1. Compatibility Testing practical NO 4 Dr: Dalia Kamal Eldien

2. Compatibility Testing Also called pretransfusion 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 Purpose: To select blood components that will not cause harm to the recipient and will have acceptable survival when transfused

3. Compatibility Testing  There are several components of compatibility testing Proper specimen collection Reviewing patient transfusion history ABO, Rh, and antibody testing Crossmatching Actual transfusion

4. Sample Identification The sample should also have the full patient name, hospital number, and physician Date and time of collection All of this should be on the request form and the sample

5. Specimen Collection Collected in tube with EDTA or no additives If the venipuncture causes hemolysis, the sample may be rejected Samples are labeled at the bedside (pre-labeling is not recommended) A record of individuals who collect (or test) the specimens should be documented in order to “backtrack” in case of an error

6. Specimen Tubes Pink Top - EDTARed Top – no additives

7. Specimen Collection 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. Getting the history Look at recipient’s records for any prior unexpected antibodies Previous transfusion reactions

9. Serological Testing 3 tests: ABO/Rh Antibody detection/identification Crossmatch

10. Antibody screening test  The antibody screen will detect the presence of any unexpected antibodies in patient serum  If antibodies are detected, identification should be performed using panel cells (with an autocontrol) IS 37° (LISS) AHG

11. Antibody screening is used to test: 1- Donor plasma to make sure no unexpected antibodies will be transfused. 2-Patient serum before transfusion to make sure patient has no unexpected antibodies to react with donor cells. 3- Maternal serum to make sure pregnant mother has no antibodies to react with fetal cells.

12. Testing of the patient sample  Abs Regarded as always being potentially clinically significant ABO-Rh-Kell-Duffy-Kidd - S s &U  Abs that may sometime be clinically Significant Le a -p - Lu a &Lu b Cartwright.  Abs that rarely, if ever, are clinically significant Le b Chido/Rodgers (Ch a /Rh a ) York, Sd Xg& Bg

13. Procedure for Antibody Screening Antibody Screening will involve various phases to allow for antibody- antigen agglutination. Phase 1: Immediate Spin:. 3 tubes – 1- Recipient serum plus saline suspension Screening Cell I 2-Screening Cell II 3- the recipient's own cells for the auto control. Centrifuge these three tubes and read for agglutination Detects IgM antibodies

14. Phase 2: 37 o C Incubation: 37 o C phase is required since IgG are warm-acting antibodies. Can add enhancement media if desired (LISS or albumin). LISS is Low Ionic Strength Solution composed of NaCl, glycine and phosphate buffer along sodium preservative. This solution speeds up antigen-antibody reaction but unfortunately enhances "nuisance" antibodies, so add after immediate spin step. Albumin is added to lower zeta potential so cells can agglutinate without Coombs step and may detect Rh antibodies. Whether adding an enhancement media or not we must do 37 o C incubation, but we do not need to read at this step. We can proceed directly to Coombs (AHG or AGT) phase.

15. Phase 3: Coombs phase The Coombs phase is required since a number of these clinically significant antibodies may only show up at this phase. After 37 o C incubation, wash the cells 3-4 times Remove the saline and add AHG. Mix and centrifuge Read for agglutination. Add Coombs Control Cells to all negative results to confirm negative reactions. This phase detects IgG antibodies, most of which are considered clinically significant and capable of causing Hemolytic Disease of the Newborn or Hemolytic Transfusion Reactions.

16. Correct ABO grouping results are much more critical to transfusion safety than Ab screening. Most Abs, other than anti-A and anti-B do not cause severe hemolytic transfusion reactions. Thus the vast majority of patients would not suffer grave consequences if transfused with blood from ABO group compatible donor without the benefit of Ab screening tests.

17. Crossmatching Purpose : Prevent transfusion reactions Increase in vivo survival of red cells Double checks for ABO errors Another method of detecting antibodies

18. Crossmatch Two types of crossmatches Major – routinely performed in labs Minor –

19. Crossmatch Donor RBCs (washed) Patient serum No agglutination ~ compatible Agglutination ~ incompatible

20. The procedure Donor cells are taken from segments that are attached to the unit itself Segments are a sampling of the blood and eliminate having to open the actual unit

21. Major Crossmatch Tests  It is done both for IgM and IgG antibodies Requirement: 1. Recipient’s serum. 2. Donor’s red cells taken from the tube attached to the bag.

22. Method 1. Label 1 tube for each donor sample to be tested. 2. Put 2 drop of patient’s serum in labeled tube. 3. Add 1 drop of 2-5% saline suspended red cells of donor 4. Mix and incubate for 5-10 min. (spin method) or incubate for 30-60 min (sedimentation method) at RT. 5. Centrifuge at 1000 rpm for 1 min. in spin method (after 5-10 min. incubation);centrifugation is optional in sedimentation method.

23. 6. Read the result, observe for hemolysis and agglulination. 7. Negative result should be confirmed under microscope. Interpretation Agglutination or hemolysis indicates a positive result (incompatible)

24. Crossmatch Test for IgG Antibody B. Anti -Human Globulin Test (IAT) Indirect anti human globulin test (IAT) is the most important and widely used serological procedure in modern blood banking to test the IgG compatibility between recipient’s serum and donor’s cells. The majority of incomplete antibodies are IgG and are detected by AHG test.

25. Method 1. Put 2 drops of patient’s serum in a labeled tube. 2. Add 1 drop of 2-5 % saline suspended red cells of donor. 3. Incubate for 30-60 min at 37° C 4. Centrifuge at 1000 rpm for 1 min, check for hemolysis/agglutination 5. If there is no hemolysis/agglutination, wash the cells three times with normal saline.

26. 6. Perform IAT test Add 2 drops of polyspecific AHG serum to washed cells Centrifuge at 1000 rpm for 1 minute See for agglutination 7. Add IgG coated red cells to negative AHG test. 8. Centrifuge and check for agglutination - if there is no agglutination test is invalid.

27. Interpretation  Hemeolysis or agglutination at any stage indicates incompatibility.

28. Good luck