Transfusion Medicine Quality Manual Standard Operation Procedure for Direct Antiglobulin Test
Standard Operation Procedure for Direct Antiglobulin Test
Provincial Blood Coordinating Program
1.0 Policy Statements
1.1 A direct antigloulin test (DAT) shall be performed for investigation of: 1.1.1 Hemolytic disease of the newborn; 1.1.2 Autoimmune hemolytic anemia; 1.1.3 Hemolytic transfusion reactions; 1.1.4 Sensitization caused by drugs; and 1.1.5 Complications during pretransfusion and/or compatibility testing. 1.2 A DAT is required if an auto control is not completed in the antibody screen and: 1.2.1 Antibody identification is required and an auto control cannot be completed (e.g., limited volume of plasma); 1.2.2 Antigen typing of the patient cells is required. 1.3 A control reagent (e.g., Saline or 6% albumin) is required when agglutination is observed with all antisera tested. 1.4 Tests shall be read immediately after centrifugation. Delay may cause bound IgG to dissociate from the red cells causing false negative results. 1.5 The antiglobulin reagent shall contain antibodies to IgG and C3d component of complement. Exception: Cord blood testing that may be performed with a monospecific anti-IgG reagent. 1.6 If a DAT performed on a clotted specimen identifies complement on the red cell surface, the result shall be verified using an EDTA sample. 1.7 An elution shall be performed on red cells from patients who have a positive DAT and: 1.7.1 Have received a transfusion or red cells or platelets in the past three (3) months; 1.7.2 Have been pregnant in the past three (3) months; or 1.7.3 The patients history is questionable or unavailable.
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2.0 Linkages
Determining Specimen Suitability. Available at: http://www.health.gov.nl.ca/health/bloodservices/pdf/determining_specimen_suitability.p df
Guidelines for Investigation of Adverse Transfusion Reactions. Available at: http://www.health.gov.nl.ca/health/bloodservices/pdf/Guidelines_for_Investigation.pdf
Patient History Check. Available at: http://www.health.gov.nl.ca/health/bloodservices/pdf/patient_history_check.pdf
Patient Identification and Specimen Labeling. Available at: http://www.health.gov.nl.ca/health/bloodservices/pdf/patient_id_and_specimen_labeling. pdf
Preparation of Red Cell Suspensions. Available at: http://www.health.gov.nl.ca/health/bloodservices/pdf/preparation_of_red_cell_suspensio ns.pdf
Quality Control of Reagents and Antisera. Available at: http://www.health.gov.nl.ca/health/bloodservices/pdf/quality_control_of_reagents_and_a ntisera_ver1.pdf
3.0 Scope
3.1 All Transfusion Medicine Laboratory Technologists
4.0 General Information
4.1 The DAT can determine if red cells have been coated in vivo with immunoglobulin, complement, or both. 4.2 The interpretation of a positive DAT result should take into consideration: 4.2.1 The patient’s recent drug, pregnancy and transfusion history; 4.2.2 Clinical data; and 4.2.3 Results of other laboratory tests. ______This document may be incorporated into each Regional Policy/Procedure Manual.
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4.3 A positive control may be due to strong cold agglutinin in the recipient’s serum/plasma. Washing of the recipient red cells with 37°C saline and repeating DAT may correct this issue. 4.4 Mixed field reaction may indicate a transfusion reaction. 4.5 Weak anti-C3d reactions may be enhanced if tubes containing polyspecific AHG/red cells or anti-C3d/red cells are incubated for five (5) minutes at room temperature (RT) after initial reading of the test. 4.6 A DAT should be performed on every patient in whom the presence of hemolysis has been established to distinguish immune from nonimmune hemolytic anemia. 4.7 False positive test results due to in vitro coating of red cells with complement may occur if testing is performed on a clotted specimen.
5.0 Process
5.1 Quality Control
5.1.1 All reagents shall be used and controlled according to the manufacturer’s written instructions. 5.1.2 The expiry date shall be checked on each reagent used. Do not use reagents beyond expiry date. 5.1.3 The addition of IgG control cells should demonstrate agglutination or the test is invalid and shall be repeated. 5.1.4 The addition of complement control cells should demonstrate agglutination or the test is invalid and shall be repeated.
5.2 Procedure 5.2.1 Procedure for Testing with Polyspecific Antiglobulin Reagent (AHG) 5.2.1.1 Determine specimen suitability. 5.2.1.2 Centrifuge specimen (speed and time as recommended by manufacturer’s directions). 5.2.1.3 Check patient history.
______This document may be incorporated into each Regional Policy/Procedure Manual.
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5.2.1.4 Wash an aliquot of red blood cells to be tested at least once with isotonic saline. Prepare a 2%–5% suspension of red cells in isotonic saline. 5.2.1.5 Label test tube with patient identifier and ‘AHG.’ 5.2.1.6 Dispense 1–2 drops of red cells into labelled tube. 5.2.1.7 Wash three (3) times with isotonic saline. Completely decant saline after each wash and re-suspend cells prior to the addition of saline for subsequent washes. 5.2.1.8 Completely decant saline after final wash, blot to remove any residual saline in order to obtain a ‘dry’ red cell button. 5.2.1.9 Add two (2) drops (or volume indicated as per manufacturer’s directions) of polyspecific AHG to labelled tube. 5.2.1.10 Mix and centrifuge (speed and time as per manufacturer’s directions). 5.2.1.11 Immediately after centrifugation re-suspend red blood cells and examine macroscopically for agglutination. If negative macroscopically, read microscopically. 5.2.1.12 Grade and record the results. 5.2.1.13 If test is negative, incubate test at room temperature for 5– 10 minutes. If test is positive, perform DAT with monospecific reagents, anti-IgG and anti-C3d, to determine which molecules are present. 5.2.1.14 After incubation mix and centrifuge, re-suspend the red cells. Read macroscopically and microscopically. 5.2.1.15 Grade and record results: If test is positive, perform DAT with monospecific reagents, anti-IgG and anti-C3d, to determine which globulins are present. If test is negative add one (1) drop (or volume indicated as per manufacturer’s directions) of IgG coated red cells to both tubes and centrifuge. 5.2.1.16 Examine the tube for macroscopic agglutination.
______This document may be incorporated into each Regional Policy/Procedure Manual.
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5.2.1.17 Grade and record results: If positive, test is valid. DAT is negative. If negative, test is not valid. Procedure must be repeated. 5.2.2 Procedure for Testing with Monospecific Anti-IgG and Anti- C3d 5.2.2.1 Perform steps 5.2.1.1 to 5.2.1.4 as in procedure 5.2.1. 5.2.2.2 Label two (2) test tubes: one with patient identifier and ‘IgG’ and one with patient identifier and ‘C3d.’ 5.2.2.3 Dispense 1–2 drops of red cell suspension into each labelled tube. 5.2.2.4 Wash three (3) times with isotonic saline. Completely decant saline after each wash and re-suspend cells prior to the addition of saline for subsequent washes. 5.2.2.5 Completely decant saline after final wash, blot to remove any residual saline in order to obtain a ‘dry’ red cell button. 5.2.2.6 Add two (2) drops (or volume as indicated as per manufacturer’s directions) of Anti-IgG to tube labelled ‘IgG’ and add two (2) drops of anti- C3d to the tube labelled ‘C3d.’ 5.2.2.7 Mix and centrifuge (speed and time as per manufacturer’s directions). 5.2.2.8 Immediately after centrifugation re-suspend red blood cells and examine macroscopically for agglutination. If negative macroscopically, read microscopically. 5.2.2.9 IgG 5.2.2.9.1 If negative: Add one (1) drop (or volume indicated in manufacturer’s directions) of IgG coated red cells. Centrifuge, examine macroscopically, grade and record results. 5.2.2.9.2 If positive: Report as positive, IgG coating red cells.
______This document may be incorporated into each Regional Policy/Procedure Manual.
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5.2.2.10 C3d 5.2.2.10.1 If negative: Incubate at room temperature for five (5) minutes, and follow steps 5.2.1.14 to 5.2.1.15. If test is then negative add one (1) drop (or volume indicated in manufacturer’s directions) of complement coated red cells. Incubate test at room temperature for 5–10 minutes. Centrifuge, read and record results. 5.2.2.10.2 If positive: Report as positive, complement coating red cells. 5.2.2.11 If C3d and IgG are both positive, perform control if not previously completed.
5.3 Guidelines (NA)
5.4 Materials 5.4.1 Reagents: 5.4.1.1 Polyspecific Anti-Human Globulin (AHG) 5.4.1.2 Anti-IgG (Monospecific) 5.4.1.3 IgG sensitized cells 5.4.1.4 Anti-C3d (Monospecific) 5.4.1.5 C3d coated cells 5.4.1.6 Isotonic saline 5.4.2 Supplies: 5.4.2.1 Test tubes (10x75mm) 5.4.2.2 Transfer pipettes 5.4.2.3 Test tube rack 5.4.2.4 Microscope slides
______This document may be incorporated into each Regional Policy/Procedure Manual.
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5.4.3 Equipment: 5.4.3.1 Serological centrifuge 5.4.3.2 Cell washer 5.4.3.3 Interval timer 5.4.3.4 Microscope 5.4.4 Specimen: 5.4.4.1 Blood sample collected in EDTA anticoagulant. 5.4.4.2 Cord blood sample (clotted), if test is positive with anti- C3d, the test must be repeated with an EDTA specimen.
6.0 Acronyms
DAT Direct anti-globulin test EDTA Ethylenediaminetetraacetic acid RBC Red blood cells RT Room temperature
7.0 Definitions
Elution A technique that dissociates antibodies from sensitized red cells In vitro Outside the living body In vivo In living body of plant or animal Macroscopically Large enough to be observed by the naked eye Microscopically Observed with the use of a microscope
8.0 Records Management
8.1 The recipient transfusion data file in the transfusion service laboratory shall be retained for 50 years.
8.2 All transfusion records in the recipient’s medical chart shall be retained in accordance with health care facility’s retention policy for medical records.
______This document may be incorporated into each Regional Policy/Procedure Manual.
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8.3 Quality control of blood components, blood products, reagents and equipment shall be retained for five (5) years.
8.4 Date and time of specimen collection and phlebotomist’s identification shall be retained for one (1) year.
8.5 Request form for serologic tests shall be retained for one (1) month.
8.6 Documentation of staff training and competency must be kept for a minimum of 10 years.
9.0 Key Words
Direct anti-globulin test, Coombs test, complement
______This document may be incorporated into each Regional Policy/Procedure Manual.
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10.0 Supporting Documents
10.1 Process Flow/Algorithm
Process Flow – Testing with Polyspecific AHG
Determine specimen suitability
Centrifuge specimen
Check patient history
Wash an aliquot of RBC once and prepare RBC suspension
Label test tube with ‘AHG’ and patient identifier
Dispense 1– 2 drops of RBC suspension into labelled tubes
Wash 3 times with saline
Add 1– 2 drops of AHG to labelled tube
Mix and centrifuge
Immediately re-suspend red cells and POS Preform DAT with monospecific examine macroscopically and reagents microscopically Report test as Negative POS POS NEG NEG
Re-suspend red cells and Incubate at room temperature Add one drop of IgG coated Examine Mix and centrifuge examine macroscopically and for 5 minutes red cells to the tube macroscopically microscopically
NEG Test invalid. Repeat
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Process Flow– Testing with Monospecific Anti-IgG and Anti- C3d
Determine specimen Immediately re-suspend and suitability examine macroscopically and microscopically
Centrifuge specimen
IgG Results C3d Results NEG POS NEG POS Check patient history
Incubate at RT for Report as Positive Add IgG coated red Report as Positive 5 minutes for C3d cells (Check Cells) for IgG Wash an aliquot of RBC once and prepare RBC Centrifuge and suspension Centrifuge and re- re-suspend suspend
Label test tubes NEG Read POS ‘C3d’ and ‘IgG’ with Read Test Invalid. macroscopically Report as Positive patient identifier macroscopically Repeat and for C3d microscopically
NEG POS Add C3d coated red Dispense1– 2 drops Mix and centrifuge cells (complement of RBC suspension control cells) into labelled tubes Report test as Negative Centrifuge and re-suspend Add 2 drops of Anti- Wash 3 times with IgG to ‘IgG’ tube and saline 2 drops of Anti-C3d to ‘C3d’ tube. POS Read Report test as macroscopically Negative NEG
______This document may be incorporated into each Regional Policy/Procedure Manual.
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10.2 Tables/Charts
Interpretation using Polyspecific AHG reagents:
Polyspecific Control Test Result Interpretation AHG Negative Negative Negative Positive Negative Positive. Test with monospecific reagents. Positive Positive Test invalid.
Interpretation using Monospecific antiglobulin reagents:
Anti-IgG Anti-C3d Control Test Result Interpretation 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 Positive Positive Positive Test invalid.
______This document may be incorporated into each Regional Policy/Procedure Manual.
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References
AABB Standards for Blood Banks and Transfusion Services, 29th edition pg 88.
Canadian Standards Association (2015). Blood and blood components, Z902- 15. Toronto (ON): Author.
Canadian Society for Transfusion Medicine. (2011). CSTM Standards for Hospital Transfusion Service. Version 3. Ottawa: Author.
Dominion Biologicals. (2011). Anti-Human Globulin NOVACLONE™ Anti-C3d: Instructions for use. Dartmouth, NS: Author.
Dominion Biologicals. (2011). Anti-Human Globulin NOVACLONE™ Anti-IgG, -C3d Polyspecific: Instructions for use. Dartmouth, NS: Author.
Dominion Biologicals. (2011). Anti-Human Globulin NOVACLONE™ Anti-IgG: Instructions for use. Dartmouth, NS: Author.
Immucor. (2011). CHECKCELL®: Instructions for use. Norcross, GA: Author.
Immucor. (2010). Complement Control Cells. Instructions for use. Norcross, GA: Author.
______This document may be incorporated into each Regional Policy/Procedure Manual.
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