Crossmatching · 5 頁
[[p.1]] AIM OF CROSSMATCHING The procedure is used to determine compatibility between donor and recipients’ blood, i.e. to check for haemolysis or any agglutination if they are mixed together. With accurate blood grouping, Rh typing of recipient and selection of donor with same blood groups, the two bloods are to be compatible. However, there are occasions when, • The recipient may have antibodies in his serum. • The donor may have antibodies in his serum. • There may be mistake in performing, reading and recording of blood groups and Rh typing. Major Crossmatch Donor’s red cells are mixed with recipient’s serum. This procedure determines compatibility between red cells of donor and serum (plasma) of the recipient. Minor Crossmatch Donor’s serum is mixed with recipient’s red cells. This proce- dure determines compatibility between serum (plasma) of donor and red cells of the recipient. However, this is not used routinely. Immediate spin crossmatch may be preferred when: 1. No clinically significant unexpected antibodies are detected in the antibody screen. 9 Crossmatching 73 [[p.2]] 74 Essentials of Blood Banking and Transfusion Medicine 2. No record of previous detection of clinically significant unexpected antibodies. This procedure requires patient’s serum to be mixed with saline suspended red cells (2–4% conc.) of donor at room temperature. The centrifuge immediately (1000 rpm for 1 minute or 3400 rpm for 20 seconds). If clinically significant antibodies are present, the crossmatch technique should include incubation at 37°C followed by the anti-globulin test. Procedure 1. Prepare 5% cell suspension of patients and donor cells in two separate test tubes. 2. Major crossmatch: In a test tube, add two drops of patient’s serum and one drop of donor cells. 3. Minor crossmatch: In a test tube add two drops of donor serum and one drop of patient’s cells. 4. Mix the contents; keep the tubes at room temp. for 30 minutes. Centrifuge at 1500 rpm × 1 minute. 5. If no agglutination, then it is compatible. Major crossmatch by matrix gel system: 1. Identify the card with recipient’s/donor’s name or number. 2. Take out the aluminium foil. 3. Add 50 µl donor cell suspension (0.8%) to the microtube. 4. Add 25 µl of recipient’s plasma/serum. 5. Incubate at 37°C × 15 minutes. 6. Centrifuge at 1200 rpm for 10 min. 7. Read the reaction. Indirect Anti-globulin Test for Major Crossmatch In the first step of anti-globulin crossmatch is similar to imme- diate spin crossmatch. Addition of albumin acts as potentiator. Procedure Initial phase 1. Label two test tubes as A (for albumin) and B (for saline), depending upon the number of donors to be crossmatched, as many pairs of such labeled tubes would be required. [[p.3]] Crossmatching 75 2. Prepare a 5% suspension of the red cells to be tested in isotonic saline. 3. Pipette two drops of recipient serum in both the labeled test tubes. 4. Pipette one drop of donor red cells in both the labeled test tubes and mix well. 5. Only to the albumin tube (A) add two drops of bovine albumin reagent and mix well. 6. Centrifuge both the tubes for one minute at 1000 rpm or for 20 seconds at 3400 rpm. 7. Observe for haemolysis. Re-suspend the cell button and observe for agglutination macroscopically. 8. Proceed to incubation phase. 9. Prepare a 5% suspension of the red cells to be tested in isotonic saline. 10. Pipette two drops of recipient serum in both the labeled test tubes. 11. Only to the albumin tube (A) add two drops of bovine albumin reagent and mix well. 12. Centrifuge both the tubes for one minute at 1000 rpm or for 20 seconds at 3400 rpm. 13. Observe for haemolysis. Re-suspend the cell button and observe for agglutination macroscopically. 14. Proceed to incubation phase. Incubation phase 1. Incubate the saline tube at room temperature and the albumin tube at 37°C for fifteen minutes (up to 30 minutes). 2. Re-suspend the cell button and observe for agglutination macroscopically. 3. Proceed to anti-globulin phase. Anti-globulin phase 1. Only the albumin tubes (A) are tested in the anti-globulin phase. 2. Wash the mixture of red blood cells and serum thoroughly with isotonic saline for minimum of three times. Decant completely after the last wash. [[p.4]] 76 Essentials of Blood Banking and Transfusion Medicine 3. Place two drops of anti-human globulin reagent into the test tubes containing the sedimented cells and mix well. 4. Centrifuge for one minute at 1000 rpm or 20 seconds at 3400 rpm. Very gently, re-suspend the cell button and observe for agglutination macroscopically. COOMBS’ TEST IN ANTIBODY IDENTIFICATION Direct Anti-globulin Test Agglutination of red blood cells is a positive test result and indicates presence of IgG antibodies or components of comple- ment on the red blood cells. No agglutination is a negative test result and indicates absence of IgG antibodies or components of complement on the red blood cells. Indirect Anti-globulin Test In all phases of the compatibility test, if no agglutination or haemolysis is observed, then the patient and the donor may be considered compatible. If haemolysis or agglutination at any point till the completion of the anti-globulin phase is observed, the patient and the donor are considered incompatible. Note: 1. If plasma is used in the indirect anti-globulin test, the complement dependent antibodies may not be detected due to the absence of calcium. 2. To all negative test results, after the antiglobulin test phase, one drop of Coombs’ control cells should be added. If Coombs’ control cells do not agglutinate, then the compati- bility test must be repeated. 3. In the indirect antiglobulin test procedure, an auto-control tube (individual’s cells in his own serum) should run. 4. Red blood cells showing a positive direct anti-globulin test cannot be used for the indirect anti-globulin test. 5. It is recommended that anti-IgG activity of the anti-human globulin reagent be tested from time to time preferably on a daily basis using Coombs’ control cells as a positive control. [[p.5]] Crossmatching 77 6. All glassware used in the test should be scrupulously clean, dry and free from contamination with human serum. 7. Contaminated bovine serum albumin, saline or glassware may inactivate anti-human globulin reagent. 8. Use of various drugs and certain diseases (such as megalo- blastic anaemia) are known to be associated with a positive direct anti-globulin test. 9. Cord cells obtained from a newborn exhibiting haemolytic disease of the newborn, especially due to ABO incompati- bility may give false negative results. 10. Anti-human globulin reagent should not contain anti-C4 and is free from anti-T activity. 11. As under centrifugation or over centrifugation could lead to erroneous results, it is recommended that each labora- tory calibrate its own equipment and the time required for achieving the desired results. Note: Addition of albumin (22%, 30%, polymerized bovine serum) prior to incubation at 37°C has been used to enhance the sensitivity of antibody detection tests. In particular, direct agglutination by Rh antibodies after incubation at 37°C appears to be enhanced.