1. Compatibility Testing
Practical Blood Bank
Compatibility Testing

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
Red Top – no additives
Pink Top - EDTA

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 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)

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. 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
Major: Patient serum crossmatched with donor red cells.
Minor: Donor serum crossmatched with patient red cells. Antibody screen testing on donor samples has replaced the minor crossmatch.

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 Donor RBCs (washed)
Patient serum
Agglutination ~ incompatible

21. 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

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

25. The type and screen consists of ABO/Rh, antibody screen, and a  records check.
This procedure is used most frequently to screen pre-operative or obstetrical patients whose risk of excessive blood loss is minimal. In case of an emergency, where blood is needed for these patients, uncrossmatched ABO and D compatible blood can be released with 99.9% assurance of safety, as long as the patient has no unexpected antibodies.
If the antibody screen is positive the patient is not a T&S candidate, the antibody present in the serum or plasma must be identified and antigen negative donor units must be crossmatched.

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

27. Incompatible crossmatches
Antibody screen
Crossmatch
Cause
Resolution
Positive
Negative
Antibody directed against antigen on screening cell
ID antibody, select antigen negative blood
Antibody directed against antigen on donor cell which may not be on screening cell OR donor unit may have IgG previously attached
ID antibody, select antigen negative blood OR perform DAT on donor unit
Antibodies directed against both screening and donor cells
Antibody ID, select antigen negative blood

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: Immediate Saline
IS detect RT reactive antibodies (Auto, Alloantibody, Naturally occuring)
IAT detect IgG antibodies (Auto & alloantibody)

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.
Low ionic media

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 bound 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

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 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).
Patient’s Type
1st Choice
Other Choices O
None A B AB
A, O, B only one of the three should be used for a given patient
Note that for AB individuals the Second Choice lists group O as the next logical choice. Group B blood is relatively uncommon, you would not wish to deprive group B patients of type specific blood, so it makes more sense to choose group O, which is usually in abundant supply.
For plasma components group O is the universal recipient, since they have all ABO antibodies present all plasma products will be compatible. Group AB is the universal donor for plasma products since they lack all ABO antibodies.

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:
Recipient’s serum.
Donor’s red cells taken from the tube attached to the bag.

49. A-Saline technique Method:
Saline technique is designed to detect compatibility of IgM antibody(ies) in patient’s serum against antigens on donor’s red cells.
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 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 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.

51. 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.

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

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

54. Interpretation
Hemeolysis or agglutination at any stage indicates incompatibility.
Note: Cross-match can be done by two tubes technique for IgM and IgG separately as described above or by one tubes in which donor’ cell and the patient’s serum after step 5 in saline technique is incubated at 37°C for minutes and then do IAT.
In major-cross for IgG antibodies albumin or enzyme or LISS can be used with IAT to increase sensitivity. For techniques see chapter on Antiglobulin Test.

55. Cross Match – Major Compatibility Test:
Label 3 tubes S1, S2 (Saline) and A1 (Albumin).
To each tube add 2 drops of fresh serum from recipient.
To each Tube add 2 drops of 5% saline suspension of donor's Cells.
To tube A1 add 2 drops of Bovine Albumin (22%).
Centrifuge both tubes S1 and A1 for 15 seconds at rpm.
Read Macroscopically for Haemolysis and/or agglutination and record results.
ABO incompatibility may be detected in this phase.

56. Incubate the Tube S1 at room temperature for 15 min (Optional).
Incubate the Tube S2 and A1 in the water bath for 30 min at 37o C.
When the incubation time finished centrifuge the tube/tubes for 15 second at 3400 rpm.
Read the tube/tubes macroscopically for Haemolysis and/or agglutination and record results.
Wash Tube A1 with saline 3 times.
Add drops of Anti Human Globulin serum and mix well.
Centrifuge tube A for 15 second at 3400.
Read for agglutination and record the results. 

57. Interpretation:
If no agglutination of Haemolysis is present in corssmatch procedure, the blood is regarded compatible and reported as crossmatch Negative.

58. Cross Match – Minor Compatibility Test:
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.
Wash the tube 3 times with saline.
Add 2 drops of anti human globulin serum to the dry cell button.
Add Check Cells to all negative tests; spin, read and record results.

59. Interpretation:
If no agglutination of Haemolysis is present in corssmatch procedure, the blood is regarded as serological compatible and reported as crossmatch Negative.

60. The Incompatible Crossmatch:
Although the majority crossmatches will indicates compatibility, problems still occur. Even if an incompatible is detected before crossmatch has been carried to the anti-globin stage, the procedure should be completed for investigational purpose. If blood is urgently needed, additional donor blood should be crossmatched before starting to investigate the problem.
Rather than continuing to crossmatch blindly, it is always advisable to try to determine the cause of the incompatibility. However, in emergency situations, it may be necessary to crossmatch many units of blood of appropriate ABO group and Rh Type, in the hope that a compatible unit will be found. In addition, the patient's blood relatives should be tested for compatibility since there is an increased chance of finding suitable donors among them.
The antibody should be identified, not only for the present transfusion, but also to protect the patient in any future transfusions when the antibody titer may have decreased or even disappeared.

61. The following Questions and Answers will help guide subsequent investigations:
Identification:
Is the "Patient's Blood Specimen" really from the intended recipient?
Was a unit of blood with the correct ABO group and Rh Type Selected?
Does both the blood unit pilot tube have the same identification?
ABO grouping:
Recheck recipient and donor from original specimens using freshly prepared red cell suspensions. If anti-A1 or anti-H is identified, blood for transfusion should be selected on the basis of A subgroup.
Rh Type:
Recheck recipient and donor, determination of the Rh phenotype may be helpful in some cases.
Auto Control:
Test Patients serum with his own cells to determine if the problems are due to blood group isoantibody, autoantibody, or nonspecific reaction. This control should be run concurrently with crossmatch or antibody identification.

62. Stage of apparent incompatibility
Stage of incompatibility:
Procedure will be determined to a large extent by the stage at which the incompatibility is most pronounced, as suggested by the following table.
If some donors are incompatible in an early stage of the crossmatch but other donors are not incompatible until a later stage, this might indicate two or more antibodies.
Stage of apparent incompatibility
Possible Cause
Saline or serum at RT
ABO Error.
Cold autoagglutinin or irregular agglutinin
Saline, Serum or High protein at 37o C
Irregular Antibody
Autoagglutinin
Rouleaux
Other serum direct Antiglobulin test
Antiglobulin or Enzyme
Autoantibody
Positive Direct Antiglobulin test

63. Percentage of incompatible donors:
The approximate percentage of incompatible donors may help in elucidate the problem, for example, with an antibody reaction n the Antiglobulin phase: six or seven bloods positive out of 10 bloods tested suggests anti- Fya; one blood positive out of 10 tested suggests anti-K.
Grading donor reactions:
Are the reactions of incompatible bloods all of the same strength? If not
There may be two or more antibodies of varying strength.
The antibody may be exhibiting a dosage phenomenon.
What was the patient diagnosis?
Is the direct Antiglobulin test of either recipient or donor positive?
If recipient has a positive direct Antiglobulin tests:
Serum may or may not contain autoantibody. If an autoantibody is present, the serum may react with all donor samples tested. The technique by which the incompatibility is detected depends upon the type of antibody (cold or warm).
All minor crossmatches will be incompatible.
If the recipient has been recently transfused, the positive Antiglobulin test may indicate incompatibility of infused donor red cells, specially if the appearance is that of a mixed filed reaction

64. If donor has a positive direct Antiglobulin test.
Major Crossmatch will be incompatible.
Minor crossmatch may or may not be incompatible.
Other donor units will crossmatch satisfactorily.
Abnormal proteins, autoagglutinin and cold agglutinins. Factors relating to disease or medication may cause agglutination or pseudo agglutination.
If Rouleaux occurs:
Check patient's diagnosis and serum protein level.
Autologous red cells and serum at 22o C and 37o C should give the same reactions as in the compatibility test.
Compatibility testing with strong Rouleaux, the saline anti-globulin crossmatch may be the only reliable test since the Antiglobulin reactions is not affected by properties of serum that cause Rouleaux. High protein techniques are affected.

65. Cold agglutinins are the most common cause of difficulty in compatibility testing. Although they react best at 4o C, they may cause agglutination in the room temperature phase of the crossmatch and on immediate centrifugation of the high protein test. They also may cause a positive Antiglobulin test, especially in autoimmune disease.
Strong cold autoagglutinin, especially those with wide thermal amplitude, must be absorbed from the patient's serum since they may mask the presence of specific blood group antibodies. If the autoantibody is active at 22o C or lower, it can usually be removed from the serum by placing a fresh recipient blood specimen in ice and allowing it to clot in the refrigerator.
After the cold active antibody is adsorbed onto autologous red cell, the absorbed serum is used for antibody detection and compatibility testing. A suspension of the red cells (For Control) should be prepared from blood that has not been refrigerated.

66. Does the serum contain irregular antibodies?
Test with reagent blood cells (DiaCell), if this has not been done as part of the compatibility test, identify any antibodies present.
If the crossmatch is incompatible only with one donor, and antibody detection tests are negative, the recipient's serum should be tested for antibodies directed against low-incidence antigens.
Technical Causes of apparent incompatibility (False Positive):
Dirty Glassware
Bacterial Contamination.
Chemical or other contamination or reagents, including saline.
Fibrin clots.
Over-centrifugation.  

67. What to Do With Crossmatch Clues

68. Crossmatch for Newborns and infants:
In case of Erythroblastosis:
The corssmatch should include a crossmatch with the mother's serum. If mother's serum is not available crossmatch with baby's serum.
In ABO incompatibility: choose blood compatible with mother's or group O cells suspended in-group specific plasma. Perform major and minor crossmatch.
In Rh incompatibility: Mother and infant are of the same blood group, transfer with compatible group specific Rh Negative
In Rh incompatibility: Mother and infant are of different blood group, choose O Rh Negative cells suspended in-group specific fresh plasma.
In Erythroblastosis due to (c): use blood which is c/c also Rho(D).
In case transfusion is to be repeated, use the same group and method as for the first transfusion
In Case of No Erythroblastosis:
When Infant's RBC is compatible with mother's serum; do crossmatch with mother's serum.
When infant's RBC's are incompatible with mother's serum use infant's serum for crossmatch.

69. Selection of Blood for Exchange transfusion