Coombs (Antiglobulin)Tests (DAT, IAT) PDF

Blood Banking & Transfusion Science Coombs (Antiglobulin)Tests (DAT & IAT) Dr. Futoon Al-Rawashde Department of Medical Laboratory Sciences Al- Balqa' Applied University (BAU)  Coombs (Antiglobulin)Tests Was first described by Robin Coombs in 1945 Divi...

Blood Banking & Transfusion Science Coombs (Antiglobulin)Tests (DAT & IAT) Dr. Futoon Al-Rawashde Department of Medical Laboratory Sciences Al- Balqa' Applied University (BAU)  Coombs (Antiglobulin)Tests Was first described by Robin Coombs in 1945 Divided into Direct Coomb’s Test (DAT) Indirect Coomb’s Test (IAT)  Principles of the Antiglobulin Test All antibody molecules are globulins. Animals injected with human globulins produce antibody to the foreign protein. After the animal serum is adsorbed to remove unwanted agglutinins, it will react specifically with human globulins and can be called AHG serum. AHG – React with human antibodies and complement molecules that are bound to red cells or are present free in serum; increase antigen-antibody reaction. Unbound globulins may react with AHG, may neutralize AHG and cause a false-negative result Unless the red cells are washed free of unbound proteins before addition of AHG serum.  Coombs Control Cells (CCC)/Check cells IgG-Coated Cells CCC (IgG-coated cells) – a quality control measures for negative antiglobulin tests and verifying that the AHG reagent was added/functional. React with the antiglobulin sera and required for antibody detection and crossmatching procedures. Reactivity with IgG-sensitized cells demonstrates that AHG was added and it had not been neutralized. Tests need to be repeated if the IgG-coated cells are not reactive (gives negative results). Testing with IgG-sensitized cells does not detect all potential failures of the antiglobulin test.  Direct Antiglobulin Test (DAT) Detects in vivo sensitization of RBC with IgG or complement / auto antibodies. Washed red cells from a patient or donor are tested directly with AHG reagents. Reporting result: positive - agglutination negative - no agglutination  Indication of DAT test The DAT is used in investigating: 1. Hemolytic disease of fetus and newborn (HDN/HDFN), (Alloantibody) 2. Hemolytic transfusion reaction (Alloantibody) 3. Autoimmune Hemolytic anemia and diagnosis (AIHA), (Autoantibody). 4. Drug induced hemolysis (Autoantibody).  + Patient’s RBCs Coombs Reagent (Antiglobulin-AHG)  DAT procedure RBC pt wash 3 X Dilute 5% with normal saline 1 drop RBC + 1 drop AHG spin 15 sec 3000 rpm yes (positive) Agglutination no (Negative) CCC  DAT Results DAT is positive when agglutination is observed after centrifugation that followed room temperature incubation. Negative when no agglutination is observed and CCC are agglutinated. A positive DAT, may result from: Autoantibodies to intrinsic red cell antigens. Transfusion Alloantibodies in a recipient's circulation, reacting with antigens on recently transfused donor red cells. Alloantibodies in donor plasma, plasma derivatives, or blood fractions that react with antigens on the red cells of a transfusion recipient. HDFN Alloantibodies in maternal circulation that cross the placenta and coat fetal red cells. Antibodies directed against certain drugs that bind to red cell membranes (e.g, penicillin).  Indirect Antiglobulin Test (IAT) / Group-screen & hold (GSH) An IAT is used to demonstrate in-vitro reactions between red cells and antibodies that sensitize. Agglutination that occurs when AHG is added indicates that antibody has bound to a specific antigen present on the red cells. Indication of test: 1. Antibody detection (screening). 2. Antibody identification (Panel Test or Ab ID). 3. Cross matching. 4. Weak D (Du) identification.  Indirect Coombs Test (IAT/GSH) Detects antibodies that have coated the red cells in vitro Step 1 + Patient’s Target Serum RBCs Step 2 + Coombs Reagent (Antiglobulin) DAT IAT Indirect Coomb’s procedure 1- Saline phase (IS- immediate spin) 2 – Incubation phase (37OC) 3 – AHG phase Indirect Coomb’s procedure 1 drop pt. serum + 1 drop screening cells - 3 sets Incubate at room temperature/18-22OC 5 minutes 1 Spin 15 sec. 3000 rpm Observe for agglutination  If agglutination- positive If not – Add LISS (Low Ionic Strength Solution) 2 Incubate 15 min, 37OC, spin 15 sec,3000 rpm Observe for agglutination If agglutination- positive If not – Wash with saline 3 x- decant  Add AHG 2-3 drops Spin 15 sec, 3000 rpm 3 Observe for agglutination If agglutination- positive If not – add CCC (Coomb’s Control Cell)- 1 drop Spin 15 sec, 3000 rpm If agglutination- valid If not – not valid test; check for AHG and CCC  Interpretation for Antiglobulin Tests IAT: The presence of agglutination after incubation at 37 C constitutes a positive test. The presence of agglutination after addition of AHG constitutes a positive test. Antiglobulin tests are negative when no agglutination is observed after initial centrifugation and CCC added afterward are agglutinated. If the CCC are not agglutinated, the negative result is invalid and must be repeated.  Task Reason Incubate RBC with antisera Allows time for antibody molecule attach to RBC antigen 3x saline washing Remove free globulin (Abs) molecules Add antiglobulin reagent Forms RBC agglutination Centrifuge Accelerates agglutination by bringing closer Examine for Interprets test as positive or agglutination negative The End