BLOOD BANK PROCEDURES

Transcription

1 6 T H ANNUAL WORKSHOP HEMATOLOGY AND BLOOD TRANSFUSION MUHARRAM 1424 (08-12 MARCH 2003) BLOOD BANK PROCEDURES TABLE OF CONTENTS Specimen Receipt 1-5 Grading of Agglutination Reactions 6-8 Reagent Quality Control 9-12 ABO Grouping Discrepancies Resolution Antibody Screen Crossmatch Antibody Identification Basic Panel Antibody Identification Ficin Panel Antigen Typing Direct Antiglobulin Testing Elusion Acid (Elu-kit) Elution Lui Freeze Emergency Transfusion Transfusion Reaction Investigations W.A.R.M Adsorption Technique Antibody Titration 86-95

2 TITLE / DESCRIPTION: INDEX NUMBER(S): SPECIMEN RECEIPT 610 EFFECTIVE DATE: REVISION NUMBER / DATE: DISSEMINATION: FEB92 REV11 / AUG02 DP XM PRINCIPLE Proper collection and labeling of Blood Bank specimens is of the utmost importance to ensure patient safety. POLICY All samples received in the Blood Bank will be labeled in accordance with AABB Standards 5.11 Samples not meeting the labeling requirements must be recollected. Although samples of insufficient quantity should generally not be received, it may be necessary to process these samples when difficulties in collection were encountered. All specimens are drawn according to Specimen Collection and Processing Policies SCP-02-05, SCP and SCP Rejection of inadequately collected or labeled samples will be handled in accordance with LAB- OM Handling and Documentation of Sample Integrity Issues. Specimen Collections and Processing will receive all Blood Bank samples initially. Where necessary they may reject samples not meeting Blood Bank criteria. A second sample receipt and acceptance will occur in the Blood Bank. At this time, a Transfusion Service staff member will confirm that all identifying information on the request is in agreement with that on the sample label. Only after this confirmation can testing begin. In certain situations it may be necessary to process an inadequately labeled sample, but only with approval of the Blood Bank Supervisor or Medical Director. Page 1 of 95

3 SPECIMEN CROSSMATCH ADULT VOL. (ml) PEDIATRIC VOL. (ml) TYPE OF TUBE T E S T (MINIMUM VOL.) (MINIMUM VOL.) Type and Screen* Red Top 10 (7) 5 (3) Crossmatch* Red Top 10 (7) 5 (3) Ab Titre Red Top 10 (7) 5 (3) ABO Iso Red Top 10 (7) 5 (3) DAT Lavender Top 5 (3) 3 (1) Red Cell Phenotype Red Top 10 (7) 5 (3) Group Confirmation Red Top 5 (3) 3 (1) Transfusion Reaction Red Top 10 (7) 5 (3) Lavender Top 5 (3) 3 (1) Cord Blood Lavender Top N/A 5 (3) Fetal Blood Lavender Top N/A 3 (1) Blood Group only Lavender Top 3 (3) 3 (3) RhIG Red Top Lavender Top 10 (7) 5 * For Type and Screens or Crossmatches on newborns (collected by heelstick), a minimum of two full bullets is required N/A DONOR PROCESSING ADULT VOL. (ml) PEDIATRIC VOL. (ml) TYPE OF TUBE T E S T (MINIMUM VOL.) (MINIMUM VOL.) HIV1/2 Red Top 7 (3) 7 (3) HTLVI/II Red Top 7 (3) 7 (3) HBsAg Red Top 7 (3) 7 (3) RIBA Red Top 7 (3) 7 (3) Delta Red Top 7 (3) 7 (3) HIV Antigen Red Top 7 (3) 7 (3) Western Blot Red Top 7 (3) 7 (3) (HIV1 & HTLVI) Viral Marker Red Top 7 (3) 7 (3) (Combination Order) PCR Green Top 7 REAGENTS, SUPPLIES, EQUIPMENT Specimen labels generated by computer Blood Bank Specimen Labeling poster (Appendix I) Specimen Revision / Rejection Form (Appendix II) SAFETY PRECAUTIONS All blood and blood products must be treated as potentially infectious. Follow universal precautions detailed in the Laboratory Safety Manual. Page 2 of 95

4 PROCEDURE 1. Upon receipt of a sample, check to ensure that the following requirements have been met: tube is firmly stoppered. label is firmly attached (sticky label is preferred). label information is legible and indelible. label information matches the information on the request. label information includes: Patient full name. Patient Medical Record Number. Date / time drawn. I.D. number of phlebotomist. When all the above criteria are met, the sample may be processed. 1.1 Log-in sample by Accession Number. This will be the default method for receiving samples and Product orders. Samples should be bar-coded into the application to avoid transcription errors. 1.2 Open the LOG-IN application and click on the button next to Accession. 1.3 Click RETRIEVE and press ENTER. Patient demographics will populate the Demographics field and the specimen information will populate the row of the spreadsheet. A red check mark will display in the row header of the specimen. 1.4 Enter the correct Collection date, Collection time and Collector ID. 1.5 If there is more than one order associated with an accession then you will see an ellipsis next to the order Click on the ellipsis button to view a list of the tests ordered Click on the ellipsis button next to Cont/Vol to view the list of containers collected Click on the DETAILS button to see all details if there is more than a single test associated with the accession. If there are tests associated with the accession that are not Blood Bank tests then it is VERY important to only receive the correct sample This can be done by only ticking the line associated with the Blood Bank specimen Clicking the first ticked line will deselect the ticks and allow you to select the desired container Click on DETAILS again to close. Page 3 of 95

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6 1.6 Multiple accession numbers may be entered on the spreadsheet. Patient demographics will change for each entry selected. If all specimens have been entered select a location from the location drop-down box where you want to log the specimen. 1.7 Click on LOG IN. The status will be changed to Collected and the specimen will be In-Lab. 1.8 The sample is ready for processing now. 2. Do not receive any sample which: the tube is cracked or broken, compromising the integrity of the sample the label is inadequately attached. the label information is incomplete. has inadequate volume. the specimen date is different than date collected. (patient samples only). the information on label does not match the information on the request. (patient samples only). 3. If patient samples must be rejected for one of the above reasons or if hemolyzed, they must be cancelled in Cerner. 3.1 Click on CANCEL ORDERS to select the patient for whom you want to cancel the order. Alternatively, you can go to TASK and select the CANCEL ORDERS option. 3.2 The top of the screen indicates the mode of Department Order Entry that you are currently in either : Result Entry (default), Cancel Orders or Accession Add On The mode for canceling orders is CANCEL ORDERS. 3.3 Enter the patient MRN. 3.4 Click on the FILTER button, located just beneath the patient s name This mechanism allows you to choose to search on orders by Laboratory or Radiology. Furthermore, it allows you to search for all encounters associated with the patient or only the encounter you chose in the Encounter Search screen when initially selecting the patient. The column headers will display. Orderable, Order Status, Departmental, Start/Time and Order Details Page 5 of 95

7 3.4.2 These can be arranged into a desired order by clicking on any of the column orders and the system will sort to your selected criteria. 3.5 Select the order you want to cancel and the cancel format will display in the Details section. All fields are highlighted and mandatory. 3.6 The Cancel Reason can be selected from the drop-down box or by typing the first letter of the reason you wish to choose At this point it is also essential to add a Chartable Cancel Comment. The cancel reason selected from the drop-down box does not pass to the on-line chart and so a cancel comment needs to be added by clicking the COMMENT icon This will open a Cancel Reason Dialogue Box, Click EDIT and add a more detailed cancellation reason. This can be viewed in the patients on-line notes attached to the cancelled order and also can be seen in Order Result Viewer. 3.7 Click ADD ORDER TO SCRATCH PAD and then SUBMIT ORDERS, which will process the cancellation. 3.8 Complete a Specimen Revision / Rejection Form (Appendix II) with all the required information. Especially important is the phlebotomist ID number, note this under other on the form. 3.9 Forward to the Quality Assurance Coordinator for follow-up. 4. If the sample does not meet criteria but must be accepted, get Medical Director or Supervisor to approve acceptance. Write a variance report (refer to IPP904). 5. If samples from donors cannot be tested, due to the above reasons, the unit must be discarded physically in and Lifeline with comment stating why the sample could not be tested. PROCEDURE NOTES 1. The sample should not be drawn from the tubing used for infusion of intravenous fluid or from the contiguous vein, but from a fresh venipuncture site. 2. Sample must be drawn from an arm without I.V. If site not available, draw below I.V. 3. Do not allow nursing staff or phlebotomy staff to re-label improperly labeled specimens. Specimens must be recollected. 4. Do not accept samples for crossmatching and / or Type and Screen if the specimen date is not the same as the date collected. Page 6 of 95

8 Exception: This does not apply to group confirm samples, cord bloods, or samples ordered and drawn close to midnight. The collection date must be handwritten if it doesn t match the specimen date. 5. If computer-generated labels are not available, imprinted labels (e.g. addressograph labels) may be used provided all required information is included. The following items must be handwritten, if they are not included on the addressograph label: The specimen number. Specimen date. Collection time. ID # of the person drawing the specimen. 6. Store crossmatch samples for 14 days, donor clot tubes for 1 month and donor segments for 3 months. 7. Store patient samples for viral marker testing at 1-6 C in walk-in refrigerator #10 in bags labeled with date of testing for at least 14 days after testing. Refer to viral marker manufacturer inserts for suitability of sample for testing. 8. If the specimen is improperly labeled, phone floor to explain what is wrong. Send floor a copy of the Blood Bank Specimen Labeling poster (Appendix I). 9. The Blood Bank Quality Assurance Coordinator will forward the Specimen Revision / Rejection Form to Laboratory and Nursing Quality Improvement Coordinators who will initiate the corrective action. REFERENCE 1. Accreditation Requirements Manual, 6th edition, Washington, DC: American Association of Blood Banks, Standards for Blood Banks and Transfusion Services, 21st Edition. Bethesda, MD: American Association of Blood Banks, Technical Manual, 13 th Edition, Bethesda, MD: American Association of Blood Banks, Cerner HNA Millenium Integrated Clinical Information System PathNet User Training Manual, 1 st Edition,, July Page 7 of 95

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10 TITLE / DESCRIPTION: INDEX NUMBER(S): GRADING OF AGGLUTINATION REACTIONS 624 EFFECTIVE DATE: REVISION NUMBER / DATE: DISSEMINATION: JAN92 REV8 / OCT01 DP XM PRINCIPLE The grading of agglutination reactions will be standardized among the members of the Blood Bank staff in the interest of uniformity and reproducibility of test results. Scoring of reactions will only be used for antibody titration. POLICY All Blood Bank staff will assign numerical values (grades) to reactions observed. Score values will be assigned for antibody titrations. Correct grading will be verified for each staff member during their annual competency assessments. REAGENTS, SUPPLIES AND EQUIPMENT Agglutination viewer Microscope SAFETY PRECAUTIONS All blood and blood products must be treated as potentially infectious. Follow universal precautions detailed in the Laboratory Safety Manual. PROCEDURE 1. Spin the cell / serum mixtures at a time appropriate to the calibration of the centrifuge (refer to time posted on the centrifuge being used). 2. Observe for presence of hemolysis. 3. Gently shake the tube and disrupt the cell button in the tube. 4. Observe the cells as they disperse. Page 9 of 95

11 5. If agglutination is present, record the reactivity by comparing the agglutinates to descriptions in the table under Reporting Results. 6. Record the results of the test immediately after observations are made. REPORTING RESULTS GRADE H SCORE VALUE APPEARANCE Red supernatant, few or no intact red cells. PH W M Pink supernatant, some intact red cells. A single agglutinate. No free cells detected. Clear background. Strong reaction. A number of large agglutinates. Clear background. Large agglutinates in a sea of smaller clumps. Clear to slightly cloudy background. Numerous small agglutinates. Cloudy red background. Very small agglutinates easily dispersed. Cloudy red background. Macroscopically: appears negative. Microscopically: A few agglutinates in most fields. PROCEDURE NOTES 1. Serum surrounding the centrifuged cell button must be observed for hemolysis. Hemolysis is generally regarded as a positive reaction, but may be the consequence of bacterial or chemical contamination and should not be interpreted as a positive result without further investigation. Most often, antibodies capable of producing hemolysis have specificity in the ABO, P, Lewis, Kidd, or Vel blood group systems. 2. Mixed field reactions (small medium aggregates in a field of otherwise unagglutinated cells) should be noted as such, as well as graded e.g. 2;MF. REFERENCES 1. Technical Manual, 13th Edition. Bethesda, MD: American Association of Blood Banks, Rudman, Sally V, Textbook of Blood Banking and Transfusion Medicine. Philadelphia, PA: W. B. Saunders Company, Page 10 of 95

12 TITLE / DESCRIPTION: INDEX NUMBER(S): REAGENT QUALITY CONTROL 760 EFFECTIVE DATE: REVISION NUMBER / DATE: DISSEMINATION: PRINCIPLE SEP91 REV4 / JAN99 (Reviewed OCT01) XM Blood Bank reagents are subject to extensive quality control testing by the manufacturer, but factors such as duration of storage, severe changes in temperature and contamination during usage may affect the integrity and performance of the product. Daily Quality Control testing is performed to confirm that reagents are giving expected results and to provide a means to recognize deteriorating potency or reactivity of reagents. POLICY In accordance with AABB Standard and Code of Federal Regulations Title 21 Part (c), quality control of reagent red blood cells and antisera will be performed on each day of use. Records of this quality control testing will be retained for five years in compliance with AABB Standard 6.0. REAGENTS, SUPPLIES, EQUIPMENT 1. Gamma RQC Kit, consisting of: A 1 B D-negative cells 3-4% in Alsevers solution O D-positive cells 3-4% in Alsevers solution Antibody reagent - polyclonal / monoclonal antibodies in 6% bovine albumin 2. Routine blood grouping reagents, consisting of: Anti-A, Anti-B Anti-D A 1 and B cells 3. Routine Antibody Detection Reagents, consisting of: Antibody screening cells Antibody enhancement media Antihuman globulin reagent Coombs control cells 6% bovine albumin 4. Equipment: 12 x 75 mm glass test tubes Serofuge and/or automatic cell washer Agglutination viewer 37C heat block or water bath 5. Reagent Quality Control Weekly Record Sheet (Appendix I) Page 11 of 95

13 SAFETY PRECAUTIONS 1. All blood and blood products must be treated as potentially infectious. Follow universal precautions detailed in the Laboratory Safety Manual. 2. Do not attempt to pick up broken glass with fingers. Use a dustpan or other scooping implement and dispose of glass fragments in sharps disposal container. PROCEDURE 1. Locate the Reagent Quality Control Weekly Record Sheet (Appendix I) specific to your reagent rack number in the Daily QC folder. If filling out a new Record Sheet, record RQC lot number, expiry date and rack number at the top. 2. Under "Reagent Identification," record the manufacturer, lot number, and expiry date of the reagents in use in your rack. 3. If any reagents are expired, replace them with in-date reagents. If no in-date reagents are available, refer to Expired Reagents/Products Certification IPP Examine the visual appearance of the reagents in use for hemolysis, turbidity, discoloration, etc. 4.1 If the visual appearance is satisfactory, under "Reagent Appearance mark a tick in the appropriate column. 4.2 If the visual appearance is NOT satisfactory, under "Reagent Appearance put an X in the appropriate column and replace the reagent. Make a note of the change in the Comment section. 5. Record any changes of lot numbers on the blank lines under Reagent Identification. 6. Label ten (10) 12 x 75 mm test tubes from 1 to 10, which will contain the following: Tube Test Reagent # drops RQC reagent # drops LISS # drops # 1 Anti-A 1 A 1 B, D negative cells 1 no 0 # 2 Anti-B 1 A 1 B, D negative cells 1 no 0 # 3 Anti-D 1 A 1 B, D negative cells 1 no 0 # 4 Anti-D 1 O, D positive cells 1 no 0 # 5 A 1 cells 1 Antibody reagent 2 no 0 # 6 B cells 1 Antibody reagent 2 no 0 # 7 Screening Cell 1 1 Antibody reagent 2 yes 2 # 8 Screening Cell 2 1 Antibody reagent 2 yes 2 # 9 Screening Cell 3 1 Antibody reagent 2 yes 2 #10 6% Albumin 1 A 1 B, D negative cells 1 no 0 Page 12 of 95

14 7. Add one drop of reagent antisera or red cells to be tested to the appropriately labeled tube. 8. Add one drop of the corresponding RQC cells or 2 drops of the RQC Antibody reagent to the appropriately labeled tube. 9. Mix all tubes and centrifuge for time indicated on centrifuge (20 seconds). Examine macroscopically for agglutination and record on the Record Sheet. Grade reactions according to Grading of Agglutination Reactions IPPD 624.) 10. Add two drops of LISS additive to tubes 7, 8 and Incubate tubes 3, 7, 8, 9 and 10 for 15 minutes at 37C. Centrifuge and examine for hemolysis and/or agglutination. Record on Record Sheet. Wash 4 times on automatic cycle in cell washer (CW2), then add 2 drops of anti-igg AHG reagent. Centrifuge and observe for agglutination macroscopically. Record results on Record Sheet. 12. Add one drop of Coombs control cells to all negative AHG tubes. Centrifuge and examine macroscopically for agglutination. Record results on Record Sheet. REPORTING RESULTS 1. Interpret results using Record Sheet Expected Rx as a guide. If any results are not acceptable, do not use the reagents. Exchange the reagent(s) in question for a new vial. Repeat the quality control test. If still not acceptable, report to the Senior in Crossmatch. 2. If QC was not done on the previous day or days, write Not In Use in the column(s). 3. When a new lot number of reagent red cells is placed in use, use a new Weekly Record Sheet rather than recording the new numbers on the previous form. On the previous form, write See Other Sheet in the blank spaces. On the new form, write See Other Sheet in the blank spaces. 4. The Senior Technologist will review, date and initial each Record Sheet at the end of each week. 5. Tests performed with these reagents are not valid unless the quality control results are acceptable. 6. Record Sheets will be retained for 5 years. PROCEDURE NOTES 1. Test each reagent rack each day of use. Make a note on Record Sheet indicating a rack is NOT IN USE for each day the rack is not used. 2. Use a new Weekly Record Sheet at the beginning of each week. Blank forms are in the front of the Daily Reagent QC binder. 3. When there are multiple components within a reagent kit, the components will only be used with kits of the same lot numbers. Page 13 of 95

15 REFERENCES 1. Accreditation Requirements Manual, 5th edition, Bethesda, MD: American Association of Blood Banks, 1994, pages Technical Manual, 13 th Edition, Bethesda, MD: American Association of Blood Banks, Product Insert, RQC Kit (Reagent Quality Control). Gamma Biologicals Inc. January Standards for Blood Banks and Transfusion Services, 20 th Edition. Bethesda, MD: American Association of Blood Banks, Page 14 of 95

16 TITLE / DESCRIPTION: INDEX NUMBER(S): ABO GROUPING DISCREPANCIES - RESOLUTION 702 EFFECTIVE DATE: REVISION NUMBER / DATE: DISSEMINATION: DEC91 REV12 / AUG02 XM PRINCIPLE Under most circumstances individuals possess ABO antibodies directed against those ABO antigens absent from their own cells. Discrepancies, however, do occur and may result from technical errors, from intrinsic properties of the red blood cells or from intrinsic properties of the serum. Resolution of ABO problems may depend upon information about the patients age, diagnosis, medication, and history of pregnancy and transfusion. POLICY In accordance with AABB Standards and blood shall not be released for a patient until any discrepancy in patient s ABO grouping is resolved. If blood must be transfused prior to resolution of the discrepancy, group O red blood cells will be issued. SPECIMEN Freshly drawn clotted whole blood (Preferred) Anti coagulated (EDTA) specimen REAGENTS, SUPPLIES, EQUIPMENT Anti-A, anti-b and Anti-AB A1, A2 and B cells Anti-Human Globulin Screening cells ABO compatible cord serum or plasma Anti-A1 (lectin) Other antisera and neutralizing substances as indicated. Refrigerator (4-6C) Resolution of ABO Discrepancies Worksheet (Appendix I) SAFETY PRECAUTIONS All blood and blood products must be treated as potentially infectious. precautions detailed in the Laboratory Safety Manual. Follow safety Page 15 of 95

17 QUALITY CONTROL 1. Routine daily quality control of antisera and cells. Refer to Reagent Quality Control IPP All other anti-sera used must be tested against cells positive and negative for the corresponding antigen the same day of testing. All reagent red cells must be checked against the corresponding antisera the same day of testing (refer to Antigen Typing IPP710). PROCEDURE 1. If the discrepancy exists between a previous blood group and the current group, order a third sample with a new accession number under DEPARTMENT ORDER ENTRY and LABORATORY ORDER ENTRY, and repeat the testing If the new sample confirms the original blood group, the second sample was incorrectly drawn. Complete a variance report with printouts of the testing results If the new sample confirms the discrepancy, refer to the Senior Technologist or Supervisor for resolution. After making printouts of the original testing results and medical record data, remove the historical ABO/Rh. Complete a variance report. 2. If the discrepancy exists between the forward group and the reverse group, obtain patient history including diagnosis, treatment, medications, and transfusions. Then proceed as follows: 3. UNEXPECTED NEGATIVE REACTIONS: 3.1. IN REVERSE GROUP Repeat the testing of both serum and cells with the following modifications to the reverse group: Add autocontrol Use three to four drops of serum per tube Incubate for 30 min. at RT, then if negative, 15 minutes at 4 o C Read microscopically, comparing result against autocontrol IN FORWARD GROUP Repeat the testing with the following modifications: Wash patient cells 2-3 times Test with different antibody source Incubate for 30 min. at RT, then if negative, 15 minutes at 4 o C. Page 16 of 95

18 Read microscopically, comparing result against negative saline control Look for mixed field and evidence that the patient has been transfused with group O blood Absorb/elute with human source reagents may help to establish true group. 4. UNEXPECTED POSITIVE REACTIONS 4.1. IN REVERSE GROUP Incubate reverse group reactions for 5 minutes at 37 o C to dissipate any cold non-significant reactivity Perform antibody screen, including auto control If all screening cells are positive including the positive control If AHG phase is negative, suspect rouleaux Repeat positive reactions and read using saline replacement technique (Technical Manual 13th Ed) If AHG phase is positive, an autoantibody may be present If cold autoantibody, prewarm the cells and serum at 37 C prior to testing Autoabsorb serum with autologous cells and repeat reverse group, antibody screen and crossmatch. [See W.A.R.M. (Warm Autoantibody Removal Medium) IPP781] If one or more screening cells are positive Suspect unexpected antibody in serum. Perform antibody identification according to Antibody Identification Basic Panel IPP Type A cells and B cells used in the reverse typing for the corresponding antigen Repeat reverse group with: If screen is negative A and B cells negative for corresponding antigen OR Serum neutralized with appropriate neutralizing substance Suspect presence of Anti-A Test serum against A2 cells. Incubate for 5 minutes Centrifuge at calibrated spin time and read macroscopically. Page 17 of 95

19 If Anti-A1 is identified type patient with anti-a1 (refer to Antigen Typing IPP 710) IN FORWARD GROUP Wash patient cells 3 times with warm saline (37 C) and repeat testing If Test Results Now Coincide With Reverse Group Results caused by cold agglutinin or rouleaux If Test Results Are Still Discrepant Repeat test with different source reagents Suspect acquired B if the patient is group A Perform DAT. Suspect protein-induced agglutination if the DAT is positive. Look for mixed field agglutination due to blood group chimera, recent transfusion, contamination of sample, recent bone marrow transplant Retest with fresh ABO compatible human adult and cord serum. If the adult serum is positive and the cord serum negative suspect polyagglutinable RBC. REPORTING RESULTS 1. Record the initial reactions observed on the Resolution of ABO Discrepancies worksheet (Appendix I) under Initial Blood Group Reactions. 2. Record any repeat testing performed, on a new specimen or treated specimen (e.g. prewarmed or absorbed) or if alternative reagents (e.g. human source vs. monoclonal, or antigen typed reverse cells) are used, on the worksheet under Repeat Testing after Sample Change / Modification. The initial results will indicate what repeat testing is required, if any. 3. Record the results of an antibody screen or anti-a1 identification on the worksheet in the appropriate places. If a further panel is required, attach the completed panel sheet to the worksheet and record the antibody identified on the worksheet. 4. Complete and leave the discrepancy worksheet and any panel sheets or antigen typing forms for Senior review. All work will be reviewed by Supervisor and Blood Bank physician prior to filing in Immunohematology binders. 5. Enter final results in computer under BLOOD BANK RESULT ENTRY and the accession number. Enter reactions with Anti-A, Anti-B, A1 Cells, and B Cells and the ABO interpretation in the computer. Enter free text comments, by selecting the COMMENTS icon, as necessary to explain the additional procedures used to obtain the final results. If anti-a1 is identified, enter results under antibody screen interpretation. 6. Enter antigen typing into computer according to Antigen Typing IPP 710. Page 18 of 95

20 PROCEDURE NOTES 1. Eliminate technical errors by repeating the test with strict adherence to proper procedure and technique. Use quality controlled reagents, appropriate positive and negative controls, careful observation, recording and interpretation of reactions. 2. Until correct ABO group can be determined, the patient must be transfused with O cells (if additional sample is required for investigation, have it collected prior to transfusion). 3. In most cases, the correct ABO group of a patient can be determined once the causes of discrepancies have been recognized and resolved or interpreted correctly. Problems that cannot be resolved after investigation should be referred to the supervisor. 4. Recorded discrepancies (e.g. following bone marrow transplant, immunosuppressed children etc.) do not require reinvestigation each time a specimen is received. 5. Enter identification of anti-a1 under antibody screen results. 6. Refer to AABB Technical Manual (latest edition) for help with difficult resolutions. REFERENCES 1. Technical Manual, 13 th Edition, Bethesda, MD: American Association of Blood Banks, Standards for Blood Banks and Transfusion Services, 21st Edition. Bethesda, MD: American Association of Blood Banks, Cerner HNA Millenium Integrated Clinical Information System PathNet User Training Manual, 1 st Edition,, July Page 19 of 95

21 TITLE / DESCRIPTION: INDEX NUMBER(S): ANTIBODY SCREEN 706 EFFECTIVE DATE: REVISION NUMBER / DATE: DISSEMINATION: DEC91 REV10 / AUG02 XM PRINCIPLE Immunization to foreign red cell antigens may occur through pregnancy, transfusion or deliberate injection with immunization materials. Antibody screening is performed on: 1. Obstetric patients: to identify women with allo-antibodies that might cause hemolytic disease of the newborn. 2. Potential candidates for transfusion: to detect allo-antibodies that might cause a hemolytic transfusion reaction. 3. Donors: to detect allo-antibodies that might cause passive transfer of antibody. Antibody screening tests allow serum of the patient or donor to react with selected red blood cells under conditions that demonstrate antibodies active at 37C. The antiglobulin phase is required. POLICY Antibody screens will be performed on all samples received with a request for transfusion, and when specifically requested. In accordance with AABB Standards , samples will be tested by a method that will demonstrate all clinically significant unexpected antibodies. Pretransfusion testing will include 37 o C incubation and an antiglobulin phase using cells that are not pooled. IgG sensitized cells will be added to all antiglobulin tests interpreted as negative. If the patient has been transfused in the preceding 3 months with blood or a blood component containing red blood cells or has been pregnant within the preceding 3 months or if the history is uncertain or unavailable, the sample must be obtained from the patient within 3 days of the scheduled transfusion. Antibodies that are known to exist need not be re-identified; it is necessary to run a selected cell panel to exclude any additional clinically significant unexpected antibodies. SPECIMEN Serum and cells from 10 ml (minimum 7 ml) clotted whole blood. Page 20 of 95

22 REAGENTS, SUPPLIES, EQUIPMENT 12 x 75 mm glass test tubes Saline (0.9%) LISS additive Anti-human serum globulin reagent Antibody Screening cells IgG sensitized RBC's Serofuge and/or automatic cell washer Agglutination viewer Microscope 37C heat block or water bath Disposable pipettes SAFETY PRECAUTIONS 1. All blood and blood products must be treated as potentially infectious. Follow universal precautions detailed in the Laboratory Safety Manual. 2. Do not attempt to pick up broken glass with fingers. Use a dustpan or other scooping implement and dispose of glass fragments in sharps disposal container. QUALITY CONTROL Reagent Quality Control IPP 760. PROCEDURE 1. Place two drops of serum to be tested into three properly labeled test tubes (one tube for each screening cell I, II, III). 2. Add one drop of 2-5% suspension of red cells to the corresponding tube and mix. 3. Add two drops of LISS additive and mix. 4. Incubate at 37C for 15 to 30 minutes in the dri-bath incubator. 5. Centrifuge immediately upon completion of incubation. Examine for hemolysis. Dislodge the cell button and observe for agglutination. Record results in computer. 6. Wash 4 times with saline. After last wash decant completely. 7. Add 2 drops of anti-human globulin reagent and mix. 8. Centrifuge. Examine macroscopically with agglutination viewer for agglutination. Record results in computer. Page 21 of 95

23 9. To all negative tests, add 1 drop of IgG sensitized red cells. Centrifuge and examine for agglutination. If no agglutination is present, test must be repeated. Record results in computer. Interpretation of results 1. A negative test indicates that the serum tested has no antibodies that react with the screening cells. 2. When a positive antibody screening result is obtained, proceed with Antibody Identification - Basic Panel IPP When there is a discrepancy between the results of the antibody screening and the crossmatch, (i.e., reagent red cells are positive and the crossmatch is negative, or vice versa) one of the following antibodies may be responsible: 3.1 Anti-H, in the serum of A 1 and A 1 B individuals, may agglutinate all group "O" reagent cells and A 2 cells. Group A 1 and A 1 B cells have very little H antigen. 3.2 Anti-Le bh reacts with group "O" Le(b+) cells, but not with A 1 or A 1 B cells that are Le(b+). 3.3 Anti-A 1 in the serum of A 2 individuals will test negative with group O cells (antibody screen), while A 1 donor cells will be positive (80% of A s are A 1 ). 3.4 Antibody reactive with a low frequency antigen. Low incidence antigens present on screening cells or donor cells cause sporadic positive results. 3.5 Antibody reactive only with cells homozygous for the corresponding antigen. 4. If a negative result is obtained when the presence of an antibody is suspected, one of the following enhancement methods may be used: 4.1 Enzymes: use Ficin Panel (see Antibody Identification Ficin Panel 708). 4.2 Increased incubation time: Do not incubate more than 30 minutes with LISS. 4.3 Increased ratio of serum to cells: Do not do this with LISS. REPORTING RESULTS 1. See procedure for Grading of Agglutination Reactions IPP Record all results as soon as they are read. 3. Record results in the computer under BLOOD BANK RESULT ENTRY Complete the New Worksheet Dialogue box. Select the accession number format, optionally name the worksheet and press OK Enter the accession number associated with the Antibody Screen procedure to be resulted. Page 22 of 95

24 3.1.3 The system will retrieve the order information and reference data for use in the worksheet When this data has been displayed, you can enter additional accession numbers Click on, or use the arrow key to move to the first cell to be resulted, represented by a white cell in the worksheet Enter or select a result from the list. To enter a result, type the first letter or number associated with the result (e.g. type 1 for 1+, n for Not Performed) or click the down arrow key to display the list of options. 3.2 Press ENTER to move to the next result cell For negative antibody screens, the system will automatically interpret the AB Screen Interpretation For positive antibody screens, or incomplete antibody screen results, the AB Screen Interpretation will not automatically interpret and a warning Pattern match not found will be displayed The result of the AB Screen Interpretation will have to be entered manually either by: Typing the first letter of the result, N-Negative or P-Positive or Using the drop down box and selecting the result The system will display the Blood Bank Exception dialogue box To accept the result, select YES in the Override group box, then select a reason for the override and Click OK to return to Result Entry Click PERFORM or VERIFY to save the results. 4. The Coombs Control cell results must be weakly positive (1+ or 2+). Repeat any test that is negative with Coombs Control cells. PROCEDURE NOTES 1. Although the minimum incubation time quoted for the Ortho Antibody Enhancement Solution (LISS) is 10 minutes at 37 o C, incubate for a minimum of 15 minutes as Dri-baths are not thought to heat as quickly as waterbaths. 2. The specimen expiration time may be extended for up to 7 days if it is certain the patient has not been transfused or pregnant in the past 3 months. Do not rely on computer transfusion history as the patient may have been transfused elsewhere or have been pregnant. This is an exception that requires the Medical Director s approval. Page 23 of 95

25 LIMITATIONS OF PROCEDURE 1. Using LISS additive, the ionic strength of the test system is dependent on the amount of serum used. Therefore it is not acceptable to increase the serum-to-cell ratio as it will increase the ionic strength and weaken the sensitivity. REFERENCES 1. AABB Technical Manual, 13th Edition. Bethesda, MD: American Association of Blood Banks, Standards for Blood Banks and Transfusion Services, 21st Edition. Bethesda, MD: American Association of Blood Banks, Cerner HNA Millenium Integrated Clinical Information System PathNet User Training Manual, 1 st Edition,, July Page 24 of 95

26 Page 25 of 95

27 TITLE / DESCRIPTION: INDEX NUMBER(S): CROSSMATCH 722 EFFECTIVE DATE: REVISION NUMBER / DATE: DISSEMINATION: DEC91 REV12 / AUG02 XM PRINCIPLE The crossmatch is an in-vitro procedure to determine serologic compatibility between the donor's red cells and the recipient's serum. This is accomplished by adding a suspension of donor cells to the recipient serum and observing for agglutination and/or hemolysis in the various phases. Massive transfusion is defined as infusion, within a 24-hour period, of a volume of blood approaching or exceeding replacement of the recipients total blood volume (approximately 10 units). So little is left of the patient s original blood that complete crossmatching has little or no benefit. POLICY Prior to issuing blood, the patient history must be reviewed, and the review documented, to compare current ABO/Rh with historical results and to check for history of antibodies or severe reactions. In accordance with AABB Standards , an immediate spin crossmatch is acceptable for patients with no currently detectable or history of previously identified clinically significant antibodies. The following antibodies are considered clinically insignificant and require immediate spin crossmatching only: P1, M, N, Le a, Le b, Sd a, Bg a, and A 1, if they are only reactive at the immediate spin phase OR if they become negative upon prewarming the 37C and the AHG phases. If these antibodies are still reactive at 37 o C or AHG phase using a prewarming technique, they will be considered clinically significant and therefore antigen negative, full crossmatch compatible units are required. Patients with currently detectable or previously identified clinically significant antibodies must receive full crossmatch compatible antigen negative units. Patients who have received RhIG will have full crossmatch compatible units while the anti-d is detectable, once the anti-d is no longer detectable, an immediate spin crossmatch is acceptable. In accordance with AABB Standards and internal policies, massively transfused patients will receive ABO compatible blood which is antigen negative for any existing or pre-existing antibodies. The unit group will be confirmed prior to issue and normal crossmatch procedure will be resumed once a 24-hour period has elapsed since the massive transfusion protocol was initiated. Page 26 of 95

28 SPECIMEN See IPP s Specimen Receipt- and Pretransfusion Testing - for specimen labeling and request requirements and acceptance criteria Serum and cells from clotted whole blood: obtained from patient within 3 days of scheduled transfusion. Donor cells from segment originally attached to the unit being crossmatched. REAGENTS, SUPPLIES, EQUIPMENT 12 mm x 75 mm glass test tubes Disposable Pasteur pipettes Saline (0.9%) Anti-Human Serum IgG Sensitized RBC's Serofuge and/or Automatic Cell Washer Agglutination Viewer Microscope 37C Heat Block or Water Bath LISS additive Polyethylene Glycol (PEG) SAFETY PRECAUTIONS 1. All blood and blood products must be treated as potentially infectious. Follow universal precautions detailed in the Laboratory Safety Manual. 2. Do not attempt to pick up broken glass with fingers. Use a dustpan or other scooping implement and dispose of glass fragments in sharps disposal container. QUALITY CONTROL See Reagent Quality Control IPP760 PROCEDURE 1. Immediate Spin Crossmatch Note: Note: If the patient has currently detectable or a history of clinically significant antibodies (see Procedure Note 1) the Immediate Spin crossmatch is NOT sufficient. Perform a full crossmatch. The immediate spin crossmatch is intended to detect ABO incompatibility. Do not use for neonates (less than 4 months of age) or patients with current reverse type interpretations of less than 2+; instead perform a Group Check on the unit. Page 27 of 95

29 1.1 Place 2 drops of recipient serum in a properly labeled test tube (1 tube for each unit to be crossmatched). 1.2 Add 1 drop of 3-5% saline suspension of donor red cells. Mix. 1.3 Centrifuge immediately. Examine for hemolysis. Dislodge the cell button and observe for agglutination macroscopically. Record the results in the computer. 2. Coombs (AHG) Crossmatch Note: If the patient has currently detectable or a history of clinically significant antibodies (see Procedure Note 1) the Immediate Spin crossmatch is NOT sufficient. Perform a full crossmatch on units lacking the corresponding red cell antigens. Refer to Antigen Typing IPP Make a 3-4% twice-washed donor red cell suspension Place 4 drops of donor whole blood from segment into a test tube Fill the test tube with 0.9% normal saline and mix gently with a pipette Centrifuge for seconds Aspirate and discard the saline supernatant, leaving donor red cells in the test tube Repeat steps to for the second wash Add 0.9% normal saline to the donor red cells making a 3-4% red cell suspension. 2.2 Place 2 drops of recipient serum in a properly labeled test tube (1 tube for each unit to be crossmatched). 2.3 Add 1 drop of 3-4% saline suspension of twice-washed donor red cells. Mix. 2.4 Spin and observe for agglutination or hemolysis. Read and record results. 2.5 Add 2 drops of LISS additive. 2.6 Incubate at 37C for 15 to 30 minutes. 2.7 Centrifuge immediately upon completion of incubation. Examine for hemolysis. Dislodge the cell button and observe for agglutination macroscopically. Record results in computer. 2.8 Wash 4 times with saline. After last wash, decant completely. 2.9 Add 2 drops of anti-human globulin and mix. Page 28 of 95

30 2.10 Centrifuge. Dislodge the cell button and examine macroscopically for agglutination or hemolysis using the agglutination viewer. Record results in the computer immediately after reading To all negative tests, add 1 drop of IgG sensitized red cells. Centrifuge and examine macroscopically for agglutination. If no agglutination is present, test must be repeated. 3. Massive Transfusion 3.1 If the patient's antibody screen is negative, and 10 units of red blood cells have been transfused in a 24 hour period, group checks (typing with anti- A and Anti-B) may be performed on ABO compatible red cells (IPP 754) for all subsequent crossmatches requested in association with the indate specimen For ABO compatible units that have been typed as in 3.1, result in the computer as GP CHK. 3.2 After a lapse of 24 hours from the time the massive transfusion protocol was initiated, a new Type and Screen must be requested and tested irregardless of there being an indate Type and Screen specimen. The first 10 units using this specimen must be tested for serological compatibility as dictated by the patient s serologic history. 3.3 If the specimen outdates before the 24 hours have elapsed, subsequent to the initiation of the massive transfusion protocol, the first 10 units crossmatched on the new specimen must be serologically compatible with the patient s serum in association with the patient s serologic history. 3.4 If the patient has a history of clinically significant alloantibodies, full crossmatch is only required on the first 10 units transfused. Additional red cells may be group checked, provided all units are ABO compatible and antigen negative. Exceptions to this must be evaluated and approved by the Blood Bank Medical Director. 3.5 Dispense the units according to Dispensing Blood Components (IPP746). REPORTING RESULTS 1. Interpretation: Both hemolysis and agglutination in immediate spin, 37C or AHG phases of the crossmatch indicate incompatibility. Perform further testing according to Antibody Identification - Basic Panel (IPP 707). 2. Enter the unit numbers in the computer for the units being crossmatched. Record reactions in the computer as they are observed. Grade reactions according to Grading of Agglutination Reactions - IPP 624. Save all computer results in the computer. 3. For each unit found to be compatible for the patient: Page 29 of 95

31 3.1 Print a Crossmatch Transfusion Tag hand write the date and the accession # of the specimen in the top right hand corner and attach it to blood bag with mini tach-it. This will be the unit identifier attached to the unit. 3.2 Place labeled units in Revco #1 in the drawer corresponding to the last digit of the patient s medical record number. 4. If an ABO retype was performed on the unit instead of an immediate spin crossmatch, result the Immediate Spin crossmatch as GP CHK. PROCEDURE NOTES 1. A clinically significant antibody is one whose specificity has been known to cause hemolytic disease of the newborn, hemolytic transfusion reactions, or unacceptably shortened survival of transfused red blood cells. The following antibodies are considered clinically insignificant: P1, M, N, Le a, Le b, Sd a, Bg a, and A1, unless they are reacting at 37C and/or AHG using a prewarm technique (Prewarm Technique IPP 759). 2. Reactions at 37C may be due to carry-over after binding of IgM agglutinins at room temperature. Prewarm crossmatch according to IPP759. If testing is now negative at 37C and AHG, the previous reactions are considered to be due to cold reacting clinically insignificant antibodies. 3. Give priority to 'STAT' crossmatch orders over routine orders. Blood should be available under normal circumstances in one hour. 4. Crossmatch all blood products containing significant amounts of red cells (greater than 2 ml) for compatibility before issue. This includes Leukapheresis products and plateletpheresis. 5. Give random platelets that are bloody to group specific patients if the inventory levels cannot justify discarding them. 6. If the inventory levels are good, discard the blood random donor platelets. Consult the Senior Technologist if there are excessive numbers of bloody random platelets before discarding. 5. Do not use an immediate spin crossmatch for neonates, or patients who have current reverse type interpretation of less than 2+, or in cases where immediate spin crossmatch are positive due to cold agglutinins or clinically insignificant alloantibodies. Since an immediate spin crossmatch is intended to detect ABO incompatibilities, the ABO must be rechecked on the unit by performing a retype (Group Check) on the unit and resulting as IS crossmatch as GP CHK. Page 30 of 95

32 6. Acceptable blood groups of red cells for transfusion: PT BLOOD TYPE FIRST CHOICE ACCEPTABLE ALTERNATE ACCEPTABLE WITH APPROVAL OF MEDICAL DIRECTOR O pos O pos O neg O neg O neg O pos A pos A pos A neg, O pos, O neg A neg A neg O neg A pos, O pos B pos B pos B neg, O pos, O neg B neg B neg O neg B pos, O pos AB pos AB pos AB neg, A pos, A neg, B pos, B neg, O pos, O neg AB neg AB neg A neg, B neg, O neg AB pos, A pos, B pos, O pos Rh negative patients may switched to RH positive red cells, only by approval from the Blood Bank Medical Director, during exceptional circumstances. 7.1 Enter a Blood Bank comment in the patient s historical records that approval was granted by the Medical Director, for the number of units approved, the date and your ID#. 7.2 The ICIS will allow the crossmatch and dispense of Rh positive units to Rh negative individuals with an override reason. 8. Rh negative patients transfused with Rh positive red cells will be serologically monitored by: 8.1 Performing all subsequent antibody screens with polyethylene glycol (PEG) and low ionic strength (LISS) in parallel until further notice. 8.2 Performing a direct antiglobulin test (DAT) using polyspecific antihuman globulin on all red cell specimens until further notice. 8.3 Performing any additional testing as requested by the Senior and/or Supervisor. 8.4 A comment will be entered into the patient s history by the Senior Technologist outlining to Page 31 of 95

33 9. In a normal adult, the volume of blood transfused which is considered to be replacement of the original volume is 10 units of packed red cells. In pediatric cases, the volume will be less but as calculation of that volume is virtually impossible; 10 units of packed red cells is considered the safe cut-off. 10. If there is insufficient serum to perform a crossmatch, perform a group check on the units until a new sample can be obtained. Order the new sample and perform a Type and Screen on the new sample prior to crossmatching units. LIMITATIONS OF PROCEDURE 1. The crossmatch has many limitations. A compatible crossmatch will not guarantee normal survival of transfused cells, prevent immunization of the recipient, or detect all unexpected red cell antibodies in the recipient s serum. 2. An immediate spin crossmatch performed on a patient with low titred or absent isohemagglutinins may or may not detect an ABO incompatibility. Perform group check on the units. 3. Insufficient washing of donor red cells for the full crossmatch procedure may result in false negative reactions due to serum protein interference. REFERENCES 1. Technical Manual, 13 th Edition, Bethesda, MD: American Association of Blood Banks, Standards for Blood Banks and Transfusion Services, 21st Edition. Bethesda, MD: American Association of Blood Banks, Cerner HNA Millenium Integrated Clinical Information System PathNet User Training Manual, 1 st Edition,, July Page 32 of 95