STANDARD OPERATING PROCEDURE FOR THE DIRECT ANTIGLOBULIN TEST

STANDARD OPERATING PROCEDURE FOR THE DIRECT ANTIGLOBULIN TEST

TITLE: Standard Operating Procedure for Performing the Direct Antiglobulin Test 1.0 Principle To detect in vivo sensitization of red blood cells (RBC) and to determine which protein 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 Policies 2.1 The facility shall develop and maintain operating procedures for each activity that affects the safety of recipients. 2.2 The facility s SOP manual shall be available to all staff at all times to cover the 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 and reporting 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 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 component of complement. The only exception is cord blood testing that may be performed with a monospecific anti-igg reagent. Page 2 of 12

2.8 If a direct antiglobulin test performed on a clotted specimen identifies complement on the red cell surface, the result shall be verified using an EDTA sample. 3.0 Specimens 3.1 Blood sample collected in EDTA anticoagulant 3.2 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 an EDTA specimen. 4.0 Materials 4.1 Polyspecific Antihuman globulin (AHG) reagent containing Anti-IgG and Anti-C3 4.2 Monospecific Anti-IgG reagent 4.3 Monospecific Anti-C3 reagent (anti -C3b, -C3d) 4.4 IgG coated red cells (Coombs control) 4.5 C3 coated red cells 4.6 0.9% Normal saline 4.7 Serologic centrifuge 4.8 Cell washer 4.9 Microscope 4.10 Test tubes 4.11 Transfer pipettes Note: A control reagent is required when agglutination is observed with all antisera tested. 5.0 Quality Control 5.1 Test polyspecific and anti-igg reagents against IgG coated cells, C3 coated cells and unsensitized cells at time of use or once daily as applicable. Page 3 of 12

6.0 Process Flowchart 6.1 Process Flow Testing with Polyspecific AHG Page 4 of 12

6.2 Process Flow Testing with monospecific reagents Page 5 of 12

7.0 Procedure 7.1 Procedure for Testing with Polyspecific AHG 7.1.1 Check specimen suitability. Ensure patient information on specimen matches requisition. (See NL2010-012 Determining Specimen Suitability) 7.1.2 Check patient history. (See NL2010-013 Patient History Check) 7.1.3 Wash an aliquot of red blood cells to be tested at least once with normal saline. Prepare a red cell suspension in normal saline (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 s directions) of patient s red cell suspension into each tube. 7.1.6 Wash 4 times with saline. Completely decant saline after each wash and resuspend cells prior to the addition of saline for subsequent washes. 7.1.7 Completely decant saline after final wash, blot to remove any residual saline in order to obtain a dry red cell button. 7.1.8 Immediately add 2 drops (or volume indicated in manufacturer s directions) of polyspecific AHG to tube labelled AHG and add 2 drops of saline to the tube labelled CTRL 7.1.9 Mix and centrifuge (speed and time as recommended by manufacturer s directions). 7.1.10 Immediately after centrifugation re-suspend red blood cells and examine macroscopically for agglutination. If negative macroscopically, read microscopically. 7.1.11 Grade and record the results. 7.1.12 If negative, incubate test at room temperature for 5 minutes 7.1.13 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 in manufacturer s directions) of IgG coated red cells to the tube labelled AHG and centrifuge. 7.1.16 Examine the tube for macroscopic agglutination. 7.1.17 Grade and record results. Page 6 of 12

NOTE: If agglutination (must be at least 2+) following the addition of IgG coated red cells is not detected the test is invalid and must be repeated. If test with polyspecific AHG is positive, sample must be tested with monospecific Anti-IgG and Anti-C3. 7.2 Procedure for Testing with Monospecific Anti-IgG and Anti C3d 7.2.1 Check specimen suitability. Ensure patient information on specimen matches requisition. (See NL2010-012 Determining Specimen Suitability) 7.2.2 Check patient history. (See NL2010-013 Patient History Check) 7.2.3 Label 3 test tubes: one with patient identifier and IgG, one with patient identifier and C3 and one with patient identifier and control (CTRL). Perform the remaining steps without interruption 7.2.4 Dispense an aliquot (volume as indicated in manufacturer s directions) of previously prepared patient red cell suspension into each tube and wash 4 times with saline. Completely decant saline after each wash and resuspend cells prior to the addition of saline for subsequent washes. 7.2.5 Completely decant saline after final wash, blot to remove any residual saline in order to obtain a dry red cell button. 7.2.6 Immediately add 2 drops (or volume indicated in manufacturer s directions) of IgG to the tube labelled IgG, 2 drops of C3 (or volume indicated in manufacturer s directions) to the tube labelled C3 and 2 drops of saline to the tube labelled CTRL. 7.2.7 Mix and centrifuge (speed and time as recommended by manufacturer s directions). 7.2.8 Immediately after centrifugation re-suspend red blood cells and examine macroscopically for agglutination. If negative macroscopically, examine the cells microscopically. 7.2.9 Grade and record the results. Page 7 of 12

IgG C3 Positive Report result Report result Negative 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 coated red cells mix and centrifuge read macroscopically grade and record results report result NOTE: If agglutination following the addition of the appropriate check cells is not detected the test is invalid and must be repeated 8.0 Reporting / Interpretation 8.1 Interpretation using Polyspecific AHG reagents: Polyspecific Control Test Result Interpretation AHG Negative Negative Negative Positive Negative Positive. Test with monospecific reagents. Positive Positive Unable to report. See note Page 8 of 12

8.2 Interpretation using Monospecific antiglobulin reagents: Anti-IgG Anti- Control Test Result Interpretation Complement Negative Negative Negative Negative Positive Positive Negative Positive IgG and Complement coating cells Positive Negative Negative Positive IgG coating cells Negative Positive Negative Positive Complement coating cells Negative TNP Negative Neonate specimens only: Negative Positive TNP Negative Neonate specimens only: Positive IgG coating cells Positive Positive or Positive Unable to report See note TNP *TNP - Test not performed 9.0 Procedural Notes 9.1 Invalid tests 9.1.1 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 a 1+ reaction or the test is invalid and shall be repeated. 9.2 False Negative test 9.2.1 Tests should be read immediately after centrifugation. Delay may cause bound IgG to dissociate from the red cells causing false negative results. 9.3 False Positive test 9.3.1 False positive test results due to in vitro coating with complement may occur if testing is performed on a clotted specimen. 9.4 Positive Control 9.4.1 A positive control may be due to strong cold agglutinin in the recipient s serum/plasma. Wash recipient cells with 37 C and repeat DAT. 9.5 Mixed Field Reaction 9.5.1 Mixed Field reaction may indicate a transfusion reaction. 9.6 Weak Anti-complement Reactions 9.6.1 Weak anti-complement reactions may be enhanced if tubes containing polyspecific AHG /red cells or anti-complement / red cells are incubated for 5 minutes at room temperature after initial reading of the test. Centrifuge and read again. Page 9 of 12

10.0 Records Management 10.1 The recipient transfusion data file in the transfusion service laboratory shall be retained indefinitely. 10.2 All transfusion records in the recipient s medical chart, shall be retained in accordance with health care facility s retention policy for medical records. 10.3 Quality control of blood components, blood products, reagents and equipment shall be retained for 5 years. 10.4 Date and time of specimen collection and phlebotomist s identification shall be 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 a minimum of ten years. 10.7 Refer to CSTM Standards, Appendix A and CSA Standards, Table 4 for additional record retention requirements. Page 10 of 12

11.0 References 11.1 American Association of Blood Banks. Standards for blood banks and transfusion services, 26 th ed, Bethesda, Maryland: American Association of Blood Banks; 2009. 11.2 Becher M. Technical manual. 15 th ed. Bethesda, Maryland: American Association of Blood Banks; 2005. 11.3 Canadian Standards Association. Blood and blood components Z902-10. Mississauga (ON): Canadian Standards Association; 2010. 11.4 Canadian Standards for Transfusion Medicine. CSTM standards for hospital transfusion services Version 2.0. Ottawa: Canadian Society for Transfusion Medicine; 2007. 11.5 Dominion Biologicals Limited. Anti-Human Globulin Novaclone TM Anti-C3d monoclonal blend manufactures instructions. Dartmouth (NS): Dominion Biologicals Limited; 2007. 11.6 Dominion Biologicals Limited. Anti-Human Globulin Novaclone TM Anti-IgG, C3d polyspecific manufactures instructions. Dartmouth (NS): Dominion Biologicals Limited; 2008. 11.7 Dominion Biologicals Limited. Anti-Human Globulin Novaclone TM Anti- IgG monoclonal blend manufactures instructions. Dartmouth (NS): Dominion Biologicals Limited; 2008. 11.8 Immucor Inc. Checkcell (Weak) antiglobulin control IgG-coated pooled red blood cells manufacture s instructions. Norcross,(GA) USA: Immucor Gamma; 2007 11.9 Immucor Inc. Complement control cells manufacture s instructions. Norcross,(GA) USA: Immucor Gamma; 2005. 11.10 Ortho-Clinical Diagnostics, Inc. Anti-human globulin Anti-C3d monoclonal blend manufactures instructions. Raritan (NJ): Ortho-Clinical Diagnostics, Inc.; 1999. Page 11 of 12

11.11 Ortho-Clinical Diagnostics, Inc. Anti-human globulin BioClone Anti- IgG, -C3d polyspecific manufactures instructions. Raritan (NJ): Ortho- Clinical Diagnostics, Inc.; 2005. 11.12 Ortho-Clinical Diagnostics, Inc. Anti-human globulin BioClone Anti- IgG manufactures instructions. Raritan (NJ): Ortho-Clinical Diagnostics, Inc.; 2005. 11.13 Ortho-Clinical Diagnostics, Inc. Reagent red blood cells Ortho Coombs Control manufacture s instructions. Raritan (NJ): Ortho-Clinical Diagnostics, Inc.; 2007 11.14 Manitoba Provincial Blood Coordinating Office. Manitoba transfusion quality manual for blood banks Version 2.0. Winnipeg (MB).Manitoba Provincial Blood Programs Coordinating Office; 2007. 11.15 Transfusion Ontario Programs Ottawa Office. Ontario regional blood coordinating network standard work instruction manual. Ottawa (ON): Transfusion Ontario Programs Ottawa Office; 2009. 11.16 TraQ Program of the British Columbia Provincial Blood Coordinating Office. Technical resource manual for hospital transfusion services, 2 nd edition. British Columbia: British Columbia Provincial Blood Coordinating Office; 2005. Page 12 of 12