Pretranfusion Compatibility Testing

1. Pretranfusion Compatibility Testing Mr. Mohammed A. Jaber

2. Blood Transfusion Process • Pre-transfusion • Transfusion • Post-transfusion

3. What is compatibility testing? • Also called pretransfusion testing • Purpose: • 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

4. Compatibility testing? • There are several components of compatibility testing • Proper specimen collection • Reviewing patient transfusion history • ABO, Rh, and antibody testing (screen/ID) • Crossmatching • Actual transfusion

5. Compatibility testing • Can be divided into 3 categories: • Preanalytical procedures • Serological testing • Postanalytical procedures

6. Pre-analytical phases • Patient identification • Specimen collection • Review of patient history

7. Patient Identification • Must confirm recipient’s ID from bracelet ON the patient • Full patient name and hospital number • Name of physician

8. Sample Identification • 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

9. Specimen Tubes Pink Top - EDTA Red Top – no additives

10. Specimen Collection • Collected in tube with EDTA or no additives • If the venipuncture causes hemolysis, the sample may be rejected • True hemolysis in the patient is the result of complement activation • 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

11. 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 hoursor else complement dependent antibodies may be missed (complement can become unstable)

12. Getting the history • Look at recipient’s records for any prior unexpected antibodies • Previous transfusion reactions

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

14. ABO/Rh Typing • In the ABO typing, the forward and reverse MUST match • In the Rh typing, the control must be negative • Both of these will indicate what type of blood should be given

15. Antibody screen and/or ID • 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 • If an antibody is present, units negative for the antigen must be given • Proceed to the crossmatch…

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

17. Crossmatch • Two types of crossmatches • Major – routinely performed in labs • Minor – not required by AABB since 1976

18. Major vs Minor Crossmatch • Why is the minor crossmatch unnecessary? • Donated units are tested for antibodies • Most blood is transfused as packed cells, having little antibodies • The plasma volume is small, and Abs will be diluted in recipient circulation

19. Crossmatches The crossmatch “shall use methods that demonstrate ABO incompatibility and clinically significant antibodies to red cell antigens and shall include an antiglobulin phase”

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

21. The procedure • Donor cells are taken from segmentsthat are attached to the unit itself • Segments are a sampling of the blood and eliminate having to open the actual unit

22. Units of whole blood with segments attached

23. Procedure • ABO/Rh typing is FIRST performed • Antibody Screen is performed next….

24. Crossmatch Procedure • if antibodies are NOT detected: • Only immediate spin (IS) is performed using patient serum and donor blood suspension • This fulfills the AABB standard for ABO incompatibility • This is an INCOMPLETE CROSSMATCH • If antibodies ARE detected: • Antigen negative units found and X-matched • All phases are tested: IS, 37°, AHG • This is a COMPLETE CROSSMATCH

26. Crossmatches… Will Verify donor cell ABO compatibility Detect most antibodies against donor cells Will Not Guarantee normal survival of RBCs Prevent patient from developing an antibody Detect all antibodies Prevent delayed transfusion reactions Detect ABO/Rh errors

27. Incompatible crossmatches

28. Additional Information on Types of Compatibility Tests • Manual (IS and IAT) • Gel Technology • Electronic (Computerized) Cross match • Red cell Affinity Column Technology (ReACT) • Solid Phase Adherence Assays (SPAA)

29. Manual (IS and IAT) • IS detect RT reactive antibodies (Auto, Alloantibody, Naturally occuring) • IAT detect IgG antibodies (Auto & alloantibody) Antibody Acquired Naturally occuring (Cold agglutinin) Alloantibody Autoantibody

30. Gel Technology • Patient serum, and 1% of suspended RBCs in LIM are dispensed into the microtube and incubated at 37oC for 15 minutes. • The card containing the microtubes is then centrifuged at a controlled speed for 10 minutes. • At the start of centrifugation the cells are separated from the serum; then they meet the AHG contained in the microtube. • Finally the cells are trapped by the gel (if agglutinated) or pellet to the bottom of the tube.

31. New Technologies… • The electronic crossmatch • According to the AABB, the following must be fulfilled: • Critical elements of the information system have been validated on-site. • No clinically significant antibodies are detected in the current blood sample and there is no record of clinically significant antibodies in the past

32. Computer crossmatch (cont’d) • The patient's ABO group and Rh type has been done twice and entered in the computer • The donor ABO/Rh have been confirmed and entered in the computer. The donor unit identification number, component name, and ABO/Rh type must also be entered in the computer • The computer system will alert the technologist to ABO & Rh discrepancies between information on the donor label and results of donor confirmatory testing

33. Red Cell Affinity Column Technology (ReACT) • Based on affinity adherence of coated red cells in an immunologically active matrix. • Antibody- sensitized red cells bind or adsorbed to ligands attached to an agarose matrix. • The main ligand is Protein G (prepared from Group C or G Streptococcus or by recombinant technology), which has high affinity for all four IgG subclasses. • Another ReACT ligand is Protein A (from Group A Staphlococcus), which binds to IgG 1, 2, and 4.

34. Red Cell Affinity Column Technology (ReACT) • Positive reaction: the coated red blood cells with IgG are boud to immunoreactive gel particles, occurs mostly at the top of the gel column. • Negative reaction: the red blood cells are not coated with antibody and pass through to the bottom of the gel column.

35. Solid Phase Adherence Assays (SPAA) • Uses red cell membrane bound to the surfaces of polystyrene microtitration strip wells, capturing IgG antibodies (if present) in patient sera. • Patient serum is added to wells coated with screen cells • Incubated at 37oC for 15 min. • Washing • anti-IgG-coated indicator red cells are added. • centrifuge

36. SPAA • Result: • Positive • Negative dispersed cells indicator cells forming distinct ring

37. Post-analytical phase • Involves labeling, inspecting, and issuing the blood unit • Labeling form includes patient’s full name, ID number, Location, ABO/Rh(D) of patient and unit, donor #, compatibility results, and tech ID • Form is attached to the donor unit and only released for the recipient • The unit is visually inspected for abnormalities, such as bacterial contamination, clots, etc

38. Issuing blood • When it’s time to release a blood product to the nurse or physician, a few “checks” must be done • Requisition form • Comparing requisition form  donor unit tag  blood product label • Name of persons issuing and picking up blood • Date and time of release • Expiration date

39. What if the unit is unused? • Blood can be returned to the blood bank if it is not needed for transfusion • Unit closure has to remain unopened • Storage temperature must have remained in the required range (1° to 10°C for RBCs) • If not at correct temp, unit must be returned within 30 minutes of issue

40. Special Circumstances

41. Emergency Release • In an emergency, there may not be enough time to test the recipient’s sample • In this case, blood is released only when signed by the physician (O negative) • The tag must indicate it is not crossmatched • Segments from the released units should be retained for X-matching • Every detail is documented (names, dates..)

42. Emergency Release • Once the specimen is received, ABO/Rh typing and antibody screening should be performed • Crossmatching the segments from the released unit should be tested • In addition, the lab may crossmatch additional units as a precaution if more blood is needed • If death should occur, testing should be complete enough to show that the death was unrelated to an incompatibility

43. What can be given in an emergency? • Group O Rh(D)-negative red cells or AB plasma • Emergency release • Women below or of childbearing age • Group O Rh(D)-positive red cells • Used as a substitution if O negative is not available • Male or elderly females

44. Massive transfusion • Defined as a transfusion approaching or exceeding the recipient’s own blood volume (about 5 liters or 10-12 units in an adult male) within 24 hour period • The original sample no longer represents the patient’s condition • Complete crossmatch not necessary (if no antibodies were detected originally) • Give ABO identical units • If antibodies were originally ID’s, continue to give antigen negative units

45. Donor Selection: Appropriate donor units to give • ABO specific blood should always be given first. • When ABO-specific blood is not available or is in less than adequate supply, alternative blood groups are chosen as summarized in the following table; (must be administered as red blood cells).

46. Selection of Appropriate Donor Units. • Rh-negative blood can be given to Rh-positive patients, however, good inventory management should conserve this limited resource for use in Rh-neg recipients. • If Rh-neg units is near expiration, the unit should be given rather than wasted.

47. Selection of Appropriate Donor Units. • Rh-pos blood should not be given to Rh(D) -neg women of childbearing age. • Transfusion of Rh-neg male patients and female patients beyond menopause with Rh-pos blood is acceptable as long as no performed anti-D is demonstrable in the sera.

48. 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. • Saline technique • Saline technique is designed to detect compatibility of IgM antibody(ies) in patient’s serum against antigens on donor’sredcells.

49. 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.

50. Read the result, observe for hemolysis and agglulination. • 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.