Coombs test

Coombs test

1. COOMBS TEST MR. MANOJ MEHTA

2. HISTORY 1908, Moreschii , described the principle of the test (the use of rabbit anti-goat serum to agglutinate rabbit RBCs that were sensitized with low non-agglutinating doses of goat anti-rabbit RBC serum .) 1945 , R R coombs and associates described the use of the antigolubulin test for the detection of weak and non agglutinating Rh antibodies in the serum . 1946, coombs and coworker describe the use of AHG to detect in vivo sensitization of the RBCs of babies suffering from HDN Kell blood grouping was described by using antiglobulin test

3. INTRODUCTION It It is based on the principle of AHG obtained from immunized non human species bind to human globulin IgG or complement either free or attached to Ag of RBCs The use of AHG serum to detect sensitization of red cells in vitro can be: One stage technique , the direct anti globulin test (DAT). Two stage technique , the indirect antiglobulin test (IAT).

4. PRINCIPLE Normal human red blood cells, in presence of antibody directed towards the antigen they possess, may fail to agglutinate when centrifuged and become sensitized . This may be due to the particular nature of the antigen and antibody involved. Sensitization of RBC’s may be with IgG or complement. In order for agglutination to occur an additional of anti-antibody or anti-complements, which reacts with the Fc portion of the IgG antibody , or with the C3b or C3d component of complement alternatively.

5. This will form a " bridge" between the antibodies or complement coating the red cells , causing agglutination. The coating (sensitization) of red cells can occur in vivo or in vitro following incubation at 37°C with serum containing antibody. PRINCIPLE

6. ANTI- HUMAN GLOBULIN REAGENT a kind of antibody that react with the human globulin AHG combines with the Fc portion of a sensitizing antibody. This completes the antigen-antibody bridge, allowing agglutination to occur. Can be of two types Polyspecific Anti-human Globulin : blend of Anti- IgG and Anti-C3b, -C3d Monospecific reagents: Anti- IgG alone or Anti-C3b,-C3d alone

7. PREPARATION OF AHG Polyclonal AHG :- mixture of Ab from different plasma cell clone Can recognize different antigenic determinants(epitope Or same epitope by different affinities Monoclonal AHG:- Detect only one epitope on the antigen They will consist of only one antibody subtype where a secondary antibody is required for the detection , an antibody against the correct subclass should be chosen

8. Preparation of polyclonal AHG

9. MONOCLONAL AHG

10. COOMB’S CELLS To show that test cells were properly washed and that no neutralization or reagent deterioration has occurred, antibody-coated cells are used as a positive indicator . In a negative antiglobulin test the anti-human globulin should remain active and this can be demonstrated by the addition of IgG sensitized cells . Agglutination of the IgG sensitized cells after mixing and centrifuging confirms that the anti-human globulin was added to the test, that the test cells were properly washed and all free globulin molecules were removed and that the anti-human globulin was active. Failure of the IgG sensitized cells to agglutinate indicates that the original negative antiglobulin test result is not valid and testing must be repeated .

11. PREPARATION SENSITIZED CELL

12. DIRECt COOMBS TEST (DCT)

13. 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. These mechanism could be: Autoimmunity Alloimmunity Or a drug-induced immune-mediated mechanism .

14. PRINCIPLE OF DAT Sensitization of RBCs occur in vivo AHG reagent is added to the washed RBCs and incubated to observe agglutination Sensitized RBCs addition of AHG agglutination

15. METHOD OF DCT Tube method :- Gel technique method :- Based on column technology RBCs are filtered through a column containing a gel which contained in strips of micro well Gel medium contain anti IgG and anti C 3 b Sensitized RBCs binds anti- glubulin and gets trapped in gel column Unsensitized cell pellet at the bottom of column Flow cytometry method :- Immunofluorescences technique is utilized Can detect and measure low level of cell bound IgG

16. PROCEDURE OF TUBE METHOD

17. RESULT INTERPRETATION Alloimmune hemolysis :- Hemolytic transfusion reaction :- The patient has an antibody in the plasma that is directed against an antigen on the donor red cells. The patient’s antibody coats the donor cells. In an acute (immediate) hemolytic transfusion reaction , complement is activated. The donor cells are lysed (intravascular hemolysis). The DAT may be positive due to IgG or complement. In a delayed hemolytic transfusion reaction , the antibody coated cells are removed via phagocytosis .( extravascular lysis ) A drop in hemoglobin usually occurs 2-10 days following transfusion. The DAT is usually positive due to IgG .

18. HEMOLYTIC DISEASE OF THE NEWBORN (ALSO KNOWN AS HDN OR ERYTHROBLASTOSIS FETALIS) Occurs in :- Rhesus D hemolytic disease of the newborn (also known as Rh disease) ABO hemolytic disease of the newborn (the indirect Coombs test may only be weakly positive) Anti- Kell hemolytic disease of the newborn Rhesus c, E hemolytic disease of the newborn Other blood group incompatibility ( RhC , Rhe , Kidd, Duffy, MN, P and others ) Occurs by Mother must have been stimulated to make an IgG antibody. Antibody must cross the placenta. Fetus must be antigen positive. Fetal cells become coated with maternal antibody & are cleared by the fetal RES. In HDFN, the infant’s cells are coated with maternal IgG antibody, resulting in a positive DAT in the infant. The DAT is THE diagnostic test for HDFN.

19. AUTOIMMUNE HEMOLYSIS Warm antibody autoimmune hemolytic anemia:- Idiopathic Systemic lupus erythematosus Evans' syndrome (antiplatelet antibodies and hemolytic antibodies) Cold antibody autoimmune hemolytic anemia:- Idiopathic cold hemagglutinin syndrome Infectious mononucleosis Paroxysmal cold hemoglobinuria (rare)

20. DRUG INDUCE HEMOLYSIS Mechanism of hemolysis Drug Adsorption Drug attaches to RBC. Antibody directed at drug only DCT positive due to IgG Immune Complex Antibody directed at a “ neoantigen ” having determinants on both the drug and the red cell membrane. Antibody activates complement DCT positive due to complement Some drugs showing positive DCT Methyldopa ( IgG mediated type II hypersensitivity) Penicillin (high dose) Quinidine ( IgM mediated activation of classical complement pathway and Membrane attack complex)

21. INDIRECT COOMBS TEST (ICT) The ICT is performed to determine in-vitro sensitization of RBCs Serum containing incomplete Ab in incubated with RBCc to sensitized and than AHG is added to observe agglutination Application :- Detection of incomplete ( nonagglutinating ) antibodies to potential donor RBCs (compatibility testing) or to screening cells (antibody screen) in serum 2. Determination of RBC phenotype using known antisera (e.g., Kell typing, weak D testing) 3. Titration of incomplete antibodies

22. PROCEDURE OF TUBE ICT

23. FACTORS AFFECTING THE IAT Serum/Cell ratio:- increasing the ratio increases the sensitivity so minimum 40:1 is selected’ Reaction medium or use of potentiaters Incubation temperature:- 37⁰c is selected as majaority of reaction occurs at this temperature Length of incubation:- saline 30-60n, LISS 10-15 Washing of RBC:- at least three time washing is required to remove unbound AB before adding AHG . Should be done as soon as possible Saline use to wash :- should of pH 7.2-7.4 , stored long time in plastic container can decrease the pH causing elution of Ab from RBCs Addition of AHG:- Centrifugation for reading

24. POTENTIATORS Some incomplete antibodies will not react in a saline environment. Potentiators are reagents that adjust the test environment. Reduce the zeta potential Promote agglutination Enhance antibody uptake

25. 22% bovine solution :- High molecular weight protein Reduces the zeta potential by dispersing some of the cations surrounding each negatively charged red cell. Increases the dielectric constant , defined as a measure of ability to dissipate a charge . Polyethylene glycol :- a low ionic strength medium. Removes water from the test system, thereby concentrating any antibody present. Antibody uptake is also increased . PEG can cause cellular aggregation, therefore, tests using PEG can not be centrifuged and evaluated following a 37 o C incubation. Testing should proceed immediately to the wash phase, with a minimum of 4 washes performed. PEG is not the potentiator of choice when the patient has elevated proteins, such as in multiple myeloma. In these cases, LISS is the preferred enhancement. POTENTIATORS

26. Improper sample(refrigerated , clotted ) may causes invitro complement attachment Autoagglutinable cells Bacterial contamination of cells or saline used in washing Cells with positive direct AhG test used for the ICT Saline contaminated by heavy metals or colloidal silica Dirty glassware Over centrifugation and over reading Polyagglutinable cells Preservative –dependent antibody in LISS reagent Contaminating antibodies in the AHG reagent Centrifugation of test with polyethylene gylcol prior to washing FALSE POSITIVE RESULTS

27. FALSE NEGATIVE RESULTS Inadequate or improper washing of cells AHG reagent nonreactive because of deterioration or neutralization AHG reagent not added Serum not added in the indirect test Serum nonreactive because of deterioration of complement Inadequate incubation conditions in the IAT Cell suspension either too weak or too heavy Undercentrifuged or overcentrifuged Poor reading technique Low pH of saline

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