NL09-005 SOP for Performing the Direct Antiglobulin Test Ver 3

STANDARD OPERATING PROCEDURE FORPERFORMING THE DIRECT ANTIGLOBULIN TEST

Provincial BloodCoordinating ProgramStandard Operating Procedure forPerforming the Direct AntiglobulinTest.TITLE: Standard Operating Procedure for Performing theDirect Antiglobulin Test1.0 PrincipleTo detect in vivo sensitization of red blood cells (RBC) and to determine whichprotein is coating red cells.A 3% saline suspension of the red blood cells to be tested is washed with saline.Antiglobulin reagents, polyspecific (anti-IgG and C3) and/or monospecific (anti-IgG and anti-C3) antisera, are added to the washed red cell button, mixed,centrifuged and then examined microscopically.2.0 Scope and Related Policies2.1 The facility shall develop and maintain operating procedures for eachactivity that affects the safety of recipients.2.2 The facility’s SOP manual shall be available to all staff at all times to coverthe activities they perform.2.3 Quality control shall be carried out as specified in an operating procedure.2.4 To provide work instruction for consistent testing, interpretation andreporting of the Direct Antiglobulin Test (DAT).2.5 The Direct Antiglobulin Test (DAT) may be performed for investigation of:• Hemolytic disease of the newborn• Autoimmune hemolytic anemia• Transfusion reactions• Sensitization caused by drugs• Complications during pretransfusion and/or compatibility testing.2.6 A DAT is required if an auto control is not done in the antibody screen and:• Antibody identification is required and an auto control cannot be done(e.g. limited volume of plasma)• Antigen typing of the patient cells is required.2.7 The antiglobulin reagent shall contain antibodies to IgG and C3d componentof complement. The only exception is cord blood testing that may beperformed with a monospecific anti-IgG reagent._______________________________________________________________________This document may be incorporated into each Regional Policy/Procedure Manual.NL09-005Version: 3.0Effective Date: 2012-09-30Page 2 of 12

Provincial BloodCoordinating ProgramStandard Operating Procedure forPerforming the Direct AntiglobulinTest.2.8 If a direct antiglobulin test performed on a clotted specimen identifiescomplement on the red cell surface, the result shall be verified using anEDTA sample.2.9 An elution must be performed on red cells from patients who have a positiveDAT and have been transfused red cells or platelets, or pregnant in the past3 months, or the patients history is questionable or unavailable.2.10 Related Standard Operating Procedures:2.10.1 NL2010-012 Determining Specimen Suitability2.10.2 NL2010.013 Patient History Check2.10.3 NL2012-033 Preparation of Red Cell Suspensions2.10.4 NL2012-042 Quality Control of Reagents and Antisera3.0 Specimens3.1 Blood sample collected in EDTA anticoagulant3.2 Clotted blood sample – Cells from a clotted specimen may be used.However, if the test is positive with anti-C3, it must be repeated with anEDTA specimen.4.0 MaterialsReagents:Polyspecific Anti-Human Globulin (AHG)Anti-IgG (Monospecific)IgG sensitized cellsAnti-C3d (Monospecific)C3 coated cellsIsotonic salineSupplies:Test tubes (10x75mm)Transfer pipettesTest tube rackEquipment:Serological centrifugeCell washerInterval timerMicroscopeNote: A control reagent is required when agglutination is observed withall antisera tested._______________________________________________________________________This document may be incorporated into each Regional Policy/Procedure Manual.NL09-005Version: 3.0Effective Date: 2012-09-30Page 3 of 12

Provincial BloodCoordinating ProgramStandard Operating Procedure forPerforming the Direct AntiglobulinTest.5.0 Quality Control5.1 Test polyspecific and anti-IgG reagents against IgG coated cells, C3 coatedcells and unsensitized cells at time of use or once daily as applicable._______________________________________________________________________This document may be incorporated into each Regional Policy/Procedure Manual.NL09-005Version: 3.0Effective Date: 2012-09-30Page 4 of 12

Provincial BloodCoordinating Program6.0 Process FlowchartStandard Operating Procedure forPerforming the Direct AntiglobulinTest.6.1 Process Flow – Testing with Polyspecific AHG_______________________________________________________________________This document may be incorporated into each Regional Policy/Procedure Manual.NL09-005Version: 3.0Effective Date: 2012-09-30Page 5 of 12

Provincial BloodCoordinating ProgramStandard Operating Procedure forPerforming the Direct AntiglobulinTest.6.2 Process Flow– Testing with monospecific reagents_______________________________________________________________________This document may be incorporated into each Regional Policy/Procedure Manual.NL09-005Version: 3.0Effective Date: 2012-09-30Page 6 of 12

Provincial BloodCoordinating ProgramStandard Operating Procedure forPerforming the Direct AntiglobulinTest.7.0 Procedure7.1 Procedure for Testing with Polyspecific AHG7.1.1 Check specimen suitability. Ensure patient information onspecimen matches requisition.7.1.2 Check patient history.7.1.3 Wash an aliquot of red blood cells to be tested at least oncewith isotonic saline. Prepare a red cell suspension in isotonicsaline (concentration as indicated in manufacturer’s directions).7.1.4 Label 2 test tubes: one with patient identifier and AHG (eg. JD-AHG) and one with patient identifier and control (eg. JD-CTRL).Perform the remaining steps without interruption.7.1.5 Dispense an aliquot (volume as indicated in manufacturer’sdirections) of patient’s red cell suspension into each tube.7.1.6 Wash 4 times with saline. Completely decant saline after eachwash and resuspend cells prior to the addition of saline forsubsequent washes.7.1.7 Completely decant saline after final wash, blot to remove anyresidual saline in order to obtain a ‘dry’ red cell button.7.1.8 Immediately add 2 drops (or volume indicated inmanufacturer’s directions) of polyspecific AHG to tubelabelled “AHG” and add 2 drops of saline to the tube labelled“CTRL”.7.1.9 Mix and centrifuge (speed and time as recommended bymanufacturer’s directions).7.1.10 Immediately after centrifugation re-suspend red blood cells andexamine macroscopically for agglutination. If negativemacroscopically, read microscopically.7.1.11 Grade and record the results.7.1.12 If negative, incubate test at room temperature for 5 minutes7.1.13 After incubation mix and centrifuge, re-suspend the red cells,read macroscopically and microscopically.7.1.14 Grade and record results.7.1.15 If test is negative add 1 drop (or volume indicated inmanufacturer’s directions) of IgG coated red cells to the tubelabelled “AHG” and centrifuge.7.1.16 Examine the tube for macroscopic agglutination.7.1.17 Grade and record results._______________________________________________________________________This document may be incorporated into each Regional Policy/Procedure Manual.NL09-005Version: 3.0Effective Date: 2012-09-30Page 7 of 12

Provincial BloodCoordinating ProgramStandard Operating Procedure forPerforming the Direct AntiglobulinTest.NOTE: If agglutination (must be at least 2+) following the addition of IgGcoated red cells is not detected the test is invalid and must berepeated.If test with polyspecific AHG is positive, sample must be testedwith monospecific Anti-IgG and Anti-C3.7.2 Procedure for Testing with Monospecific Anti-IgG and Anti-C37.2.1 Check specimen suitability. Ensure patient information onspecimen matches requisition.7.2.2 Check patient history.7.2.3 Label 3 test tubes: one with patient identifier and IgG, one withpatient identifier and C3 and one with patient identifier andcontrol (CTRL).Perform the remaining steps without interruption7.2.4 Dispense an aliquot (volume as indicated in manufacturer’sdirections) of previously prepared patient red cell suspensioninto each tube and wash 4 times with saline. Completely decantsaline after each wash and resuspend cells prior to the additionof saline for subsequent washes.7.2.5 Completely decant saline after final wash, blot to remove anyresidual saline in order to obtain a ‘dry’ red cell button.7.2.6 Immediately add 2 drops (or volume indicated inmanufacturer’s directions) of IgG to the tube labelled “IgG”, 2drops of C3 (or volume indicated in manufacturer’s directions)to the tube labelled “C3” and 2 drops of saline to the tubelabelled “CTRL”.7.2.7 Mix and centrifuge (speed and time as recommended bymanufacturer’s directions).7.2.8 Immediately after centrifugation re-suspend red blood cells andexamine macroscopically for agglutination. If negativemacroscopically, examine the cells microscopically.7.2.9 Grade and record the results._______________________________________________________________________This document may be incorporated into each Regional Policy/Procedure Manual.NL09-005Version: 3.0Effective Date: 2012-09-30Page 8 of 12

Provincial BloodCoordinating ProgramStandard Operating Procedure forPerforming the Direct AntiglobulinTest.IgGC3PositiveReportresultReportresultNegative• add 1 drop IgG coated red cells• mix and centrifuge• read macroscopically• grade and record results• report result• incubate “C3” and “CTRL”tubes at room temperature 5 minutes• mix and centrifuge• read macroscopically and microscopically• grade and record result▪ if still negative, add 1 drop C3 coatedred cells▪ mix and centrifuge▪ read macroscopically▪ grade and record results▪ report resultNOTE: If agglutination following the addition of the appropriate checkcells is not detected the test is invalid and must be repeated.8.0 Reporting / Interpretation8.1 Interpretation using Polyspecific AHG reagents:Polyspecific Control Test Result InterpretationAHGNegative Negative NegativePositive Negative Positive. Test with monospecific reagents.Positive Positive Unable to report. See Procedural Note 9.4_______________________________________________________________________This document may be incorporated into each Regional Policy/Procedure Manual.NL09-005Version: 3.0Effective Date: 2012-09-30Page 9 of 12

Provincial BloodCoordinating ProgramStandard Operating Procedure forPerforming the Direct AntiglobulinTest.8.2 Interpretation using Monospecific antiglobulin reagents:Anti-IgG Anti-Control Test Result InterpretationComplementNegative Negative Negative NegativePositive Positive Negative Positive – IgG and Complement coatingcellsPositive Negative Negative Positive – IgG coating cellsNegative Positive Negative Positive – Complement coating cellsNegative TNP Negative Neonate specimens only: NegativePositive TNP Negative Neonate specimens only: Positive – IgGcoating cellsPositive Positive or Positive Unable to report See Procedural Note 9.4TNP*TNP - Test not performed9.0 Procedural Notes9.1 Invalid tests9.1.1 The addition of IgG control cells should demonstrate a 2+reaction or the test is invalid and shall be repeated.9.1.2 The addition of complement control cells should demonstrate a1+ reaction or the test is invalid and shall be repeated.9.2 False Negative test9.2.1 Tests should be read immediately after centrifugation. Delaymay cause bound IgG to dissociate from the red cells causingfalse negative results.9.3 False Positive test9.3.1 False positive test results due to in vitro coating withcomplement may occur if testing is performed on a clottedspecimen.9.4 Positive Control9.4.1 A positive control may be due to strong cold agglutinin in therecipient’s serum/plasma. Wash recipient cells with 37°C andrepeat DAT.9.5 Mixed Field Reaction9.5.1 Mixed Field reaction may indicate a transfusion reaction.9.6 Weak Anti-complement Reactions9.6.1 Weak anti-complement reactions may be enhanced if tubescontaining polyspecific AHG/red cells or anti-complement/ redcells are incubated for 5 minutes at room temperature afterinitial reading of the test. Centrifuge and read again._______________________________________________________________________This document may be incorporated into each Regional Policy/Procedure Manual.NL09-005Version: 3.0Effective Date: 2012-09-30Page 10 of 12

Provincial BloodCoordinating ProgramStandard Operating Procedure forPerforming the Direct AntiglobulinTest.10.0 Records Management10.1 The recipient transfusion data file in the transfusion service laboratory shallbe retained indefinitely.10.2 All transfusion records in the recipient’s medical chart shall be retained inaccordance with health care facility’s retention policy for medical records.10.3 Quality control of blood components, blood products, reagents andequipment shall be retained for 5 years.10.4 Date and time of specimen collection and phlebotomist’s identification shallbe retained for 1 year.10.5 Request form for serologic tests shall be retained for one month.10.6 Documentation of staff training and competency must be kept for aminimum of ten years.11.0 References11.1 Canadian Standards Association. Blood and blood components Z902-10.Mississauga (ON): Canadian Standards Association; 2010.11.2 Canadian Standards for Transfusion Medicine. CSTM standards for hospitaltransfusion services Version 3.0. Ottawa: Canadian Society for TransfusionMedicine; 2011.11.3 Dominion Biologicals Limited. Anti-Human Globulin Novaclone TMAnti-C3d monoclonal blend manufactures’ instructions. Dartmouth (NS):Dominion Biologicals Limited; 2011.11.4 Dominion Biologicals Limited. Anti-Human Globulin Novaclone TMAnti-IgG, –C3d polyspecific manufactures’ instructions. Dartmouth (NS):Dominion Biologicals Limited; 2011.11.5 Dominion Biologicals Limited. Anti-Human Globulin Novaclone TMAnti- IgG monoclonal blend manufactures’ instructions. Dartmouth (NS):Dominion Biologicals Limited; 2011._______________________________________________________________________This document may be incorporated into each Regional Policy/Procedure Manual.NL09-005Version: 3.0Effective Date: 2012-09-30Page 11 of 12

Provincial BloodCoordinating ProgramStandard Operating Procedure forPerforming the Direct AntiglobulinTest.11.6 Immucor Inc. Checkcell® (Weak) antiglobulin control IgG-coated pooledred blood cells manufacture’s instructions. Norcross,(GA) USA: ImmucorGamma; 2011.11.7 Immucor Inc. Complement control cells manufacture’s instructions.Norcross, (GA) USA: Immucor Gamma; 2010.11.8 Ortho-Clinical Diagnostics, Inc. Anti-human globulin Anti-C3d monoclonalblend manufactures’ instructions. Raritan (NJ): Ortho-Clinical Diagnostics,Inc.; 2010.11.9 Ortho-Clinical Diagnostics, Inc. Anti-human globulin BioClone® Anti-IgG,-C3d polyspecific manufactures’ instructions. Raritan (NJ): Ortho-ClinicalDiagnostics, Inc.; 2010.11.10 Ortho-Clinical Diagnostics, Inc. Anti-human globulin BioClone® Anti-IgGmanufactures’ instructions. Raritan (NJ): Ortho-Clinical Diagnostics, Inc.;2010.11.11 Ortho-Clinical Diagnostics, Inc. Reagent red blood cells Ortho® CoombsControl manufacture’s instructions. Raritan (NJ): Ortho-ClinicalDiagnostics, Inc.; 2010.11.12 Manitoba Provincial Blood Coordinating Office. Manitoba transfusionquality manual for blood banks Version 2.0. Winnipeg (MB).ManitobaProvincial Blood Programs Coordinating Office; 2007.11.13 Roback, J., Grossman, B., Harris, T & Hillier, C. Technical manual 17 thed. Bethesda, Maryland: AABB; 2011.11.14 Transfusion Ontario Programs Ottawa Office. Ontario regional bloodcoordinating network standard work instruction manual. Ottawa (ON):Transfusion Ontario Programs Ottawa Office; 2009.11.15 TraQ Program of the British Columbia Provincial Blood CoordinatingOffice. Technical resource manual for hospital transfusion services, 2 ndedition. British Columbia: British Columbia Provincial BloodCoordinating Office; 2005._______________________________________________________________________This document may be incorporated into each Regional Policy/Procedure Manual.NL09-005Version: 3.0Effective Date: 2012-09-30Page 12 of 12