standard operating procedure for the direct antiglobulin test

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Provincial Blood Coordinating Program Standard Operating Procedure For Performing The Direct Antiglobulin Test. 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. _______________________________________________________________________ This document may be incorporated into each Regional Policy/Procedure Manual. NL09-005 Version: 2.0 Effective Date: 2011-02-14 Page 6 of 12
Provincial Blood Coordinating Program Standard Operating Procedure For Performing The Direct Antiglobulin Test. 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. _______________________________________________________________________ This document may be incorporated into each Regional Policy/Procedure Manual. NL09-005 Version: 2.0 Effective Date: 2011-02-14 Page 7 of 12
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standard operating procedure for the direct antiglobulin test

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