Antiglobulin Testing PDF

The Antiglobulin Test Unit 1 – Chapter 5 Preamble  PowerPoints are a general overview and are provided to help students take notes over the video lecture ONLY.  PowerPoints DO NOT cover the details needed for the Unit exam  Each student is responsible for READING the...

The Antiglobulin Test Unit 1 – Chapter 5 Preamble  PowerPoints are a general overview and are provided to help students take notes over the video lecture ONLY.  PowerPoints DO NOT cover the details needed for the Unit exam  Each student is responsible for READING the TEXTBOOK for details to answer the UNIT OBJECTIVES  Unit Objectives are your study guide (not this PowerPoint)  Test questions cover the details of UNIT OBJECTIVES found only in your Textbook! 1 Specimen Requirements for Blood Bank  Collection  6 ml PINK top tube (EDTA) with blood bank band ID label  Stability and Storage:  Tubes should be transported at room temp to the laboratory.  If there is a delay in testing, tubes should be refrigerated (1-6°C).  Specimen can be used for routine testing for 3 days following collection.  Causes for rejection of specimen  Hemolyzed  Clotted specimen  Wrong tube or container  Tube not labeled or mislabeled  Inadequate quantity Phases of laboratory testing Three phases of laboratory testing include Pre-analytical Analytical Post-analytical Specimen Testing testing results collection, transmission, transport and interpretation, processing follow-up, retesting. 2 Types of Errors Pre-Analytic Errors Analytic Errors Post Analytic Errors Patient Identification Results reported when control Interpretation of results Phlebotomy Technique results out of range Critical value not called Test Collection Procedures Reagents stored Transcription errors in inappropriately or used after reporting Specimen Transport expiration date Specimen Processing Report sent to the wrong Contamination of cells or location saline used in washing Report illegible Report not sent The Antiglobulin Test Coombs’ Test (Antihuman Globulin Test-AHG) Obtained from immunized nonhuman species to bind to human globulin IgG Complement Two major blood group antibodies IgG IgM 3 Coombs’ Test  IgM binds to corresponding antigen and directly agglutinates RBCs suspended in saline  IgG (non-agglutinating) will not bind agglutinate sensitized RBCs directly  Coombs, containing anti-IgG, will bind RBCs sensitized with IgG antibodies allowing agglutination.  Some blood group antibodies have ability to bind complement to the RBC membrane History of Antihuman Globulin Prior to AHG only IgM antibodies could be detected 1945 – Coombs described the AHG for detection of weak and nonagglutinating Rh antibodies 1946 – detected in vivo sensitization of newborn babies with antibodies Robin Coombs, British immunologist, co-discoverer of the Coombs test (1945) used for detecting antibodies in various clinical scenarios, such as Rh disease and blood transfusion. 4 AHG Reagents  Polyspecific AHG  IgG  C3d component of complement  Other antibodies many be present  Anti-C3b, anti-C4b, Anti-C4d  Little if any activity against IgA and IgM heavy chains  May contain antibody activity to kappa and lambda light chains on all immunoglobulins  Monospecific AHG  Contains only one antibody specificity  IgG  C3b  C3d Anti-IgG and Anti-complement Anti-IgG Contains no anti-complement activity Contains anti-IgG specific for Fc fragment of IgG molecule If not labeled “gamma heavy-chain specific, may contain anti-light chain specificity and react with cells sensitized with IgM and IgA as well Anti-complement Reactive against designated complement components only No activity against human immunoglobulins 5 Preparation of AHG  Polyspecific  Rabbits, goats, sheep  Immunize with IgG, C3  Hyperimmunized to produce high-titer, high avidity antibodies  Absorbed with A1, B, and O cells  Purified Preparation of AHG  Monoclonal  Immunization of mice with purified human globulin  Spleen cells (lymphocytes) are fused with Myeloma cells  Results in hybridomas”  Screened for specificity and affinity  Clones are propagated in tissue culture  Inoculated into mice – antibody collected in ascites  No need for absorption 6 Antibodies Required in AHG Anti-IgG Antibody activity to nonagglutinating blood group antibodies IgG1 IgG3 Nonagglutinating IgM antibodies may be found, but fix complement, therefore detected with anti-complement Rare IgA alloantibodies – Only anti-Pr Antibodies Required in AHG  Anti-complement  Some antibodies fix complement to RBC after complexing of antibody with antigen  Can be detected by anti-complement activity in AHG  Not clinically significant  Anti-Lea, anti P1  Clinically significant  Anti- Jka 7 Principles of AHG Test  Antibody molecules and complement components are globulins  Injecting an animal with human globulin simulates the animal to produce antibody  Antihuman globulin reacts with human globulin molecules either bound or free in serum  Washed RBCs coated with human globulin are agglutinated with AHG AHG and Direct Antiglobulin Test (DAT)  DAT detects in vivo sensitization of RBCs with IgG and/or complement  C3 and C4 split into C3b and C4b, bind to RBC membrane  Further degradation of membrane bound C3b and C4b occurs by removal of C3c and C4c leaving C3d and C4d firmly attached  Degradation occurs in vitro with both warm and cold autoimmune hemolytic anemia's if incubation is greater than 1 hour 8 Direct Antiglobulin Test DAT detects in vivo sensitization of RBC's Clinical conditions resulting in vivo coating Hemolytic disease of newborn (HDN) Hemolytic transfusion reaction (HTR) Autoimmune and drug induced hemolytic anemias (AIHA) DAT Panel  Test one drop of a 3 to 5% suspension of washed RBC’s with polyspecific AHG (anti-IgG and anti-C3d reagent)  Testing with monospecific AHG can help differentiate: Anti-IgG Anti-C3d Type___________ + + WAIHA + - WAIHA - + CAS, PCH, WAIHA 9 Evaluation of Positive DAT Interpreting significance of positive DAT Patient’s diagnosis Drug therapy Recent transfusion history May occur without clinical manifestations of immune mediated hemolysis Table 5-4 – page 110 DAT does not require incubation phase – antigen/antibody complexes in vivo Evaluation of Positive DAT  AABB (American Association of Blood Banks) manual states  “Results of serological tests are not diagnostic of their significance, they can only be assessed in relationship to the patient’s clinical condition” 10 Evaluation of Positive DAT When evaluated a positive DAT (except for neonates) look at the following to determine further testing: Has patient Is the patient Does the received blood receiving any Is there Has patient patient’s serum Is patient products antilymphocyte evidence of in been transfused contain receiving any containing globulin or vivo hemolysis? recently? unexpected drugs? ABO- antithymocyte antibodies? incompatible globulin plasma? Principle of Indirect Antiglobulin Test  IAT is performed to determine in vitro sensitization of red cells and used: 1. Detection of incomplete antibodies to potential donor RBC’s (compatibility tests) or screening cells in serum 2. Identification of antibody specificity using a panel of RBC’s with known antigens 3. Determination of RBC phenotype using know antisera (Kell typing or weak D testing) 4. Titration of incomplete antibodies 11 Tasks in IAT  Incubate RBC’s with antisera to allow time for antibody molecule attachment to RBC antigen  Perform at lease 3 saline washings to remove free globulin molecules  Add anti-globulin (AHG) reagent to form RBC agglutinates  Centrifuge to accelerate agglutination by bringing cells closer  Examine for agglutination to interpret test as positive or negative  Grade agglutination reactions to determine the strength of rection  Add antibody coated RBC’s (Check cells) to negative reactions  Checks for neutralization of antisera by free globulin molecules (Coombs control cells are D-positive RBC’s that are coated with anti-D IAT (see chart) Application Tests In Vitro Sensitization Antibody Compatibility Recipient antibody detection Testing,anti- reacting with donor cells. body screens Antibody reacting with screening cells Antibody Antibody Antibody reacting with Identification panel panel cells Antibody Rh antibody Antibody and selected Rh titration titer cells Red cell RBC antigen Specific antisera + RBC’s phenotype detection to detect antigen (weak D, K, Fy) 12 Factors Affecting the Antiglobulin Test  Detects 150 to 500 IgG molecules per RBC  Ratio of serum to cells  Ratio of serum to cells increases sensitivity  40:1 ratio minimum  1 drop of serum and 1 drop of 4% of RBC’s  Reaction Medium  2 drops of albumin –  zeta potential  2 drops of LISS – enhances antibody uptake -  2 drops of PEG – increase antibody uptake – removes water – Anti-IgG is AHG of choice Factors Affecting the Antiglobulin Test  Temperature  37oC – optimal for IgG  Incubation Time  Between 30 and 120 minutes  With LISS – 15 minutes  Washing of cells required for DAT and IAT  Minimum of three saline washings  Removes unbound globulins  Inadequate results in false-negative reactions  Performed in as short a time as possible  Cell pellet should be completely resuspended before next wash 13 Factors Affecting the Antiglobulin Test  Saline  Fresh  Buffered to a pH of 7.2 to 7.4  Bacterial contamination can cause false positives  Addition of antihuman globulin (AHG)  Added immediately after washing cells to minimize the chance of antibodies eluting from cells and neutralizing the AHG reagent  Add amount recommended by manufacturer Factors Affecting the Antiglobulin Test  Centrifugation and resuspending cells for reading  Crucial step  500 to 1000 RCF – higher RCF yields more sensitive results depending on how resuspended  For 20 seconds  Weak false-positive if inadequately resuspended  Negative results is too vigorous resuspended 14 Sources of Error – False Positive 1. Improper specimen (refrigerated, clotted specimen) may cause in vitro complement attachment 2. Autoagglutinable cells 3. Bacterial contamination of cells or saline used in washing 4. Cells with a positive DAT used for IAT 5. Saline contaminated by heavy metals or colloidal silica 6. Dirty glassware 7. Over centrifugation and over reading 8. Polyagglutinable cells 9. Preservative-dependent antibody in LISS reagent 10. Contaminating antibodies in AHG reagent Sources of Error – False Negative 1. Inadequate or improper washing of cells 2. AHG reagent nonreactive – deterioration of neutralization 3. AHG reagent non added 4. Serum not added in the indirect test 5. Serum nonreactive because of deterioration of complement 6. Inadequate incubation conditions in the indirect test 7. Cell suspension too weak or too heavy 8. Undercentrifuged or overcentrifuged cells 9. Poor reading technique 15 Modified and Automated AHG Techniques  Liss, PEG, Albumin  Enzymes  LIP – Low Ionic Polybrene  Microplates  Gel Test  RBC’s are centrifuged through a gel contained in microtube  Easy to read  Can save to be reviewed  Gel acts as a trap  Free unagglutinated RBC’s form pellet in bottom  Agglutinated RBC’s trapped in gel Postamble  READ the TEXTBOOK for the details to answer the UNIT OBJECTIVES.  USE THE UNIT OBJECTIVES AS A STUDY GUIDE  All test questions come from detailed material found in the TEXTBOOK (Not this PowerPoint) and relate back to the Unit Objectives 16