Compatibility testing.pptx

Compatibility testing,Cross match-major and minor,LISS,Procedure.

1. Compatibility testing Dr.Sumitha.J M.Sc.,M.Phil.,PGDBI,Ph.D.

2. Learning objectives Compatibility testing Cross match-major and minor LISS Procedures of cross match methods Factors affecting compatibility testing.

3. The aim of Blood transfusion i s to provide safe, compatible blood for transfusion to each individual patient. The steps necessary for safe transfusion are: Accurate ABO and Rh typing of the patient. Accurate ABO and Rh typing of the donor. Compatibility Testing. Accurate completion of paperwork and labels Compatibility testing -Blood transfusion

4. COMPATIBILITY TESTING Each compatibility test is a unique experiment in which an unknown (patient) serum and (donor) red cells are tested for the detection of unexpected antibodies which are directed against antigens found on the cells. Negative results indicate compatibility. This is one of the most important tests performed by a transfusion service.

5. The purposes of compatibility testing are: To select blood components that will not cause harm to the recipient and will have acceptable survival when transfused If properly performed, compatibility tests will confirm ABO compatibility between the component and the recipient and will detect the most clinically significant unexpected antibodies To detect irregular antibodies in the recipient serum that are directed against the donor’s cells.

6. Components of CT Proper specimen collection Reviewing patient transfusion history ABO, Rh, and antibody testing (screen/ID) Crossmatching Actual transfusion

7. Specimen collection The sample should also have the full patient name, hospital number, and physician Date and time of collection, phlebotomist’s initials All of this should be on the request form and the sample Collected in tube with EDTA or no additives If the venipuncture causes hemolysis, the sample may be rejected If the sample is drawn from an IV line, the IV infusion should be stopped 5-10 minutes prior to blood drawing and the first 10 mL discarded Testing should be performed on samples less than 72 hours or else complement dependent antibodies may be missed (complement can become unstable

8. Cross matching The cross-match became part of a series of pre-transfusion test known as compatibility testing . The compatibility test includes an ABO and Rh grouping performed on the donor and recipient samples, screening of the donor ’ s and patient ’ s sera for unexpected antibodies, and a cross-match.

9. Cross matching The two main functions of the cross-match test can be cited as, It is a final check of ABO compatibility between donor and patient. It may detect the presence of an Ab in the patient ’ s serum that will react with Ags on the donor RBCs but that was not detected in the Ab screening.

10. Major and Minor cross-match tests Major cross-match test , consisting of mixing the patient ’ s serum with donor RBCs. Minor cross-match test , consisting of mixing the donor ’ s plasma with patient ’ s RBCs The minor cross-match test has been completely eliminated in most blood banks, because donor samples are screened beforehand for the more common Abs.

11. The  major  crossmatch involves testing the patient's serum with donor cells to determine whether the patient has an antibody which may cause a hemolytic transfusion reaction or decreased cell survival of donor cells.  Of greatest importance is detection of ABO incompatibilities.  ABO problems quickly become apparent when the mixture of patient's serum and donor cells is examined after centrifugation (immediate spin crossmatch). Unexpected cold reacting antibodies may also become apparent at this step, but once identified as such, they are usually considered insignificant.  Basic Principles

12. The crossmatch is incubated at 37C to detect  clinically significant  agglutinating or hemolyzing antibodies. After incubation and washing, antiglobulin serum (Coomb's serum) is employed to detect antibodies that have attached to cells without causing agglutination (sensitization).

13. Additional solutions such as albumin, polymerized albumin or low ionic salt solutions (LISS) are frequently employed to increase the sensitivity of the crossmatch or to reduce the incubation time. By lowering the ionic strength or increasing the dielectric constant of the test medium, these solutions increase antibody uptake and enhance the strength of the antigen-antibody reaction.

14. The  minor  crossmatch  involves testing the patients cells with donor plasma to determine whether there is an antibody in the donor's plasma directed against an antigen on the patient's cells. Since all donors are routinely screened at the collection facility for irregular antibodies this procedure is no longer routinely performed. There may be rare instances when this procedure may need to be performed so be familiar with it.

15. Major cross matching-Procedure 1.Collect one tube from each recipient and possible donor(s). 2. Centrifuge tube(s) at 1000rpm for 5 min. to separate plasma from red blood cells (RBCs). 3. Remove plasma from each sample with a clean pipette and transfer to clean, labeled glass or plastic tubes. 4. Wash RBCs 3 times with a normal saline solution; resuspend to make a 3-5% RBC suspension.

16. 5. Prepare for each donor 3 tubes labeled with Major, Minor, and Recipient control. 6. Add to each tube 2 parts of plasma and 1 part of RBC suspension as follows: Major BCM(Blood Cross match): recipient plasma + donor cells Minor BCM(Blood Cross match): donor plasma + recipient cells Recipient control: recipient plasma + recipient cells 7. Mix gently and incubate for 15 min. at room temperature.

17. 8. Centrifuge for 15 sec. at 1000 x 9. 9. Examine supernatant for hemolysis. 10. Gently resuspend button of cells by tapping tube with a finger and examine for macroscopic agglutination. 11. If macroscopic agglutination is not observed, transfer a small amount onto a glass slide and examine for microscopic agglutination. 12. The absence of hemolysis or agglutination indicates compatibility.

18. cross matching methods Saline method-Open Slide method,Tube method-Complete cold antibodies. Immediate spin (IS) cross-match-Complete warm antibodies Albumin Tube method-incomplete immune antibodies(Rh) Coomb’s Cross matching- incomplete immune antibodies(Rh)

19. Saline method Saline suspension of cells is mixed with serum .this is done at RT. Saline technique is designed to detect compatibility of IgM antibody(ies) in patient’s serum against antigens on donor’s red cells. Only Complete, saline active, cold antibodies will be detected.

20. Method Label 1 tube for each donor sample to be tested. Put 2 drop of patient’s serum in labeled tube. Add 1 drop of 2-5% saline suspended red cells of donor Mix and incubate for 5-10 min. (spin method) or incubate for 30-60 min (sedimentation method) at RT. Centrifuge at 1000 rpm for 1 min. in spin method (after 5-10 min. incubation);centrifugation is optional in sedimentation method.

21. Read the result, observe for hemolysis and agglutination. Negative result should be confirmed under microscope. Interpretation Agglutination or hemolysis indicates a positive result (incompatible) Note: In emergency spin technique is acceptable. Saline technique is inadequate as a complete compatibility test because it is inadequate to detect clinically significant IgG antibodies.

22. Is method In Immediate Spin method ,the patient ’ s serum with donor cell are centrifuged at 500-1000 rpm for 2 minutes,Incubate at 37degrees for 60 minutes

23. Albumin tube method Set up the tubes as in other methods and incubate the cells for 60 minutes at 37 Allow a drop of BSA to each of the tube so that it forms a layer between the cells and the serum Incubate for an additional 30 mins Look for agglutination microscopically.

24. Coomb’s cross match The procedure begin in the same manner as the IS cross-match, continues to 37 C incubation and cells are washed thrice in saline and combos serum is added, Centrifuged at 500-1000 rpm for 2 minutes.

25. Minor cross match -procedure Label a test tube with donor number and recipient's initials. Add one drop of 2-5% suspension Recipient cells. Add 2 drops of Donor serum and 1 drop of 22% bovine albumin to the tube. Centrifuge immediately 1 min at 1000 rpm. Read macroscopically for Haemolysis and agglutination. Incubate at 37o C for 30 minutes. Centrifuge 1 min at 1000 rpm.

26. Read macroscopically for Haemolysis and agglutination. Wash the tube 3 times with saline. Add 2 drops of anti human globulin serum to the dry cell button. Centrifuge 1 min at 1000 rpm. Read macroscopically for Haemolysis and agglutination. Add Check Cells to all negative tests; spin, read and record results.

27. Factors leading to false results Auto-agglutination Cold antibodies Bacterial contamination Drying

28. Questions??