Presented by DR. NUSRAT SIDDIQUA Phase B student. Department of Microbiology & Immunology, BSMMU

Transcription

1 Presented by DR. NUSRAT SIDDIQUA Phase B student Department of Microbiology & Immunology, BSMMU

2 Introduction Agglutination is a laboratory diagnostic test based on the reaction between a particular antigen and the matching specific antibody, wherein the antigen is insoluble, or represent an integral part of a large insoluble particle (e.g. red blood cells, bacteria or inert particles). Definition: the interaction between antibody and particulate soluble antigen results in visible clumping called agglutination. Antibodies that produce such reaction are called agglutinin. It is most commonly used Ag-Ab reaction.

3 History and discovery Two bacteriologists, Herbert Edward Durham and Max von Gruber, discovered specific agglutination in The clumping became known as Gruber-Durham reaction. Gruber introduced the term agglutinin. French physician Fernand Widal,in 1896, used the reaction as the basis for a test for typhoid fever. Austrian physician Karl Landsteiner, in 1900, discovered ABO blood grouping.

4 Types of agglutination Active (direct) agglutination: Antibody directly binds with a particular antigen to form a visible clump. Ex- blood grouping, Widal test Now these are done by latex agglutination test Weil felix test

5 Passive (Indirect) agglutination: the soluble antigens are coated on particulate substances (latex, carbon, RBC etc) to make the Ag-Ab reaction visible. Latex agglutination test Hemagglutination test Antiglobulin test Coagglutination test Antigens (Bacteria) RBCs Antigens

6 Haemagglutination Antigen is coated on surface of RBC that is agglutinated by specific antibody this is called haemagglutination Coated Ag +

7 Types of haemagglutination Passive haemagglutination Haemagglutination inhibition Reverse passive haemagglutination

8 Passive Haemagglutination It is so called, because it is not antigens of the RBC themselves, but rather the passively attached antigens that are being bounded by Ab. Here antigen is coated on the surface of the RBC that is agglutinated by specific antibody. Examples : TPHA Rose- Waller test IHA test for E-granulosus.

9 TPHA Principle : Pt s diluted serum samples are mixed in the wells of a microtitration plate with sheep red cells coated (sensitized) with T. pallidum antigen. If antibody is present the sensitized red cells are agglutinated and they settle in a charecteristic pattern of cells in the bottom of a microtitration plate.

10 Turkey chicken s RBC are usually used as carrier because they are nucleated and therefore sediment rapidly. RBC are formalin fixed, treated with tanic acid to make the antigen adhere. Antigen coated RBC are called sensitized RBC. These sensitized RBCs are added to dilutions of patient s serum in microtitre well If the serum contain specific Ab, RBC will be agglutinated and settle to form an even covering of the bottom of the well. If serum don t contain specific Ab, sensitized RBC wil not be agglutinated and form a red button in the bottom of the well.

11 interpretation : Agglutinated cells form an even layer over the bottom of the well. Non-agglutinated cells form compact button in the center of the well. Agglutination of the test cells but not the control tests indicates presence of specific antibody to T. pallidum. Agglutinaiton of both test & control cells indicates anti-cell antibody. The test is not valid & should be repeated.

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13 TPHA is often negative in early (Primary) Syphilis but may become positive at con. titer (80-320) towards the end of the primary stage. Positive in the 4 th week of infection. Titer rise sharply during the secondary stage and commonly reaches 5120 or greater. A negative TPHA in blood-excludes active neurosyphilis.

14 Advantages : It is simple to perform. can be performed in field level. Used as a confirmatory test, with routine screening test VDRL It is more sensitive and more specific. Limitation : The test can not discriminate between antibody due to T. pallidum infection and antibody due to T. Careteum infection. It is negative in early active and late latent syphilis. False positive result from heterophil antibody in patient with infectious mononeucleosis, HBV, Leprosy, various auto- immune diseases. Has no prognostic value.

15 Rose waller test It is a haemagglutination slide test for rapid detection of IgM Rheumatoid factor in the serum of the patient. Sheep RBC are coated with rabbit anti-sheep IgG. Treated with patient s serum Slowly rotate for 2 minutes.

16 Negative test Positive test

17 Haemagglutination Inhibition Test Certain viruses are able to agglutinate RBC because the posses haemagglutinin (HA) on their surface. Haemagglutinin Ag is added to each well in a microtitre plate Pt s serum is placed in the 1 st well and serially diluted. Incubate 1 hr. RBC added to each well. If pt s serum contain corresponding Ab no agglutination positive test. If pt s serum contain no Ab agglutination occur negative test.

18 Hemagglutination-Inhibition (HI) Test + + RBC Suspension Hemaggutinin Antibody Settling Pattern

19 Hemagglutination phenomenon is almost commonly used for diagnosis of infection produced by Orthomyxovirus, paramyxovirus and the arbovirustogavirus ( rubella), flavivirus and bunyavirus. The presence of virus infected cell cultures can be detected by hemagglutination, the identity of the virus can be determined by specific inhibition of that haemagglutination. Although influenza virus can be detected by hemadsorption test, typing of the isolate is done most effectively by hemagglutination inhibition. Reagents and conditions for the test vary by virus.

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21 Reverse passive haemagglutination These test are performed to detect those viruses which do not agglutinate RBC. It perfoemed by reacting viral Ag with RBC coated with specific viral Ab If corresponding Ag are present in serum, RBC will be agglutinated. It is used to detect HBsAg in serum.

22 Haemagglutination and Latex agglutination Haemagglutination Need more time for settle down of RBC 3-4 hrs. Agglutination spread pattern may not be uniform, sometimes unstable. shelf life of RBC is less. Few Ag can be coated with RBC. Latex agglutination Less time 3-5 min. Consistent, uniform, stable. Ag coated latex bead can be used immediately or stored. A variety of Ag can be coated with latex.

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