Moderator:Moderator:Respected S.K. Sharma (Lecturer) Respected A.P. Chauhan (Tutor) Department of Haematology
History
The antiglobulin test was discovered by Coombs et al.(1945) for detecting incomplete antibodies in serum. He was traveling on an ill-lit wartime train from London to Cambridge, trying to read some papers by Ehrlich on the side-chain theory and speculating idly( Kekule 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!
Coombs
et al in 1947 showed that 'incomplete' antibody mainly responsible for positive anti-globulin test results is IgG antiwhile the 'complete' antibody is IgM. on,Dacie et al.(1957) showed that complement components attached to RBCs could also be detected by this test 1964,same workers performed DAT to detect in vivo RBCs sensitization in HDN.
Later
In
Introduction
For the detection of non-agglutinating Abs, especially nonIgG1 and IgG3 or complement components (C3d) affixed to RBCs in vivo or in vitro with the use of AHG reagent and Anti C3d or Anti-C3b. Anti-
Principle of Anti-globulin Tests AntiWashed red cells which are coated with immune incomplete anti-bodies (IgG) and anticomplement 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 37C with serum containing antibody.
Antibodies
Antibodies (also known as immunoimmuno-globulins) 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 unitseach with units two large heavy chains and two small light chains. Antibodies are produced by activated B-cell called as Bplasma cell.
destruction by: Antibody-mediated adhesion of Fc portion of IgG with Fc receptors on Macrophage and then, Phagocytosis of sensitized red cells occur or Macrophage pits the anitgen- antibody complex after continued pitting cell becomes rigid and is phagocytized No Agglutination in saline at 37C,So use of HG is required,which causes their agglutination. Incomplete complement Activation.
Antihuman globulins (AHG) from immunized animals bind to human globulins either free in serum or attached to RBCs Polyclonal - Animals hyperimmunized with human globulins; bleed for antisera to obtain high titered, high avidity, specificity antibodies to human Monoclonal - Hybridoma cells from mice; collection of culture or ascites fluid yields antisera.
mice hyperimmunized with human globulins prepare cell suspension from spleens; fuse with immortalized myeloma cells screen hybridoma clones for desirable specificities and affinities maintain cultures of clones in vivo or in vitro.
Polyclonal Abs
Monoclonal Abs
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 complement-dependent Abs complementon RBCs Disadvantage - more nuisance positives
Monospecific AHG
Abs to human IgG only or human C3d only Fewer nuisance positives may miss an important Ab
Anticomplement Antibodies
The anticomplement antibodies are directed against the complement components such as C3b and C3d. The roles of anticomplement antibodies in AHG reagent are: 1)To detect complement binding antibodies of kidd,kell and Duffy systems(not picked by Anti-IgG). Anti2)To enhance of complement binding antibodies. 3)To detect IgM antibodies which invariably bind to the complement. 4)To detect IgM which elute off the red cells with increase in temperature. Most commonly used are anti-C3b and anti- C3d. antianti-
IgG or IgM
Sample Requirements:
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
Requirements
Appartus
12 x 75 mm test tubes Pasteur pipettes Cenrifuge Marking pen Normal saline Microscope
Reagents
Polyspecific AHG AntiAnti-D AntiAnti-C3d Coombs Control Cells
Procedure:Procedure:
Add ml of patient blood from the EDTA to a tube labelled with the patients initials. Wash this tube 3 times with N/S.
Prepare a 2-5% suspension from the washed 2red cells. Label four tubes with the patients test, C3, +ve & -ve control tubes. Add one drop of the 2-5% washed cells to each 2of the tubes labelled in step 4 above except +ve control tube . Add 2-5% suspension of O+ve sensitized cells in 2+ve control tube. Wash cell suspension in four tubes one more time, decant well and blot the last drop of saline. Add one drop of Anti-IgG to Patients test tube & Anti+ve control tube, 1 drop Anti-C3d to the C3 tube, Antiand 1 drop saline to the ve control tube. Centrifuge all tubes at 1000 rpm for 1 min.
Incubate C3 tube for 5-10 mins. 5Resuspend the cell button gently and examine for agglutination in all tubes microscopically. Read, grade and record results in the appropriate columns of the worksheet. Confirm any negative Patients test and C3 reactions with the Coombs Control cells.
INTERPRETATION:INTERPRETATION:DCT is +ve when agglutation is seen in Patients test and Anti C3 d tube. DCT is ve when no agglutination is seen in Patients test and Anti C3d tube except using coated red cells. A ve DCT does not necessarily mean absence of coating antiglobulin antibody. +ve Control - agglutination in +ve Control tube -ve Control - No agglutination in ve
Procedure:Procedure:a)Place 2-4 drops of the test serum in a tube. 2b)Add 1 drop of 2-5% suspension of O+ve 2cells. c)Mix and incubate at 37 C for 60-90 mins. 60-
g)Add 1-2 drops of AHG serum. 1h)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 Patients test Anti-IgG coated AntiCoombs Control cells.
Interpretation: Interpretation:ICT is +ve when agglutation is observed either after immediate spin or after spin following RT incubation. ICT is ve when no agglutination is seen at either phase except using coated red cells. Controls are same as for DCT
Methods ::a) b)
One Stage Method (Albumin- Additive) (AlbuminProcedure:Procedure:Add 2-3 drops of serum to a labelled tube. 2Add 1 drop of 2-5% O+ve red cells suspensions 2in the tube. Add 2 drops of 22% of Bovine Albumin. Mix and incubate 37*C for 15-20 min. 15Centrifuge at 1000rpm for 1-2 min. 1Add 1-2 drops of AHG serum. 1Gently resuspend the cell button and observe for agglutination. Confirm all negative results under microscope.
Two stage Method(Albumin Layering) Procedure:Procedure:1)Add 2-3 drops of serum to a labelled tube. 22)Add 1 drop of 2-4% red cell suspension in the tube. 23)Mix and incubate at 37C for 30 mins. 37 4)Centrifuge at 1000 rpm for 1-2 mins. 15)Add two drops of 22% albumin in the tube.Aibumin will form a layer on the top of the cells.Do not mix. 6)Incubate at 37C for 10-20 mins. 37 107)Add 1-2 drops of AHG serum. 18)Gently resuspend the cell button and observe for agglutination/haemolysis. 9)Confirm all negative results under microscope.
Enzymes Techniques
Principle:Principle:The action of enzymes on red cell potentiates agglutination in two ways: 1) Enzyme removes sialic acid from the red cell surface and thus,reduces the surface charge(Zeta potential) and allowing the cells to come closer to one another. 2)Enzyme may potentiate the agglutination by removing structures which sterically interfere with the access of antibody molecule.
Procedure:Procedure:PrePre-treatment of RBCs
1)Take 0.1 ml of 3 times RBCs washed in N/S in a test tube. 2)Add 0.2 ml of Papin working solution. 3)Incubate at 37C for 15-30 mins. 37 154)Wash the red cells three times with N/S. 5)Make 2-4% cells suspension in N/S. 2-
PostPost-treatment of RBCs
a)Take 2 vol of serum in a test tube. b)Add 1 vol of papainised red cells. c)Add 1-2 drops of AHG serum. 1d)Incubate at 37C for 30 mins. 37 e)See for agglutination.
One Stage Enzyme Technique Reagents: Reagents:Preparation of Papain Cysteine Reagent Papain - 10gm Cystein HCl - 4.85 gm Phosphate buffer PH 6.2 - 1000 ml Store in small aliquots at -20C. 20
Method:Method:1)Take 1 VOL of serum in a test tube. 2)Add 1 VOL of 2% cell suspension. 3)Incubate at 37C for 1hr. 37 4)Add 1-2 drops of AHG serum. 15)See for agglutination.
LISS(Low Ionic Strength Salt Solution) Solution) Principle:Principle:In N/S Na+ and Cl- ions form ionic clouds around oppositely Clcharged sites on Ag and Ab molecules respectively and partially neutralizes them.When the ionic strength of the reaction mixture is reduced,the no. of ions available to form ionic clouds around cells or protein molecules is decreaesd and they unit more effectively and they unit more effectively.It results increased 2-4 folds in comparison with 2N/S.
Method:Method:1)Wash the RBCs twice in N/S. 2) Wash these cells once in LISS. 3)Make 2-3% cell suspension in LISS. 24)Take equal volume of serum and LISS suspened cells in a tube. 5)Incubate at 37C for 10-15 mins. 37 106)Add 1-2 drops of AHG serum. 17)See for agglutination.
7)Low speed of centrifugation 8)Failure to check ve rxns microscopically 9)Improper storage of test cells,resulting loss of sensitivity. False +ve Results 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 False +ve Results 1)Inactivation of AHG reagent may be due to improper storage. 2)In bacterial contamination of test cells or septicemia in the patient, Anti-T in the AHG serum react with TAntiTactivated red cells. False -ve Results 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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