Transfusion Medicine Quality Manual Standard Operating Procedure for Weak D Typing
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1 Transfusion Medicine Quality Manual
2 1.0 Policy Statements 1.1 The Rh type shall be determined by testing red cells with anti-d reagent. An Rh control system, appropriate to the anti-d reagent in use must be included. 1.2 Testing for weak D shall be performed in the case of an Rh negative neonate (less than 4 months of age) of an Rh negative mother with no evidence of Rh alloimmunization. 1.3 Weak D shall be performed if there is a discrepancy in previous and current results obtained with the Rh anti-sera. 1.4 If an Rh typing problem is detected or in an emergency situation: Rh negative recipients should be transfused with Rh negative red cells. Each facility shall have an established policy for the transfusion of Rh positive red cells to an Rh negative recipient when Rh negative red cells are in diminished supply; Rh negative women of child bearing age and children must receive Rh negative red cells, except for a life-threatening situation and when Rh negative red cells are not available; and Rh positive recipients may receive red blood cells that are either Rh positive or Rh negative. 1.5 Each facility shall have a policy for the administration of RhIg for Rh negative recipients who receive blood components containing Rh positive red cells. 2.0 Linkages Determining Specimen Suitability. Available at: suitability.p df Patient History Check. Available at: Patient Identification and Specimen Labeling. Available at: labeling. pdf Page 2 of 11
3 Preparation of Red Cell Suspensions. Available at: ns.pdf Procedure for Rh Typing Tube Method. Available at: Quality Control of Reagents and Antisera. Available at: ntisera_ver1.pdf Resolving ABO & Rh Blood Group Discrepancies. Available at: crepancies.pdf 3.0 Scope 3.1 All Transfusion Medicine Laboratory Technologists who are responsible for testing and reporting transfusion medicine samples. 4.0 General Information 4.1 Not every anti-d reagent is suitable for the weak D test. Consult the manufacturer s package insert for test procedures and appropriate controls. 4.2 The anti-human globulin reagent may be either polyspecific AHG or anti- IgG. 4.3 Positive Weak D results are valid only if a Direct Antiglobulin Test (DAT) is performed and the results are negative. 4.4 IgG coating on the red cell may sometimes be removed by treating the cells with glycine/edta (i.e. EGA) or chloroquine. If the DAT is negative on the treated cells they may then be used to perform weak D typing. 4.5 Pre-washing the red cells is not necessary, however, if a discrepancy is detected the cells should be washed and the tests repeated. 4.6 Tests should be read and interpreted immediately after centrifugation. Delay may cause bound IgG to dissociate from the red cells causing false negative results. Page 3 of 11
4 4.7 Weak D should not be performed if the recipient has been transfused within the last 3 months with Rh positive cellular donor unit(s). 4.8 The addition of IgG control cells should demonstrate a positive reaction or the test is invalid. 4.9 False positive or negative results can be caused by variables such as: 5.0 Process Improper technique; Contaminated materials; Omission of reagents or anti-sera; Delays in testing; Inadequate incubation time and temperature; Inappropriate centrifugation; Inappropriate or prolonged storage of red cells; Immunoglobulins coating the red cells (positive DAT); and Rouleaux. 5.1 Quality Control All reagents shall be used and controlled according to the manufacturer s written instructions All anti-sera must be visually inspected for contamination such as discoloration, cloudiness, turbidity and/or particulate matter All reagent red cells must be visually inspected for hemolysis and/or discoloration The results of the visual inspection, reagent lot number, expiry date, date of the inspection and the individual performing the inspection must be documented The expiry date should be checked on each reagent used. Reagents must not be used beyond expiry date A control consisting of 6-8% bovine serum albumin or a diluent control may be used with the recipient s red cell suspension. Page 4 of 11
5 5.1.7 The reactivity of blood grouping reagents shall be confirmed each day of use by control tests with known antigen positive and negative red cells. Positive control cells should be selected to represent weak expression of the specific antigen. 5.2 Procedure Note: Procedure is for use with a limited specificity Anti-D reagent (i.e. Immucor series 4 or series 5.) There may be variations in test procedure if other anti-d reagents are used. Refer to manufacturer s instructions to ensure correct procedure is used Determine suitability Centrifuge. (Speed and time as recommended by manufacturer s directions) Check after centrifuging (e.g. hemolysis) Perform a patient history check Ensure patient information on the sample corresponds with the patient information on the worksheet Label 2 tubes, one with the recipient s identifier and Weak D and the other with the recipient s identifier and Weak D control Add 1 drop of appropriate anti-d to the labelled Weak D Add 1 drop Rh control to the tube labelled Weak D control Prepare a 3-5% recipient red cell suspension Add 1 drop of the recipient s cell suspension to each tube Mix the contents of the tubes Incubate tubes at 37 (±1) º C for minutes or according to manufacturer s instructions. The temperature of the water bath and time should be recorded After incubation wash tubes with isotonic saline. (Refer to manufacturer s instructions for number of washes). Decant after each wash Add 2 drops of AHG to the dry red cell button in each tube Mix the tubes gently to re-suspend the red cell button. Page 5 of 11
6 Centrifuge immediately (speed and time as recommended by manufacturer s instructions) Remove tubes from centrifuge and re-suspend the red cell button and examine macroscopically for agglutination. Do not read microscopically Grade and record results Verify that the recipient s name on the tubes, s and worksheet correspond Confirm the validity of negative tests with IgG sensitized control cells. Centrifuge, re-suspend cells, read macroscopically and record results. Agglutination must be present as per manufacturers package insert or the test must be repeated Perform a direct antiglobulin test (DAT) if Weak D results are positive Review previous and current results Interpret and report the Rh type using the chart in Update the patients transfusion history for transfusion rerquirements. Neonates positive for weak D are considered Rh negative for transfusion purposes. 5.3 Guidelines (N/A) 5.4 Materials Reagents: Anti-D reagent Rh control (suitable for the anti-d reagent used) Anti-human globulin (AHG) IgG sensitized cells Isotonic Saline Supplies: Test tubes (10x75mm) Transfer pipettes Test tube rack Page 6 of 11
7 6.0 Acronyms Equipment: Serological centrifuge Waterbath 37(±1) C Cellwasher Interval Timer Specimen Blood sample collected in EDTA anticoagulant; or Red cells from clotted samples (SST tubes with gel separator should not be used); or Venous or capillary blood sample from neonates. Cord blood must not be used for pre-transfusion testing. AHG DAT EDTA RhIg SST Antihuman globulin Direct Antiglobulin Test Ethylenediamine tetracetic acid (anticoagulant) Rh immune globulin Serum-separating tubes 7.0 Definitions neonate rouleaux Less than four (4) months of age The stacking up of red blood cells caused by extra or abnormal proteins in the blood that decrease the normal distance red cells maintain between each other. 8.0 Records Management 8.1 The recipient transfusion data file in the Transfusion Medicine Laboratory shall be retained indefinitely. Page 7 of 11
8 8.2 All transfusion records in the recipient s medical chart shall be retained in accordance with health care facility policy. 8.3 Quality control of blood components, blood products, reagents and equipment shall be retained for 5 years. 8.4 Date and time of collection and phlebotomist s identification shall be retained for 1 year. 8.5 Request form for serologic tests shall be retained for one month or as per facility policy. 8.6 Documentation of staff training and competency must be kept for a minimum of ten years. 9.0 Key Words Weak D, Partial D, Rh typing Page 8 of 11
9 10.0 Supporting Documents 10.1 Tables/Charts IAT Interpretation ANTI-D CONTROL Neg Neg Rh negative Rh positive A canned text may be reported along with this result stating: Pos Pos Neg Pos This patient sample exhibits a weakened or partial expression of the D antigen. Mother is a candidate to receive Rh immune globulin if not already administered. For transfusion purposes the patient will be considered Rh negative. Future testing may indicate a change in Rh status from Rh negative to Rh positive. Invalid results Further investigation is required. 1. Agglutination in the anti-d tube, combined with a smooth suspension in the control tube, indicates that the red cells are D positive. It is incorrect to report the results as weak D positive or D negative, weak D positive. 2. No agglutination of the red cells in both the anti-d and the control tubes is a negative test result Agglutination at any phase in the control tube invalidates the test, and no interpretation can be made. IgG removal from the red cells may be helpful. Page 9 of 11
10 10.2 Process Flow/Algorithm Weak D typing Determine suitability NL Label Test Tubes Mix well and centrifuge immediately No Add A/D reagent to tube labeled Weak D Resuspend gently read macroscopically Is suitable? Yes Collect new Add Rh control to tube labeled Weak D Control Grade and record Centrifuge Prepare RBC suspension NL Verify recipient s identification on tubes, s and worksheets match Check suitability after centrifugation Add RBC suspension to tubes Confirm validity of negative result with IgG sensitized cells Is suitable? No Collect new Mix contents of tubes Perform DAT if Weak D results are positive (NL09-005) Yes Perform patient history check NL Incubate tubes Compare to previous results Check name on matches requisition & worksheet Wash 4 times with saline Add Anti-Human Globulin Rh Discrepancy No Report Results Yes Resolve Discrepancy before reporting Page 10 of 11
11 References American Association of Blood Banks, (2011). Standards for Blood Banks and Transfusion Services, (27 th ed.). Bethesda, MD: Author. Canadian Society for Transfusion Medicine, (2011). Standards for Hospital Transfusion Services, Version 3.0. Ottawa, ON: Author. Canadian Standards Association, (2010). CAN/CSA-Z902-10, Blood and Blood Components. Mississauga, ON: Author. Cooling L. ABO, H, and Lewis blood groups and structurally related antigens. In: Fung M, Grossman BJ, Hillyer CD, Westhoff CM, eds. Technical manual. 18th edition. Bethesda, MD: AABB, 2014: Immucor Gamma. (2012) Blood Grouping Reagent: Package Insert. Immucor Gamma. (2012) Reagent Red Cells; Package Insert. Page 11 of 11
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