10
  11. 11. Steps in Agglutination• Primary phenomenon (SENSITIZATION)  First reaction involving Ag-Ab combination  Single antigenic determinant on the surface particle 1) Initial reaction: rapid and reversible 2) Cross link formation  visible aggregates (stabilization)1/3/2013 Dr.T.V.Rao MD 11
  12. 12. SENSITIZATION1/3/2013 Dr.T.V.Rao MD 12
  13. 13. 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 • temperature1/3/2013 Dr.T.V.Rao MD 13
  14. 14. 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, precipitation1/3/2013 Dr.T.V.Rao MD 14
  15. 15. 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
  16. 16. 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 ofMD1/3/2013 Dr.T.V.Rao blood groups 16
  17. 17. 1/3/2013 Dr.T.V.Rao MD 17
  18. 18. 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
  19. 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 agglutination1/3/2013 Dr.T.V.Rao MD 19
  20. 20. 1/3/2013 Dr.T.V.Rao MD 20
  21. 21. Slide Agglutination Test1/3/2013 Dr.T.V.Rao MD 21
  22. 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 Testing1/3/2013 Dr.T.V.Rao MD 22
  23. 23. Tube Agglutination Test1/3/2013 Dr.T.V.Rao MD 23
  24. 24. Tube Agglutination Test Agglutination No agglutination1/10 1/20 1/40 1/80 1/160 1/320 Neg. ctrl In this case, the titre is 1/40
  25. 25. 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 – charcoal1/3/2013 Dr.T.V.Rao MD 25
  26. 26. 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 spiralis1/3/2013 Dr.T.V.Rao MD 26
  27. 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 LATEXe.g. Lancefield grouping in Streptococci.1/3/2013 Dr.T.V.Rao MD 27
  28. 28. The latex particles are coated with IgG and mixed with the patients serum1/3/2013 Dr.T.V.Rao MD 28
  29. 29. Quantitative MicroHemagglutination Test (HA) Haemagglutination Test (HA)1/3/2013 Dr.T.V.Rao MD 29
  30. 30. Haemagglutination1/3/2013 Dr.T.V.Rao MD 30
  31. 31. Haemagglutination RBC1/3/2013 Dr.T.V.Rao MD 31
  32. 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 32
  33. 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 this1/3/2013 Dr.T.V.Rao MD 33 manner agglutination becomes visible
  34. 34. Equivalence point:(suitable proportion between the virus particles and RBCs)1/3/2013 Dr.T.V.Rao MD 34
  35. 35. Negative control well (only RBCs+ buffer) (no Haemagglutinnins)Positive control well (contains haemagglutinin)
  36. 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 4096The HA titer of this virus in this row is 256 or 28(1:256 dilution contains (1 HA unit) (one haemagglutinatingDr.T.V.Rao MD 1/3/2013 unit) 36
  37. 37. Example of readings1/3/2013 Dr.T.V.Rao MD 37
  38. 38. Hemagglutination test: method 1:8 1:2 1:2 1:2 1:2 1:2 virusserial dilution 8 16 32 64 128 256 mix with red blood cells side view top view Titer = 32 HA units/mlOne HA unit :minimum amount of virus that causes complete agglutination of RBCs
  39. 39. WHAT DO WE NEED?1/3/2013 Dr.T.V.Rao MD 39
  40. 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 40
  41. 41. WASHING RBCs1/3/2013 Dr.T.V.Rao MD 41
  42. 42. 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 Abs1/3/2013 Dr.T.V.Rao MD 42
  43. 43. 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)1/3/2013 Dr.T.V.Rao MD 43
  44. 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 44
  45. 45. In the absence of anti-virus antibodies Erythrocytes Virus Virus agglutination of erythrocytes1/3/2013 Dr.T.V.Rao MD 45
  46. 46. In the presence of anti-virus antibodies Erythrocytes Virus Anti-virus antibodies Viruses unable to bind to the erythrocytes1/3/2013 Dr.T.V.Rao MD 46
  47. 47. 1/3/2013 Dr.T.V.Rao MD 47
  48. 48. What is Antibody Titer• Is the lowest concentration of antibodies against a particular antigen. 1/3/2013 Dr.T.V.Rao MD 48 Figure 18.6
  49. 49. 1/3/2013 Dr.T.V.Rao MD 49
  50. 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 (29).(the lowest concentration of Abs which inhibit HA caused by the virus ) 1/3/2013 Dr.T.V.Rao MD 50
  51. 51. 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).1/3/2013 Dr.T.V.Rao MD 51
  52. 52. Coombs (Antiglobulin)Tests • Incomplete Ab • Direct Coombs Test – Detects antibodies on erythrocytes + ↔Patient’s RBCs Coombs Reagent (Antiglobulin)1/3/2013 Dr.T.V.Rao MD 52
  53. 53. Coombs Test Direct ant globulin test (also called the Coombs’ test,1/3/2013 Dr.T.V.Rao MD 53
  54. 54. Coombs (Antiglobulin)Tests • Indirect Coombs Test – Detects anti-erythrocyte antibodies in serumStep 1 + ↔ Patient’s Target Serum RBCsStep 2 + ↔ Coombs Reagent1/3/2013 (Ant globulin) Dr.T.V.Rao MD 54
  55. 55. Application of Coombs (Antiglobulin)Tests• Applications –Detection of anti-Rh Ab –Autoimmune hemolytic anemia1/3/2013 Dr.T.V.Rao MD 55
  56. 56. 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 albicans1/3/2013 Dr.T.V.Rao MD 56
  57. 57. REVERSE PASSIVE Agglutination Tests• PRINCIPLE: latex particles coated with antibody are reacted with a patient sample containing suspected antigen1/3/2013 Dr.T.V.Rao MD 57
  58. 58. 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 proteins1/3/2013 Dr.T.V.Rao MD 58
  59. 59. Agglutination Inhibition Tests• Pregnancy Testing -classic example of agglutination inhibition – Human chorionic gonadotropin (hCG) • Appears in serum and urine early in pregnancy1/3/2013 Dr.T.V.Rao MD 59
  60. 60. Agglutination Inhibition Urine Antiserum No hCG in urine: hCG in urine: Anti-hCG free Anti-hCG neutralized Carriers coated with hCG added Carriers coated with hCG added AGGLUTINATION of carriers: NO AGGLUTINATION of carriers: Negative test for hCG Positive test for hCG1/3/2013 NOT PREGNANT Dr.T.V.Rao MD PREGNANT 60
  61. 61. 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 reactions1/3/2013 Dr.T.V.Rao MD 61
  62. 62. 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 antibody1/3/2013 Dr.T.V.Rao MD 62
  63. 63. Highly specific but not very sensitive in detecting small quantities of antigen1/3/2013 Dr.T.V.Rao MD 63
  64. 64. 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
  65. 65. 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 test1/3/2013 Dr.T.V.Rao MD 65
  66. 66. 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
  67. 67. 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
  68. 68. 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
  69. 69. 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
  70. 70. 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 saline1/3/2013 Dr.T.V.Rao MD 70
  71. 71. GRADING AGGLUTINATION REACTIONSGRADE DESCRIPTION AppearanceNegative (-) No aggregatesWeak (+/-) 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
  72. 72. 2+ Medium-sized aggregates; clear supernatant3+ Several large aggregates; clear supernatant4+ One solid aggregate; clear supernatant1/3/2013 Dr.T.V.Rao MD 72
  73. 73. 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 typhus1/3/2013 Dr.T.V.Rao MD 73
  74. 74. 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
  75. 75. 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
  76. 76. 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
  77. 77. 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 antibody1/3/2013 Dr.T.V.Rao MD 77
  78. 78. 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 results1/3/2013 Dr.T.V.Rao MD 78
  79. 79. 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
  80. 80. 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
  81. 81. 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
  82. 82. 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
  83. 83. • Programme Created By Dr.T.V.Rao MD for Medical Microbiologists in the Developing World • Email • doctortvrao@gmail.com1/3/2013 Dr.T.V.Rao MD 83