Antiglobulin Test (Coomb’s Test)

The antiglobulin test The antiglobulin test was first introduced into clinical medicine in 1945 by R.R. Coombs He showed that it could be used to detect non-agglutinating red cell antibodies or sensitized red cells Most 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.

Antihuman Globulin Definition: Antihuman: antibodies against human antigens Globulin: all antibody molecules are globulins Therefore: Antihuman Globulin is antibody directed against the Fc portion of human antibodies and/or complement components.

AHG 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.

ANTIGLOBULIN TEST Detection 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 vivo Indirect antiglobulin test (IDAT) - detection of sensitization of RBCs (coated with Abs and/or complement components) in vitro

Antiglobulin Test Principle - Antihuman globulins (AHG) from immunized animals bind to human globulins either free in serum or attached to RBCs Pentameric 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 RBCs AHG acts as a bridge molecule

Reagent Preparation Polyclonal or monoclonal sources Polyclonal - Animals immunized with human globulins (IgG & complement (C3); bled for antisera to obtain high titered Monoclonal - Hybridoma cells To 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

Reagent Preparation Polyspecific AHG Monospecific 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 on RBCs Disadvantage - more nuisance positives Monospecific AHG Abs to human IgG only or human C3d only Fewer nuisance positives; may miss an important Ab

Antiglobulin Test

Direct Antiglobulin Test Detects in vivo sensitization of RBCs The 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 an Immune etiology OR Non-immune etiology of hemolysis

Direct Antiglobulin Test

Non-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 DAT PNH = Paroxysmal nocturnal hemoglobinuria

DAT Uses Have a positive DAT Immune causes of hemolysis Autoimmune hemolytic anemias, Drug induced hemolysis, Delayed or acute hemolytic transfusion Hemolytic Disease of the new born Have a positive DAT

Indirect Antiglobulin Test (IAT) Detection of in vitro sensitization of RBCs Most 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

Indirect Antiglobulin Test

IAT 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 Ag Phenotyping: Ab of known specificity is incubated with patient’s RBC to identify specific blood group Ag on the cells Crossmatch: The patient’s serum is incubated with donor red cells to detect Abs that might reduce the survival of transfused red cells

Modifications of IAT Modifications can be done to increase sensitivity & to decrease time to perform IAT Modification of suspending media Modification of RBCs Each modification has advantages and disadvantages

Modification of suspending medium Mechanism of action Advantages Disadvantages Saline Not applicable Inexpensive Long incubation time required LISS Facilitates sensitization but diminishes agglutination Short incubation time expensive Albumin Facilitates sensitization and agglutination Short incubation time, enhances certain clinically significant Abs Enhances the reaction caused by cold autoantibodies

Modification 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 & Kidd Enzyme treatment destroy certain Abs including M, N, S & Duffy

Importance 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 test Ab sensitization below this level will not be detected by anti-IgG alone If antiglobulin reagent contains both anti-IgG & anti-C3b will increase probability of detection of sensitization

False Positive Reactions Mechanism Cold Autoantibody “in vitro” complement fixation & auto-agglutination Technical Dirty tubes, colloidal silica Anti-species Ab Heterologous Ab in the antiglobulin reagent Polyagglutinable red cells Presence of anti-T or anti-Tn in the antiglobulin reagent neuraminidase-treated red blood cells

False Negative Technical problems Failure to add reagent Neutralization of the antiglobulin reagent due to insufficient washing of the test cells Contamination or neutralization of antiglobulin reagent False –ve test can be detected by addition of IgG sensitized cells to –ve antiglobulin tests. These cells should be agglutinated by the anti-IgG