Introduction - Antibodies involved in transfusion reactions are of two types, namely the complete and the incomplete. - whereas the complete antibodies agglutinate red cells in saline medium, the incomplete type of antibody sensitizes red cells without agglutination. IgM class of antibodies and IgG1 and IgG3 type of IgG antibodies fix complement. Cell lysis, in vivo, is mediated through the complement system and the complement component C3b is further acted upon to produce C3d.
Mechanisms of extravascular destruction of red cells coated with IgG1 or IgG3 (± C3b).
Antiglobulin Test (Anti-humanglobulin or AHG) Discovered by Coombs, Mourant and Race in 1945 who found that RBC can become sensitized without visible agglutination. AHG identifies IgG antibodies and Complement proteins
Principle of AGT The antiglobulin test (AGT) or Coombs test is based on the principle that antihuman globulins (AHGs) bind to human globulins such as IgG or complement, either free in serum or bound to RBC antigens. IgG antbodies are too small to cause direct agglutination of synthesised red blood cells. The addition of the AHG reagent (which contain anti-igg) to RBC synthesised with IgG antibodies allows for hemagglutination of theses synthesised RBC. Furthermore, AGT can detect complement synthesised RBC.
AHG REAGENT Anti human globulin is a balanced ready to use blend of highly purified immunoglobulins. It contains Anti human IgG antibodies and / or antibodies reactive with human complement components C3b and C3d.
AHG Antihuman globulin reagents may be polyspecific or monospecific. Polyspecific AHG contains antibody to human IgG and to the C3d component of human complement. (other anticomplement antibodies such as anti-c3b, and anti C4b may also be present). Monospecific AHG contain only one antibody specificity. (either anti-igg or anticomplement).
Preparation of AHG The classical method involves injecting human serum or purified globulin into lab animals, such as rabbits. The rabbit immune system react to the foreign Ag and produce antibody to human globulin. (AHG). This type of response produce a polyclonal antibodies recognise different epitopes.
Monoclonal antibodies -Hybridoma technology can be used to produce monoclonal antiglobulin serum. - Monoclonal antibodies are derived from one clone of plasma cells and recognise a single epitope.
Antiglobulin test In the direct antiglobulin tests (DAT), Anti human globulin reagent is used to detect antibodies adsorbed to the red blood cells in vivo. DAT is also called direct Coombs test. In the indirect antiglobulin tests (IAT), Anti human globulin reagent is used to detect antibodies adsorbed to the red blood cells in vitro. IAT is also known as indirct Coombs test.
AHG reagent will react with bound and/or free IgG and Complement proteins. Must get rid of the free antibodies in the serum to identify the bound forms that can cause hemolytic reaction. RBC should be washed properly before carrying out the AHG 2 Types of AHG test: 1. Direct antiglobulin test (DAT) 2. Indirect Antiglobulin test (IAT or antibody screen)
DAT Direct antiglobulin Test (DAT): Detects antibodies bound to RBC in vivo Example: new born baby RBC synthesised with maternal anti D. (+) DAT indicates an immune response; patients cells have attached IgG and/or Complement) EDTA is sample of choice for DAT Use of various drugs and certain diseases (such as megaloblastic anaemia) are known to be associated with a positive direct antiglobulin test.
DAT
Examples of diseases that give a positive direct Coombs test A positive Coombs test indicates that an immune mechanism is attacking the patient's own RBC's. This mechanism could be autoimmunity, alloimmunity or a drug-induced immune-mediated mechanism.
Examples of alloimmune hemolysis Hemolytic disease of the newborn (also known as HDN or erythroblastosis fetalis) Rh 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 Rh c hemolytic disease of the newborn Rh E hemolytic disease of the newborn Other blood group incompatibility (RhC, Rhe, Kidd, Duffy, MN, P and others) Alloimmune hemolytic transfusion reactions
Examples of 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
Autoimmune & alloimmune haemolytic anaemia
Drug-induced immune-mediated hemolysis Methyldopa (IgG mediated type II hypersensitivity) Penicillin (high dose) Quinidine (IgM mediated activation of classical complement pathway and Membrane attack complex, MAC)
Drug-induced immune-mediated hemolysis
Indirect Antiglobulin Test (IAT) Detects in vitro sensitization of RBC 2 stage-test: 1. Incubation phase: incubation of test serum at 37 C with donor cells. 2. Antiglobulin Phase: Addition of Anti human globulin reagent into the cell mixture INTERPRETATION OF RESULTS: - Agglutination reported as positive result. - Positive IAT indicates a specific reaction between donor RBC antigen and antibody in the patient serum. - Sample of choice: IAT -Serum, not more than 48 hours old, should be used. -Complement dependent antibodies are not detected in plasma samples.
Applications of IAT 1- Antibody screening. 2-Cross matching 3- Antenatal antibody screening 4- determination of RBC phenotype using known antisera (eg., Kell typing, weak D testing).
DAT using the microcolumn (gel) system Gel microcolumn assay (GMA) has been shown to be more sensitive than the conventional tube test for detecting potentially significant antibodies coating red blood cells in vivo. See: Novaretti et al (2004) Positive Negative Journal of Clinical Laboratory Analalysis. 18:255-258. What is the principle of the gel method?
Advance techniques in Ag-antibody detection
Use of AHG test in HDN: Direct Coombs test: Fetus red cells and human IgGspecific antibody. Indirect Coombs test: Maternal serum, Rh+ red cells and humaniggspecific antibody
Red cell antigens Lutheran ABO Ii Gerbich Knops Diego Yt Duffy MNS Cromer Indian Kell Rh LW