2The antiglobulin testThe antiglobulin test was first introduced into clinical medicine in 1945 by R.R. CoombsHe showed that it could be used to detect non-agglutinating red cell antibodies or sensitized red cellsMost non-agglutinating (incomplete) antibodies are IgG,These antibodies do not spontaneously cause agglutination due to a strong electronegative charge on the red cell surface that prevents the cells from coming into close proximity.The antiglobulin reagent is able to bridge these negative forces.
3Antihuman Globulin Definition: Antihuman: antibodies against human antigensGlobulin: all antibody molecules are globulinsTherefore: Antihuman Globulin is antibody directed against the Fc portion of human antibodies and/or complement components.
4AHG Technique: What is the importance? Some very clinically significant unexpected antibodies (eg. Kidd, Duffy) attach to red cell but do NOT cause agglutination at immediate spin or 37o phase.Yet, these antibodies are capable of causing severe hemolytic transfusion reactions or hemolytic disease of the newborn.
5ANTIGLOBULIN TESTDetection of non-agglutinating Abs, especially IgG or complement components (C3) attached to RBCs in vivo or in vitro.Direct antiglobulin test (DAT) - detection of sensitization of RBC s (coated with Abs and/or complement components) in vivoIndirect antiglobulin test (IDAT) - detection of sensitization of RBCs (coated with Abs and/or complement components) in vitro
6Antiglobulin TestPrinciple - Antihuman globulins (AHG) from immunized animals bind to human globulins either free in serum or attached to RBCsPentameric IgM Abs are so large that, when bound to RBC Ags, the RBCs agglutinate (usually at RT)IgG Abs usually need a little help, a bridge molecule, to agglutinate RBCsAHG acts as a bridge molecule
7Reagent Preparation Polyclonal or monoclonal sources Polyclonal - Animals immunized with human globulins (IgG & complement (C3); bled for antisera to obtain high titeredMonoclonal - Hybridoma cellsTo produce monoclonal antibodies, B-cells are removed from the spleen of an animal that has been challenged with the relevant antigen. These B-cells are then fused with myeloma tumor cells that can grow indefinitely in culture (myeloma is a B-cell cancer). This fusion is performed by making the cell membranes more permeable
8Reagent Preparation Polyspecific AHG Monospecific AHG Abs to human IgG, andAbs to human C3d (C3b breaks down to C3c and C3d)Advantage is that polyspecific AHG may detect complement-dependent Abs on RBCsDisadvantage - more nuisance positivesMonospecific AHGAbs to human IgG only or human C3d onlyFewer nuisance positives; may miss an important Ab
10Direct Antiglobulin Test Detects in vivo sensitization of RBCsThe DAT is used to detect IgG or C3 bound to the surface of the red cell.In patients with hemolysis, the DAT is useful in determining whether there is anImmune etiologyOR Non-immune etiology of hemolysis
12Non-immune causes of hemolysis Mechanical hemolysis such as those due to artificial valves or burns,Hemoglobinopathies (sickle cell, thalassemia),Red cell enzyme deficiencies (G6PDP, pyruvate kinase),and red cell membrane defects (hereditary spherocytosis, PNH)Have a negative DATPNH = Paroxysmal nocturnal hemoglobinuria
13DAT Uses Have a positive DAT Immune causes of hemolysis Autoimmune hemolytic anemias,Drug induced hemolysis,Delayed or acute hemolytic transfusionHemolytic Disease of the new bornHave a positive DAT
14Indirect Antiglobulin Test (IAT) Detection of in vitro sensitization of RBCsMost clinically significant alloantibodies are IgG antibodies that react best at 37oC and are formed as a result of previous exposure via transfusion or pregnancy.Examples include antibodies to Rh, Kell, Kidd, and Duffy red cell antigens.Same as DAT, except:Step 1 entails incubating RBCs (reagent or unknown) with antisera (reagent or unknown) allowing time for in vitro attachment of Abs to RBCs
16IAT Uses When the unknown is sera: Ab Screening: The patient’s serum is incubated with red cells of known phenotype to detect Ab in patient’s serum against a specific red cell AgPhenotyping: Ab of known specificity is incubated with patient’s RBC to identify specific blood group Ag on the cellsCrossmatch: The patient’s serum is incubated with donor red cells to detect Abs that might reduce the survival of transfused red cells
17Modifications of IATModifications can be done to increase sensitivity & to decrease time to perform IATModification of suspending mediaModification of RBCsEach modification has advantages and disadvantages
18Modification of suspending medium Mechanism of actionAdvantagesDisadvantagesSalineNot applicableInexpensiveLong incubation time requiredLISSFacilitates sensitization but diminishes agglutinationShort incubation timeexpensiveAlbuminFacilitates sensitization and agglutinationShort incubation time, enhances certain clinically significant AbsEnhances the reaction caused by cold autoantibodies
19Modification of RBCs RBCs used in the IAT can be treated with enzymes This increases sensitivity of test for detection of certain auto & allo-antibodies including Rhesus & KiddEnzyme treatment destroy certain Abs including M, N, S & Duffy
20Importance of Anti-complement in antiglobulin reagents Anticomplement increases detection sensitivity for certain Abs (Anti-Kidd)Approx. 200 molecules of cell bound IgG required for +ve antiglobulin testAb sensitization below this level will not be detected by anti-IgG aloneIf antiglobulin reagent contains both anti-IgG & anti-C3b will increase probability of detection of sensitization
21False Positive Reactions MechanismCold Autoantibody“in vitro” complement fixation & auto-agglutinationTechnicalDirty tubes, colloidal silicaAnti-species AbHeterologous Ab in the antiglobulin reagentPolyagglutinable red cellsPresence of anti-T or anti-Tn in the antiglobulin reagentneuraminidase-treated red blood cells
22False Negative Technical problems Failure to add reagent Neutralization of the antiglobulin reagent due to insufficient washing of the test cellsContamination or neutralization of antiglobulin reagentFalse –ve test can be detected by addition of IgG sensitized cells to –ve antiglobulin tests. These cells should be agglutinated by the anti-IgG