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å He was traveling on an ill-lit wartime train from London to Cambridge, trying to read some papers by ! on the side-chain theory and speculating idly( +,on the tetravalent carbon atom) on the behaviour of red cells and antibodies, when he visualized the cells, already coated with molecules of incomplete antibody, which was of course a globulin, but still floating free, becoming linked together by molecules of another antibody, an antiglobulin antibody!

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 Œor the detection of nonnon-agglutinating Abs, especially IgG1 and IgG3 or complement components (C3d) affixed to RBCs £ £or £or £ £ with the use of AHG reagent and Anti C3d or Anti Anti--C3b.

|)) -!-| - detection of sensitization of RBC s (coated with Abs and complement components) in vivo

! | - detection of sensitization of RBCs in vitro

¦ Vashed red cells which are coated with immune incomplete antianti-bodies (IgG) and complement components(C3d & C3b) will show agglutination with broad spectrum AHG reagents.The sensitization of red cells can occur in vivo or in vitro following incubation at 37°C with serum containing antibody.

! ! (also known as ## are ## gamma globulin proteins that are found in blood or other bodily fluids of humans and are used by the immune system to identify and neutralize antigens such as bacteria and viruses. They are typically made of basic structural units² units²each with two large heavy chains and two small light chains. Antibodies are produced by activated BB-cell called as plasma cell.

!# ! $! -). Interactions between antigen and antibody involve non-covalent binding of an antigenic determinant (epitope) to the variable region (complementarity determining region, CDR) of both the heavy and light immunoglobulin chains. These interactions are analogous to those observed in enzymesubstrate interactions and they can be defined similarly. Õ Lock and Key Concept Non-covalent Bonds ± Hydrogen bonds ± Electrostatic bonds ± Van der Vaal forces ± Hydrophobic bonds Õ Reversible Õ Multiple Bonds

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ize ± Large. Occur in pentameric form. RBCs sensitized with IgM are not directly removed from circulation b¶coz macrophages don¶t have receptors for Œc portion of IgM. IgM antibodies cause intravascular hemolysis through complement activation. IgM antibodies may also cause extravascular hemolysis. Agglutination in saline at 37°°C. Complete complement Activation

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Õ ize - Small Õ Occur in monomeric form. Õ  ££ £ £

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£ Antibody-mediated adhesion of Œc portion of IgG with Œc receptors on Macrophage and then, Phagocytosis of sensitized red cells occur or Macrophage ³pits´ the anitgen- antibody complex ± after continued pitting cell becomes rigid and is phagocytized No Agglutination in saline at 37°°C,So use of HG is required,which causes their agglutination. Incomplete complement Activation.

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Antihuman globulins (AHG) from immunized animals bind to human globulins either free in serum or attached to RBCs ± ¦" ¦" Animals hyperimmunized with human globulins; bleed for antisera to obtain high titered, high avidity, specificity antibodies to human ± Hybridoma cells from mice; collection of culture or ascites fluid yields antisera. â mice hyperimmunized with human globulins â prepare cell suspension from spleens; fuse with immortalized myeloma cells â screen hybridoma clones for desirable specificities and affinities â maintain cultures of clones £ £ ££ £

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Polyspecific AHG ± Abs to human IgG, and ± Abs to human C3d (C3b breaks down to C3c and C3d) ± Advantage is that polyspecific AHG may detect complementcomplement-dependent Abs on RBCs ± Disadvantage - more nuisance positives

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Monospecific AHG ± Abs to human IgG only or human C3d only ± Œewer nuisance positives ± may miss an important Ab

" %""%" Anti-IgG antibodies react with the IgG isotype of human Antiimmunoglobulins. The Anti IgG recognizes the Œab region of IgG and reacts with all subclasses of IgG. IgG is the most abundant and the longest lived immunoglobulin in humans. The monomeric 150 kDa molecule is produced by B cells and is expressed in secreted and membranemembrane-associated forms. Œunctions of IgGs are:are:1)Promotion of the phagocytosis of antibodyantibody-coated particles 2)Œeedback inhibition of B cell activation 3)Complement activation, 4)Antibody--dependent cell4)Antibody cell-mediated cytotoxicity

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incomplete antibodies or AntiAnti-complement antibodies that are bound to the surface of red blood cells; a blood sample is taken and the RBCs are washed and then incubated with antihuman globulin and look for agglutination.

#*/0!#. 1)EDTA sample(prevent the uptake of complement in vitro)Testing should be performed within 48 hours of collection. 2)Clotted should be as fresh as possible(not more than 24 old). 3)Cord blood sample

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g)Add 11-2 drops of AHG serum. h)Centrifuge at 1000 rpm for 1 min. i)Gently shake the tube to dislodge the button and examine for agglutination. j)Leave an ±ve test at RT for 5 min,centrifuge and read again. K)Confirm any negative Patient¶s test AntiAnti-IgG coated Coombs Control cells.

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| is 2 when no agglutination is seen at either phase except using coated red cells. Controls are same as for DCT

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7)Low speed of centrifugation 8)Œailure to check ±ve rxns microscopically 9)Improper storage of test cells,resulting loss of sensitivity. Œ1 / 1)Enzyme treated cells may react with residual non specific antibody in AHG serum. 2)Over centrifugation packs red cells so tightly that can not be dispersed completely. 3)Use of agglutinated RBCs before washing & so on their persistence throughout the washing.

Non Technical Errors Œ1 / 1)Inactivation of AHG reagent may be due to improper storage. 2)In bacterial contamination of test cells or septicemia in the patient, AntiAnti-T in the AHG serum react with TTactivated red cells. Œ / Œ 1)Improper storage of test cells,resulting in the loss of sensitivity 2)Use of plasma sample 3)Inaccurate temperature of water bath or incubator 4) Inaccurate control of speed and temp. of centrifuge

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