COOMBS TEST direct and indirect coombs antisera.pptx

Coombs Sera direct snd indirect

1. Subtitle COOMBS TEST DR TANISHA

2. What is Coombs test? The antiglobulin test, which is also referred to as the anti-human globulin test(AHG) or the Coombs test , is the cornerstone of detecting clinically significant unexpected antibodies that have coated cells either in vivo or in vitro. OR The anti-human globulin test is a test using anti-human globulin(AHG) reagents to detect the presence of human globulin on sensitized RBCs.

3. PRINCIPLE OF COOMBS TEST The antiglobulin test can be used to detect red cells sensitized with IgG alloantibodies, IgG autoantibodies or complement components. The incomplete antibodies (IgG) attach to red cell membrane by the Fab portion of the immunoglobulin molecule (IgG). The IgG molecules attached to the red cells are unable to bridge the gap between sensitized red cells which are separated from each other by the negative charge on their surface and the sensitized red cells do not agglutinate.

4. In order for agglutination to occur an additional antibody , which reacts with the Fc portion of the IgG antibody, or with the C3b or C3d component of complement, must be added to the system. This will form a "bridge" between the antibodies or complement coating the red cells, causing agglutination.

5. There are two types of antiglobulin tests Direct Antiglobulin Test (DAT) - Detects antibodies or complement coating patient's cells in vivo. Indirect Antiglobulin Test (IAT) - Uses a 37 degree incubation step so antibodies in serum can react with antigens on cells in vitro, After washing the cells antiglobulin reagent is used to detect antibody coating of cells.

6. Production Methods of Anti-Human globulin (AHG or Coombs) Reagent May be made by injecting rabbits with purified human IgG or C3, then harvesting the antibodies produced by the rabbit. Monoclonal technology may be used to make monoclonal antiglobulin reagent Specificity types: Poly-specific Anti-human Globulin: blend of Anti-IgG & Anti-C3b, -C3d Monospecific reagents: Anti-IgG alone or Anti-C3b, C3d alone 

7. Interpretation of antiglobulin test Whether the cells have been coated, or sensitized, in vivo or in vitro the final interpretation is based on the following: Positive antiglobulin test Negative antiglobulin test

8. 2. Washing Only antibody attached to the cells remain. Wash cells three times with saline to remove unbound antibody 3. Addition of antihuman globulin Anti-human globulin reagent(AHG ) is added to the washed cells and cells are coated with IgG antibodies (or C3b component of complement), so they will be agglutinated by the anti-globulin reagent.

9. 2. Washing   Wash cells three times with saline to remove unbound antibody . 3. Addition of antihuman globulin Anti-human globulin reagent(AHG ) is added to the washed cells but the cells are not coated with IgG antibodies (or C3b component of complement),so they will not agglutinated by the anti-globulin reagent.

10. Role of Coombs control check cells (CCC) CCC are cells coated with IgG antibody. Will react with antibodies in Coombs serum still "floating around" in the tube. Agglutination will now result . Agglutination following addition of CCC verifies negative result.

11. Preparation of Coombs control check cells (CCC) Wash the group O positive cells three times in saline. Add an equal volume of anti-D IgG antibodies to the packed cells. Incubate at 37˚C for 30 minutes. Wash the cells four times. Suspend in saline to a 3-5% suspension. Take 1 volume of the 3-5 % suspension ,add 2 volumes of the Coomb’s reagent mix gently and centrifuge the tube. The agglutination reaction should be(+++). If this reaction is too strong or too weak, these cells will not properly control the anti-human globulin test. These sensitized cells can be stored for 48 hours.

12. False Negative Reaction False-negative reactions can occur when antigen-antibody reactions have occurred but WASHING IS INADEQUATE and free antibody remains when the anti-human globulin is added. Anti-human globulin (Coombs) antibody prefers to react first with free antibody and then with antibody-coated cells. If the free antibody has already reacted with the anti-human globulin, no free Coombs serum to react with Coombs Control Check Cells (CCC).

13. False negatives that are detected by negative Coombs control cells includes inadequate cell washing delay in adding antiglobulin reagent after the washing step presence of small fibrin clots among the cells inactive, or forgotten, antiglobulin reagent

14. Inadequate cell washing - unbound antibody remaining in the red cell suspension that are available to neutralize the AHG (Coombs serum) so it will not react with red cells bound with antibody. Delay in adding Coombs serum after washing step will lead to antibody eluting off, detaching from, cell while cells are sitting in saline. Now free antibody present in the saline neutralizes the AHG, Coombs, serum so it will not be able to react with the cells bound with antibody .

15. Small fibrin clot among the cells that were not washed away will have immunoglobulins and complement present. The antibodies and complement in the fibrin clot neutralizes AHG, Coombs serum leading to a negative test. Inactive AHG (Coombs serum) or the failure to add AHG (Coombs serum) will also be detected by a negative reaction when adding Coombs Control Check Cells.

16. False Positive Reaction False positive reactions can also occurred when performing this test. These would not be detected by the use of Coombs Control Check Cells. Reasons for a false positive reaction could be the following: Using improper sample (clotted cells instead of EDTA for Direct Antiglobulin Test, DAT). Spontaneous agglutination (cells heavily coated with IgM). Non-specific agglutination ("sticky cells") All of these reactions would be the result of cells appearing to agglutinate, or actually agglutinating. Using a clotted tube for the DAT may allow complement to become activated in the test tube since calcium ions are free to be part of the complement cascade .

17. Direct antiglobulin test (DAT) The direct antiglobulin test (DAT) detects sensitized red cells with IgG and/or complement components C3b and C3d in vivo. In vivo coating of red cells with IgG and/or complement may occur in any immune mechanism is attacking the patient's own RBC’s. This mechanism could be autoimmunity, alloimmunity or a drug-induced immunemediated mechanism .

18. This test is performed to detect anti-D antibody or other antibodies attached to the red cell surface within the blood stream. This occurs in the following circumstances: • When there is a Rh-positive baby in the womb of a sensitized Rh-negative women; the antibodies produced in the mothers serum cross the placenta and after entering the baby's blood stream, these antibodies will attach to the baby's Rh-positive red blood cells. These coated (or sensitized) cells are clumped and removed from the circulation, causing hemolytic anemia (Hemolytic Disease of the Newborn: Erythroblastosis Fetalis). When the baby is born, the baby's blood is collected (or cord blood is collected from umbilical cord) and tested by the anti globulin Coombs test (direct) to detect anti D antibodies coated on red blood cells.

19. Indications Hemolytic disease of the newborn Transfusion reactions Drug induced red cells sensitization Autoimmune hemolytic anemia

20. Requirements Test tubes: (10x75 mm) Pasteur pipettes Incubator Centrifuge Specimen: EDTA sample

21. Procedure Prepare a 5 % suspension in isotonic saline of the red blood cells to be tested. With clean Pasture pipette add one drop of the prepared cell suspension to a small tube. Wash three times with normal saline to remove all the traces of serum. Decant completely after the last washing Add two drops of AHG serum. Mix well and centrifuge for one minute at 1500 RPM. Resuspend the cells by gentle agitation and examine macroscopically and microscopically for agglutination.

22. Indirect Coombs test (ICT) The purpose of the indirect antiglobulin test is to detect In vitro sensitization of red cells. This is done when sensitization does not lead to direct agglutination. This occurs when there are too few antigens on the red cell, too few antibodies in the serum and those antibodies are in the IgG class.

23. Indications Weak D's in donor bloods and pregnant females of individuals who type D (- ) at room temperature when doing ABO and Rh typing. The presence or absence of antigens on a person cells from particularly the Kell, Kidd, and Duffy Blood Group systems. Unexpected, clinically significant antibodies in the patient's serum during the antibody screening procedure and the antibody identification procedure.

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