Ain t Nobody Got Time for

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1 Ain t Nobody Got Time for DATs Lindsay Peters MLS(ASCP)SBB Reference Laboratory Supervisor Mississippi Valley Regional Blood Center OBJECTIVES Define and describe the antiglobulin test. Identify the reagents used in the antiglobulin test. Compare direct and indirect antiglobulin testing. Describe DAT performance. Discuss uses of the DAT. Investigate positive DAT results. Explore the causes of a positive DAT. Antiglobulin Test Definition: Antiglobulin Test Determines if the RBC is coated by IgG/complement or both. Described by Coombs, Mourant and Race in What is detected? RBC bound IgG/C3 How is it detected? AHG Reagent Polyspecific/ Monospecific 1

2 Also known as What Reagents are Used? Polyspecific Anti-IgG + Anti-C3 Screening tool Monospecific Anti-IgG Anti-C3 Globulin coated reagent cells (Check Cells) Where do the Reagents Come From? Polyclonal Rabbits, Sheep, Goats Immunized with antigen High titer, high avidity antibodies to the human antibody Monoclonal Immunization of laboratory animals Spleen cells harvested and fused with myeloma cells High titer antibodies with well defined specificity 2

3 Antiglobulin Test- What can we do with it? Direct Testing (DAT) IN VIVO sensitization NO incubation Applications: HDN AIHA HTR/DHTR Indirect Testing (IAT) IN VITRO sensitization Incubate of aby and ag Applications: Aby detection Aby ID Crossmatch Antigen typing 3

4 DAT- How is the Test Performed? Patient Cells Washed Antiglobulin Added Centrifuge Read agglutination If Negative, Add Check Cells Can detect: (sensitivity) molecules of IgG attached to the red cell molecules of C3d attached to the red cell Is it always significant? (specificity) Up to 0.1% of Blood Donors 1-15% of hospital patients Most have no signs of clinical hemolysis 4

5 False Positives Improper sample Bacterial contamination Over centrifugation False Negatives Inadequate WASHING Cell suspension too weak /too heavy Under centrifugation Delay/ Interruption in testing Indicate Hemolysis Immune response to transfused blood Potentially serious drug interaction Suspected Hemolysis Neonates Suspected Transfusion Reaction Performed as part of an antibody investigation Evaluation of possible Drug Hemolysis 5

6 Which type of DAT do I use? SCREENING TOOL Polyspecific Anti-IgG Detect Bound IgG Antibody Anti-C3 Detect Bound C3 component C3 attached by an antibody ABO, Le, Jk, P The DAT is positive, now what? 1 st - What do I do Next? Elution Aby Screen 2 nd - Is it causing cell destruction? Other Labs to Evaluate for Hemolysis Clinical Symptoms 3 rd -Why is it positive? What is an Elution and How Do I use It? Elution Disrupt bonds between antibody and RBC Collect antibody to test Physical or Chemical Eluate Test against panel of reagent cells Can determine if there is a specificity 6

7 Lui Freeze/ Thaw Used mostly for ABO HDN Heat (56C) Used mostly for IgM antibodies, ABO HDN Acid IgG Antibdies Most commonly used Chemical (Ether, Digitonin) Time Consuming Dangerous What causes the positive DAT? Hemolytic Disease of the Newborn Maternal aby crosses the placenta and attaches to baby s RBCs Aby MUST be IgG Ag must be well developed on Baby s red cells Anemia, Jaundice, Enlargement of Liver and Spleen, Edema DAT on Cord cells Must WASH!! IgG only Eluate? Considered Unnecessary 7

8 What causes the positive DAT? Hemolytic Disease of the Newborn Most Common: ABO Most Severe: Anti-D Treatment (after birth) O mom s higher levels of IgG Usually not severe UV Light Monitor and intervene Transfusion UV Light What causes the positive DAT? AutoImmune Hemolytic Anemia Cannot be diagnosed with Blood Bank Testing ALONE Warm AIHA Cold Agglutinin Syndrome/ Cold Agglutinin Disease Mixed-type AIHA Paroxysmal cold hemlgbinuria Alloimmune Drug Induced Determine Treatment AIHA- How do we know which one? DAT Ig Eluate Plasma Specificity WAIHA IgG IgG+C3 C3 IgG Pos IAT Pos Broad Rh CAS/CAD C3 IgM Neg RT or colder I Mixed IgG+ C3 C3 IgG, IgM Pos IAT + RT or colder?? PCH C3 IgG Neg Pos Biphasic Hemolysin P 8

9 WAIHA, What do we do? Pan Reactive plasma and eluate May show Rh specificity most commonly e Ficin Enhanced, PeG Enhanced Antibody Panel Cell 1 Cell 2 Cell 3 Cell 4 Cell 5 Cell 6 Cell 7 Cell 8 Cell 9 Cell 10 Auto Plasma Eluate Plasma-Ficin WAIHA- How Do We Deal? Adsorption Autologous Allogenic Time Consuming Requires more sample Complex interpretation What causes a positive DAT? Transfusion Reaction Patient has antibody Donor unit is positive for antigen Aby + Ag (In vivo)= positive DAT Acute vs. Delayed STEP 1 RECOGNIZE THAT A TRANSFUSION REACTION IS OCCURING 9

10 Transfusion Reactions- What do we need to do? Pre- transfusion sample: ABO/RH Visual Check for hemolysis/ icteric Antibody Screen (ABID) DAT Post- transfusion sample: ABO/RH Visual check for hemolysis/ icteric DAT Other testing as indicated Pre Post Clerical OK OK ABO/RH O pos O pos Aby Scr Neg Neg Visual OK Hemolysis + DAT Neg POS Transfusion Reaction- How do we find the aby? Plasma Eluate Pre-transfusion Post-transfusion Enhancements Remove aby bound to donor cells Concentrate Test like plasma Medications Positive DAT with and without Hemolysis MANY Drugs Severe Hemolysis is RARE 1 in 1 million 10

11 4 Different Mechanisms Drug Dependent Antibodies Reactive with Drug-Treated Cells Reactive with Untreated Cells in the Presence of the Drug Drug Independent Antibodies Nonimmunologic Protein Adsorption How do Medications cause a positive DAT? Drug Dependent Aby Reactive w/drug Treated RBC IgG (+C3) DAT +++ Plasma antibody/ Antibody eluted- ONLY reacts with drug treated cells Drug Dependent Aby Reactive w/untreated RBCs in Presence of Drug C3 (+IgG) Antibody IgM, IgG or both How do Medications cause a positive DAT? Indistinguishable from AUTOANTIBODY Drug Independent Antibody Non immunologic Protein Adsorption (NIPA) RARELY hemolytic Positive DAT on RBCs that have been in the presence of the drug but eluate is non-reactive with RBCs in the presence of the drug Proteins stick to the RBC membrane 11

12 How do we know what the cause is? 12