Indirect Coombs Test PDF

Indirect Coombs test Presented by T.A/ Mai seif Supervised by Prof/ Amany Abou El-Ella Table of CONTENTS 01 Introduction 06 procedure 02 Types 07 Result 03 Indication 08 Interpretation 04 Principle...

Indirect Coombs test Presented by T.A/ Mai seif Supervised by Prof/ Amany Abou El-Ella Table of CONTENTS 01 Introduction 06 procedure 02 Types 07 Result 03 Indication 08 Interpretation 04 Principle 09 Clinical application 05 Sample 06 Equipment  Complete vs. Incomplete Antibodies  Red blood cells contain various antigens on their surface.  Blood group ‘O’ does not contain both antigen ‘A’ and antigen ‘B’ on its surface.  These blood group antigens are carbohydrate molecules. Our immune system usually makes IgM class of antibodies against carbohydrate antigen  These IgM antibodies (which are pentameric in nature) can bind specific antigens present on the surface of RBCs and can agglutinate RBC.. So, antibodies against ABO blood group antigens are called complete antibodies  Complete vs. Incomplete Antibodies  RBC also contains another antigen on its surface, which is called the ‘Rh’ antigen. Rh antigens are protein in nature. As this Rh antigen is a protein, its exposure generates IgG (class switching occurs).  IgG antibodies thus formed bind specifically to the Rh antigens present on the surface of RBCs but they become unable to agglutinate RBCs (there is no cross-linking or clumping) perhaps due to the presence of insufficient antigenic determinants (epitope) on the RBCs to permit the antibody to overcome normal electrostatic repulsion that exists among RBCs and/or because of the inherent property of IgG (monomeric form). So, the antibodies formed against Rh antigens are called incomplete antibodies  Introduction Co o m b s t e s t (An t ig lo b u lin t e s t in g ) immunology laboratory procedure used to detect the presence of antibodies against circulating red blood cells (RBCs) in the body, which then induce hemolysis. The destruction of these red blood cells (RBCs) by antibodies directed against them is described diagnostically as autoimmune hemolytic anemia (AIHA) Types 01 Direct antiglobulin test or DAT 01 DAT is to detect the presence of antibodies attached directly to the RBCs, RBC-binding antibody (IgG) or complement (C3) is present on RBC membranes. 02 Indirect antiglobulin test or IAT 02 is used to detect unbound antibodies to RBCs, which present in the patient’s serum.  Indication  Blood transfusion compatibility: To ensure donor RBCs won't be destroyed by recipient's antibodies.  Pregnancy: To check if an Rh -negative mother has developed antibodies against Rh -positive fetal blood. Principle of the Coombs Test 1 Antibody Detection 2 Agglutination The patient's serum Antibodie s a tta che d to re d blood is incuba te d with RBCs of known a ntige nicity; ce lls ca us e a gglutina tion, or the n Coombs ' s e rum clumping toge the r. (a ntibodie s to huma n IgG) is (IgG a ntibodie s (a utoa ntibodie s or a dde d a lloa ntibodie s ) a ga ins t RBCs ). a re Visual Identification pre s e nt. 3 Agglutina tion is vis ua lly obs e rve d unde r a micros cope , indica ting a pos itive Coombs te s t re s ult SAMPLE Equipment & Materials for Indirect Coombs Test:  Patient Serum Group O + RBCs Test Tubes Test Tube Rack Saline Solution (0.9% Centrifuge NaCl): Incubator  take 200µꙆ from O+ blood into the test tube and then fill it with normal saline wash the RBCs of O+ to ensure that there are no antibodies bound to RBCs  4 prepare 12 Wassermann After three times of tubes ,put 200µꙆ saline washing make in the tubes from 3 to 10 suspension 5% (0.5 ml then add 200µꙆ of of RBCs + 9.5 ml of patient serum in the saline). 1tube 200µL serum  Add 300µꙆ saline +100µꙆ repeat to do patient serum in the 2tube serial dilution mix, transfer 200µꙆ of the as shown in Discard mixture to the following table 1 mix, transfer 200µꙆ 200µꙆ tube 200µL serum  7 8 add 200µꙆ of the Mix and prepared 5% group incubate at o+ RBCs in each 37°c 1 hour 10tube  Prepare control Positive control Negative control 200µꙆ washed (200µꙆ washed RBCs +antiD RBCs+ saline )  after take 2 drops from 1 and 2 incubation wash , into 2 new Wassermann with saline three times all 2 drop from first tubes and add 2 drops 10 tubes tube +2 drop from Coombs reagent to from coombs reagent each 2 drop from second tube +2 drop from coombs reagent ,  13 14 Incubate for 10 mix and , take a drop from compare with minutes at 37 °C centrifuge 5 min each tube separately positive and on a slide and negative control examine under the microscope , if the first tubes are + ve , repeat steps for each tube separately Another method Experimental laser chamber and gel card indirect antiglobulin test (IAT). (1) RBC and antibody solutions are added to the gel card upper chamber. (2) RBC-antibody suspension is incubated by laser photons entering from above. (3) Gel card is centrifuged to mix the antibody-bound or unbound RBCs with the anti-antibody (IgG) and pass through the gel column. (4) Agglutinates and RBC positions are observed. (c) Scoring the result: positions of the agglutinates indicate strength of result.  Positive Test Negative test Indicates that the No antibodies. patient has antibodies detected in the against foreign RBCs. serum Result Interpretation Positive Test Negative test indicate sensitization from a no risk for immediate antibody - previous blood transfusion or, in mediated reactions in transfusions pregnancy, an Rh incompatibility or fetal RBC attack...  Positive indirect coombs test Erythroblastosis fetalis Autoimmune or Incompatible hemolytic disease drug -induced blood match hemolytic anemia Causes of a Positive Coombs Test Autoimmune Disorders He molytic a ne mia Tra ns fus ion Re a ctions ABO incompa tibility Drug-Induce d He molys is P e nicillin, ce pha los porins Infe ctions Mycopla s ma pne umonia e Clinical Significance of the Coombs Test 1 Diagnosis Helps diagnose various conditions like hemolytic anemia and a utoimmune dis orde rs. 2 Monitoring Us e d to monitor the e ffe ctive ne s s of tre a tme nts for conditions involving re d blood ce ll de s truction. 3 Blood Transfusion Compatibility De te rmine s the compa tibility of blood for s a fe tra ns fus ion. Limitations of the Coombs Test False-Positive Results  Type of antibody - most commercial antiglobulin testing screens for antibodies to IgG, complement C3, or both. As such, false-negative results may occur in cases of AIHA caused by autoantibodies other than IgG or C3, such as IgM or IgA  High serum protein - certain diseases, such as myeloproliferative diseases, may cause a falsely positive agglutination due to abnormally high protein levels unrelated to antibody-RBC agglutination  Infection - the serum of individuals infected with certain microorganisms. Examples include human immunodeficiency virus (HIV), malaria, hepatitis C virus (HCV), and in rare cases, the hepatitis E virus (HEV). Low Sensitivity  The test may not be able to detect all types of antibodies that can destroy red blood cells.