1. Compatibility Testing
Ahmad Shihada Silmi Msc, FIBMS
IUG

2. 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

3. 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

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

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

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

7. 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

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

9. 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

10. 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)

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

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

13. 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

14. 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…

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

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

17. History

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
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 AABB and FDA develop the standards for blood banking
According to the AABB Standards:
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 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

26. Crossmatches… Will Will Not 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
Antibody screen
Crossmatch
Cause
Resolution
Pos
Neg
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) Antibody Acquired Naturally occuring
(Cold agglutinin)
Alloantibody
Autoantibody
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. X Match Phases PHASE PURPOSE DETECTS IS detection IgM cold agglutinin
clinically insignificant such as anti- M, -N, -Lea, -Leb, and -I.
PHASE
PURPOSE
DETECTS IS
detection IgM cold agglutinin
Most ABO incompatibilities
37oC, LISS
IgG Abs
Potent Rh Abs
AHG
Rh, Duffy, Kidd, others
OCC
AHG absence, inadequacy, or neutralization
Sensitized RBCs
This phase is usually read only macroscopically
for agglutination
This phase is read microscopically for agglutination `
added to all negative tests and must produce a positive result

32. Limitations of Pretransfusion Testing
Hemolytic transfusion reaction, if the patient's antibody is too weak to be detected.
Standard antibody detection methods such as the indirect antiglobulin test require several 100 antibody molecules per red cell to produce detectable reactions.
A hemolytic transfusion reaction due to patient misidentification. For example, group A red cells (meant for transfusion to a group O recipient) will be compatible in vitro tests with an incorrect specimen drawn from a group A person.

33. Limitations of Pretransfusion Testing
Hemolytic transfusion reaction if donor red cells are inadvertently hemolysed before entering the patient, e.g., red cells hemolysed by an improperly functioning blood warmer or red cells hemolysed by contact with an ice pack in a transport container.
Nonhemolytic transfusion reactions such as allergic, febrile, and other reactions.
Pretransfusion test are meant to detect only red cell antibodies.

34. Incompatible cross match
ABO incompatibility
Recheck patient and blood unit ABO group
Clinically Significant Ab
DAT & IAT

35. Antibody Detection

36. IAT IAT is used to detect clinically significant
IgG antibodies bound to RBCS in vitro.
Detection of free Abs in patient's serum.
Clinically significant IgG Abs cause hemolysis or reduce survival of transfused RBCs, like D, c, E, K, k, JKa, JKb, FYa, FYb , S, s.
Unimportant Abs: I, P1, Lea, Leb, M, N.
Those Abs are Unimportant due to
Mostly cold reactive (IgM)
Could be neutralized

37. IAT is principally used for Ab detection
Ab investigation (identification)
Ab titration
Compatibility testing
Phenotyping for some RBCs antigens

38. Reagent used in IAT Characteristics of RBCs reagent:
Three cell reagent sets, Serocyte I, II, III.
RBCs antigens corresponding to important and clinically significant Abs.
The reagent cells are suspended in saline and 2%-5% antibiotic.
Expired reagent should not be used.
An antigram defining phenotype of reagent cell must included with every cell.
All reagent should be stored at 1-6oC.

39. Performance IAT Most clinically significant Abs react at 37oC,
RT and IS are not required.
AHG is usually monospecific IgG
No need for polyspecific AHG.
Because
Anti C will detect Abs during the incubation phase.
Anti C enhance the detection of cold agglutinin such as anti I, anti P that are non significant Abs and not important in transfusion.
Delaying transfusion in critical situations.

40. Interpretation Result of IAT
Negative IAT
OCC Positive: Report IAT negative
Positive IAT
Perform:
Ab identification.
Ab titration.
OCC: O control cells

41. Interpretation Result of IAT
Low titer, and weak reactive Abs are failed to be detected
Using commercial reagents include RBCs that express important antigens in double dose, as JK (a + b -), JK Fy and Fy (a + b -), Fy (a + b -) cells.
Avoiding use of LISS
LISS enhance reactivity of cold reactive Abs causing
difficulties in detection clinically significant Abs .
Increase amount of serum to increase amount of Abs.

42. Its not a life threatening situation when an unexpected clinically important Abs to an RBCs Ags in a blood unit is not detected,
Because
The plasma volume is small, and Abs will be diluted in recipient circulation
Unlikely to cause significant destruction to recipient's RBCs.

43. Antibody Identification

44. 0.3% - 2.8% of the population are Ab makers, they produce clinically significant Ab.
Pregnancy and transfusion are the common cause of immunization to RBCs Ags.

45. Serological Properties
Abs of blood groups vary in importance and significance due to serological properties
Temperature phase
37oC reactive Abs as anti D, c, E, K, k, JKa, JKb, FYa, FYb , S, s.
Cold agglutinin as anti Lea, Leb, I, P1, M, N.
Inducing HDN and HTR
Activation complement.
Neutralizing by soluble blood group Ags

46. Problems Encountered Autoantibody directed against own cell Ags
solution
Elution
IgG Abs can be dissociated from RBCs membrane Ags by physical or chemical means, some procedures destroy membrane, some leave it intact.
Supernatant fluid (elute) contain the autoantibodies which can be identified..

47. Problems Encountered Combination of auto and alloantibody
solution
Treatment with ZZAP (Adsorption)
ZZAP effectively removes autoantibodies (digestion) allowing more complete absorption of autoantibodies from patient's serum, allowing detection and identification of alloantibody.
Chemically treated allogenic RBCs
Treated RBCS with proteolytic enzymes, alter some Ags

48. Problems Encountered
Weak or non reacted with any or some cell reagent.
Variable reaction
Variable expression of Ags (P1, Lewis)
Ags deteriorate on storage (Duffy, P1, M, Lewis)
Abs is reacting with more than one Ag.
DDCCee
Ddccee
Solution
Use double dose of corresponding Ags, (Rh, Kidd,
Duffy, MNS)
Use new cell reagent

49. Post-analytical phase

50. Post-analytical phase
Involves labeling, inspecting, and issuing the blood unit
Labeling form includes patient’s full name, ID number, ABO/Rh 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

51. Bacterial contamination
This unit shows bacterial contamination and should NOT be given to the patient
The plasma in the segments is fine, but the plasma in the unit shows heavy hemolysis from bacteria

52. 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

53. What if the unit is unused?
Blood can be returned 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

54. Infusion Device

55. Blood Warmer

56. Special Circumstances

57. Emergency Release
In an emergency (ER or OR), 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 should be retained for X-matching
Every detail is documented (names, dates..)

58. 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

59. What can be given in an emergency?
Group O Rh-negative red cells or AB plasma
Emergency release
Women below or of childbearing age
Or if in doubt
Group O Rh-positive red cells
Used as a substitution
Male or elderly females

60. 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

61. Appropriate units to give
ABO compatible should always be given first
Patient’s Type
1st Choice
Other Choices O
None A B AB
A, B, O
Group O individuals are “universal donors”, they can donate to any blood group because they have no A or B antigens
Group AB individuals are “universal recipients”, they can receive blood from any group because they do not have A or B antibodies

62. Red blood cell compatibility table  
AB+
AB- B+ B- A+ A- O+ O-
Donor
Recipient

63. Plasma compatibility table
Recipient
Donor O A B AB AB    A       B       O            

64. Type & Screen Used to conserve blood inventory
On average, a surgical procedure uses about 1 unit of RBCs, however, many times the units are on “hold” in the lab and will not be needed (reducing inventory)
For this reason, only a type & screen are performed and if any blood is needed, the sample can be retrieved for crossmatching (only the IS phase is required)
If antibodies are identified, then antigen negative blood is reserved or crossmatched

65. Neonatal Transfusion
A neonate who is <4 months old does not have antibodies
ABO/Rh compatible blood is given
However, if clinically significant antibodies are detected, they are usually maternal and antigen negative units are given
Either infant or maternal serum can be used for the crossmatch
Pedipacks are small aliquots of larger units and prepared by the donor facility or hospital lab for infant transfusion

66. Autologous crossmatching
Autologous refers to a donation from the recipient for later use
Special procedures/protocols must be available so that the autologous unit is found and transfused to the recipient
Pretransfusion testing procedures vary

67. Other components
Other components to be given do not need to be crossmatched because they have been thoroughly screened for antibodies
Frozen plasma
Platelet concentrate
Cryoprecipitate
Platelets pheresis
Granulocyte concentrates
Give ABO compatible units

68. 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

69. 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

70. June, 1938