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DAT AC Eluate SLIDES.Pdf Handouts http://www.immucor.com/en-us/Pages/Educational- Program-Handouts.aspx 2 All Content © Immucor, Inc. 2019 Advanced Track Webinars 9 January 2019 ADAMTS-13 Testing, TTP, & MORE 6 March 2019 Advanced Antibody Work-Up Techniques: MMA & Molecular Testing 10 July 2019 Platelet Refractory Patients 7 August 2019 Monoclonal Therapy & the Blood Bank 11 September 2019 ImmuLINK for Laboratories of ALL Sizes 6 November 2019 Sickle Cell: In the transfusion service and in real life! Link to register: https://immucor.webinato.com/register 3 All Content © Immucor, Inc. 2019 Advanced Track Webinars Link to register: https://immucor.webinato.com/register 4 All Content © Immucor, Inc. Link to register: https://immucor.webinato.com/register 5 All Content © Immucor, Inc. learn.immucor.com 6 All Content © Immucor, Inc. Continuing Education • PACE, Florida and California DHS • 1.0 Contact Hours • Each attendee must register to receive CE at: https://www.surveymonkey.com/r/DATAutoControlsAndEluates • Registration deadline is 9 August, 2019 • Certificates will be sent via email only to those who have registered 23 August, 2019 7 All Content © Immucor, Inc. Questions? • You are all muted • Q&A following session - Type in questions 8 All Content © Immucor, Inc. • Course content is for information and illustration purposes only. Immucor makes no representation or warranties about the accuracy or reliability of the information presented, and this information is not to be used for clinical or maintenance evaluations. • The opinions contained in this presentation are those of the presenter and do not necessarily reflect those of Immucor. 9 All Content © Immucor, Inc. DAT, Auto Controls & Eluates: They are Not Just for the Reference Lab Presented by Jayanna Slayten, MS, MT(ASCP)SBB Supervisor, Indiana University Health Transfusion Service and Adjunct Faculty, University of Texas Medical Branch SBB Program 2019 10 Objectives • Compare and contrast the indirect antiglobulin test (IAT) and direct antiglobulin test (DAT) • DAT reagents – Polyspecific, IgG with C3 – Monospecific, IgG or C3 • Possible causes of a positive DAT • Elution types with application – Lui – Acid 2019 11 History of AHG Testing • It started with anti-D Hemolytic Disease of the Fetus and Newborn (HDFN) • In 1945 Coombs, Mourant and Race described the use of antihuman globulin, AHG/IgG, for the detection of Rh antibodies in serum to investigate HDFN. –Indirect Antiglobulin Test - IAT Coombs RRA, Mourant AE, and Race RR: (1945) A new test for the detection of weak and ‘incomplete’ Rh agglutinins. Br J Exp Pathol 26: 255 2019 12 History of AHG Testing • In 1946, Coombs and associates described the use of anti-human globulin (AHG) to detect in vivo cell sensitization of babies with hemolytic disease of the fetus and newborn (HDFN). –Direct Antiglobulin Test = DAT Coombs RRA, Mourant AE, and Pace RR: (1946) In vivo isosensitization of red cells in babies with haemolytic disease. Lancet i: 264 2019 13 History of AHG Testing • AHG testing was implemented for antibody identification procedures. • Because Coombs and associates described the test, the AHG tests is often referred to as the Coombs Test. • However, Coombs was not the first to describe the principle of the test. 2019 14 History of AHG Testing • Moreschi in 1908 – Studies involved the use of rabbit anti-goat serum to agglutinate rabbit red cells which were sensitized with low non-agglutinating doses of goat anti-rabbit red cell serum. – Demonstrating that immune serum may be used for enhancing agglutination – Could have been called the Moreschi Test AHG Testing was Re-discovered by Coombs. Moreschi, C: Neue Tatsachen über die Blutkorperchen Agglutinationen. Zentralbl Bakteriol 46:49, 1908. 2019 15 Anti-Complement • Dacie, JV, et al: Br J Haematol 3:77, 1957. – Reported antibody types demonstrated differing reaction patterns – Lewis antibodies did not react at 37C and IAT-IgG method but reacted well in IAT-IgM method – They suggested components of complement may be involved and coated on the red cells • Polley MJ, Mollison PL, and Soothill JF: Br J Haematol 8: 149 – Reported these antibodies react best with anti-complement • Jenkins et al 1960, Pondman et al 1960 and Harboe et al 1963 – Verified the work form Dacie et al – The main component being detected were C3 and C4 2019 16 Definition • Anti-Human Globulin Test (AGT) or (AHG) – Test to ascertain the presence or absence of red cell coating by immunoglobulin (IgG) and/or complement. – This test uses a xenoantibody (rabbit anti- human serum) to act as a bridge between sensitized cells; thus; yielding agglutination as a positive result. AABB Technical Manual, current edition. 2019 17 Polyspecific AHG Reagents • Polyspecific regents – Rabbit polyclonal •Contains anti-IgG and anti-C3d – Rabbit/murine monoclonal blend •Contains monoclonal blend of rabbit polyclonal antihuman anti-IgG and murine anti-C3bd – Murine monoclonal •Contains murine monoclonal anti-IgG,-C3d, -C3b Polyspecific AHG Reagents • Used in DAT and IAT testing • Contains human source anti-IgG and -C3d • Other anti-complement (anti-C4d, -C4b and - C3b may be present • Other antiglobulins may also be present (anti- IgM, - IgA, IgD) Polyspecific AHG Reagents • Polyspecific reagents are prepared to detect IgG and complement • FDA requires that products marked as polyspecific-AHG have anti-C3d reactivity which is greater than or equal to the FDA anti-C3d reference sample. IgG AHG Reagents • Anti-IgG – Rabbit polyclonal •Contains anti-IgG with no anti-complement activity (not only Gamma chain specific) – IgG Heavy Chain •Contains only antibodies reactive against human gamma chains – Monoclonal IgG •Contains murine monoclonal IgG only ANTICORPI MONOCLONALI . slideplayer.it/slide/579877/2/images/1/ANTICORPI+MONOCLONALI.jpg . Title"ANTMONOCLONALI“, Adapted from Milstein (1980) Scientific American, Oct. p.58 IgG AHG Reagents • Prepared by Hybridoma technique to produce monospecific reagent IgG AHG Reagents • Used more than Poly AHG for IAT and DAT • Able to detect IgG for interpretation of clinical significance • Does not react with anti-complement bound to red cells. – Cold reactive antibodies not as detectable – Cold reactive antibodies are not considered clinically significant. Anti-complement AHG • Anti-C3d and anti-C3b – Contains only antibodies reactive against the designated complement component with no immunoglobulin (anti-IgG) reactivity Anti-complement AHG • Prepared by animal immunization or hybridoma technique • Anti-complement reagents are helpful in the evaluation of – Clinical hemolysis – Cold reactive antibodies (allo or auto antibodies) – Complement dependent antibodies (Kidd system) Definition • Indirect Antiglobulin Test (IAT) – Used to detect antigen-antibody reactions that occur in vitro – Sensitization and Lattice Formation (2 step) •Antibody Screen •Antibody Identification •Antigen Typing •Crossmatching AABB Technical Manual, current edition. 2019 26 Definition • Direct Antiglobulin Test (DAT) –Used to detect in vivo cell sensitization (1 step) •Immunoglobulins (IgG) •Complement Image from: https://www.med-ed.virginia.edu/courses/path/innes/rcd/antibody.cfm AABB Technical Manual, current edition. 2019 27 Positive DAT Positive IAT 2019 28 Principle of DAT • DAT will detect – 100-500 molecules of IgG/red cell – 400 - 1100 molecules of C3d / red cell • Detection of lower levels of antibody may require more sensitive methods – Flow Cytometry – ELISA AABB Technical Manual, current edition. 2019 29 Principle: Specimen • EDTA anticoagulated blood specimen – EDTA chelates Ca++ and Mg++ preventing the in vitro binding of complement – Using EDTA specimens with not interfere with complement bound in vivo • A clotted sample is not ideal – For Column Agglutination Test – EDTA or Red Top Tube – For Tube DAT •If a clotted tube (red top tube) is used for DAT the results should be confirmed with an EDTA tube. AABB Technical Manual, current edition. 2019 30 Principle: Reagents • Polyspecific AHG – Contains rabbi/murine polyclonal or monoclonal anti-IgG and anti-C3d • IgG AHG – Contains rabbit or murine polyclonal/monoclonal anti-IgG only. No source of complement • Anti-C3bd – Contains antibodies reactive to complement AABB Technical Manual, current edition. 2019 31 Principle : Tube Reagents • Coombs Control Cells – IgG Sensitized •Weakly IgG Coated Cells •Used as a control to validate that IgG or Poly was added to the test system – Complement Sensitized • Weakly Complement Coated cells •Used as a control to validate that Anti-C3d was added to the test system. AABB Technical Manual, current edition. 2019 32 Tube Method • Wash the 3% cell suspension 4 times manually with saline or automatically using a cell washer. • After washing, add the appropriate reagent. • Spin and read using an agglutination viewer. – Agglutination = Positive – No agglutination = Negative DAT Control? AABB Technical Manual, current edition. 2019 33 Poll the Audience If one is testing a IgG-DAT and achieve the following results, IgG DAT= 3+, Saline DAT control = 2+), then what is the interpretation? • Valid the DAT is positive • Invalid the DAT is positive and the control is positive 2019 34 Testing a DAT Control • Run one tube for each AHG reagent, in parallel – Reason for control is to verify the positive DAT detected is not spontaneous agglutination Example: a cold agglutinin • Control is valid – Testing is valid and may be interpreted appropriately • Control is invalid – If patient and control are positive, then the testing cannot be interpreted
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