1. Unit 8 Pretransfusion Testing Part 2
Terry Kotrla, MS, MT(ASCP)BB

2. Compatibility Testing - History
Early 1980’s started to question utility of:
Routine use of anti-A,B and A2 cells in ABO grouping
Repeat D typing of D positive donor units
Weak D testing
Repeat antibody screen on donor units
DAT testing
Performance of elutions
Significance of antibodies reactive at RT or below.
Usefulness of albumin in antibody detection tests
Use of AHG in both antibody screen AND crossmatch

3. Compatibility Testing - History
During FDA and AABB allowed the AHG phase of the CROSSMATCH to be deleted if the patient’s antibody screen was negative.
In 1984 Judd recommended deleting the autocontrol as part of routine pretransfusion testing.
By 1986 the minor crossmatch was of historic interest only.

4. Compatibility Testing – Coomb’s Crossmatch
Who needs a crossmatch? Patients who are:
experiencing clinical signs and symptoms of anemia.
actively bleeding.
having a surgical procedure where possibility of excessive bleeding is high.
What is the “major” crossmatch
Patient serum/plasma added to donor cells
Read at three phases: IS, 37C and AHG.
Set up and read as part of antibody screen procedure
Agglutination and/or hemolysis are positive.
Donor cells reacting with patient sample at 37C, AHG or causing hemolysis are “incompatible” CANNOT be transfused.

5. Compatibility Testing - IS
When NO CLINICALLY SIGNIFICANT antibodies are detected in the antibody screen AND there is no history of antibodies, the AHG phase of testing is NOT required.
Rarely are AHG incompatible crossmatches obtained when antibody screen is negative.
MUST demonstrate ABO incompatibility by performing IS crossmatch.
Policy change requires medical director approval.

6. Compatibility Testing - IS
Decision to omit AHG phase based on the following:
Incidence of incompatible crossmatches when antibody screen is negative and the reason.
Sensitivity of antibody detection procedure used.
Benefits of omitting AHG phase in the laboratory.
Expertise of individuals working in the transfusion service.
If clinically significant antibodies are present the AHG phase of the crossmatch is required.

7. Compatibility Testing - Computer
Antibody screen negative and computer validated on site to prevent release of ABO-incompatible blood it may be used to detect ABO compatibility instead of serologic testing.
Following conditions MUST be met:
TWO determinations of patients ABO group by: second type on same sample OR second current sample, or comparing to previous records.
Donor ABO/D type, unit number, component name and confirmatory type.
Patient ABO/D type, antibody screen result and two unique patient identifiers.
Method to ensure correct data entry.
Computer logic to alert to ABO/D discrepancies on unit label and testing and ABO incompatibility between recipient and donor.

8. Optional Pretransfusion Testing
ABO Grouping
Testing RBCs with anti-A,B
Serum/plasma tested against A2 cells
D typing
Weak D test
Rh control with chemically modified reagents unless AB pos
Antibody Screen (IAT)
RT incubation
Additives such as albumin or LISS
Enzymes
Polyspecific AHG in IAT

9. Optional Pretransfusion Testing
Autocontrol or DAT
Published data indicate performance is of limited value even in recently transfused patients.
Standards does not require autocontrol or DAT
Microscopic reading of tests, magnifier viewing lamp adequate.
Crossmatch
37C and AHG when antibody screen and history is negative.
RT incubation
Enzyme tests
Polyspecific AHG
Minor crossmatch

10. Selection of Donor Group
When possible ABO identical.
D positive should be selected for D pos, although D neg is acceptable but should be reserved for D neg except:
D neg short date unit can be given to D pos “sure give”.
Multiple antibodies present and D neg more likely to lack.
D negative should be selected for D neg to avoid immunization to D antigen.
Consult with medical director and patient’s physician if need is urgent.
Use D negative first.
Weigh risk of patient death versus immunization to D.
May be appropriate to administer Rh immune globulin especially after platelet transfusion.

11. Selection of Donor Group
Recipient ABO
Compatible Donor ABO In Order of Selection O B
B, O A
A, O AB
AB, A, O, B – why O before B??

12. Blood Administered after Non-Type Specific Transfusion
Determine presence of anti-A and/or anti-B in patient.
When serum from freshly drawn sample is compatible at AHG with recipient’s own blood group may return to group specific.
If AHG incompatible must continue with alternative ABO group.
If change involved D only return to D type specific.

13. Other Blood Groups
Unnecessary to select units based on other blood groups UNLESS patient has clinically significant unexpected antibody.
If antibody strongly reactive use patient serum to screen then confirm with specific typing sera.
Weakly reactive screen units with specific typing sera.
If commercially prepared typing sera is not available use patient sample or plasma from donor with antibody.

14. Antibody Detection Techniques
Use 2 to 3 commercially prepared group O cells.
Relatively short shelf life, two weeks.
Antigen profile (antigram) provided with analysis of antigens present on each cell.
MAKE SURE lot number of screen cell matches antigram.
Add patient serum/plasma to screen cells and observe at:
RT/IS
After incubation at 37C with enhancement media.
After washing and addition of AHG reagent.
Agglutination and/or hemolysis is POSITIVE.
Phase of reactivity of positive reaction extremely helpful.

15. Antibody Detection Techniques
Antibody detection procedure used determined by what is considered “significant” antibody.
Carefully considered if AHG crossmatch not performed.
Once adopted method written in SOP and must be followed exactly by all staff members.
Detection method chosen should:
Detect as many clinically significant antibodies as possible.
NOT detect clinically insignificant antibodies.
Allow prompt delivery of blood to the patient.

16. Antibody Detection Techniques
Method should be sufficiently sensitive to detect low level of antibody in patient serum or plasma.
Undetected low levels of patient antibody may result in rapid production of antibody if antigen positive cells transfused.
Antibody present in donor plasma will not harm recipient.
Methods for antibody screen and crossmatch may be the same or different.
RT tests such as IS crossmatch required to detect ABO incompatibilities, may not be required for antibody screen.
Antibody screen MUST include AHG to detect clinically significant antibodies, crossmatch may be IS only.

17. Antibody Detection Techniques
Lab personnel should use same interpretations, notations and consistency in grading reactions.
Consistency in grading reactions crucial.
Hemolysis and/or agglutination constitutes visible endpoint of antigen-antibody reaction and must be observed accurately and consistently.
Use light source or optical aid to enhance sensitivity and consistency.
Microscopic observation is not required but is useful to
Distinguish rouleaux from true agglutination
Detect mixed field agglutination seen in anti-Sda.

18. Antibody Detection Techniques
Reactions must be observed for hemolysis then agglutination IMMEDIATELY after centrifugation.
Manner in which RBCs are dislodged is crucial.
Hold tube at angle so fluid cuts across cell button as tube is tilted.
Reaction is not interpreted until ALL cells resuspended.
Over shaking will result in weak or negative reactions.
Reactions are recorded IMMEDIATELY with tube held in hand in front of column to record in.

19. General Considerations
Labeling tubes
Each tube labeled properly BEFORE use.
Recipient’s initials (or other identifying information) and donor unit number or reagent RBC identification.
System must allow for accurate, rapid labeling.
NEVER rely on the position of a tube in a rack or centrifuge head to identify the contents of the tube.
ALWAYS place tubes in the serofuge head in the order they will be read.
Use the SAME organizational techniques when labeling and arranging tubes in rack to improve organization and speed.

20. General Considerations
Volume of serum or plasma.
Most procedures call for 2 drops.
Research has shown 2 drops provide optimal antibody to antigen ratio.
Some alloantibodies detected only when 3 to 4 drops used.
High variability in delivery in transfer pipettes.
Standardize volumes based on equipment used in your lab.
Low ionic reagents require ration of 2 drops serum/plasma to 2 drops LISS, cannot vary.

21. General Considerations
Cell suspension
RBCs used for crossmatching obtained from sealed segment of original tubing attached to blood container.
Wash once and prepare 2-4% suspension, some workers prefer 2% as it increases sensitivity of the test system.
Best to use weakest suspension that can be observed for agglutination.
Too heavy of a cell suspension can cause weak antibodies to be missed.

22. Testing Techniques – Saline Tube
Simplest to perform.
Mix serum or plasma with saline suspended RBCs, centrifuge and read, incubate at RT or 37C.
Used in crossmatching to detect ABO incompatibility.
In antibody tests used to detect IgM antibodies which react preferentially at RT: anti-M, -N, -P1, -Le and –I.
Rare examples of antibodies of other specificities may be observed at RT but more often will be reactive at 37C and/or AHG as well.

23. Testing Techniques – Bovine Albumin Tube
Utilized to enhance agglutination of IgG antibodies since
Decreases amount of time required for incubation.
Controversy: Decrease zeta potential (affects second stage of agglutination) or due to function of ionic strength of albumin diluent does it increase uptake of antibody onto cells?
Many antibodies have enhanced reactivity when albumin is added to test system.

24. Testing Techniques – LISS Tube
Low Ionic Strength Saline shortens incubation time.
Increases antibody uptake onto cell, enhancing agglutination.
Several important factors to consider:
Incubation time and sensitivity subsequent to AHG depends upon desired ionic conditions.
Adding additional serum will increase ionic strength, must not be done.
MUST adhere to manufacturer’s instructions.

25. Testing Techniques – PEG Tube
Polyethylene Glycol (PEG) is a water soluble, neutral polymer which is an effective potentiator of antigen-antibody reactions.
Advantages over albumin include:
Increases rate of detection of clinically significant antibodies.
Decreases detection of clinically insignificant antibodies.
May decrease need for other enhancement techniques.
Procedure
Serum or plasma added to RBCs, perform IS.
Add PEG and incubate at 37C – IS NOT READ AFTER 37C
Wash and add AHG.

26. Testing Techniques – Enzymes Tube
More appropriate for antibody ID than routine testing.
GREATLY enhance reactivity of Rh antibodies.
CANNOT be only method used as M, N, S, Fy and other antigens are destroyed, those antibody specificities would not be detected.
Enzymes used include
Papain
Bromelain
Trypsin
Ficin – MOST POPULAR

27. References AABB Technical Manual, 16th edition, 2008
CAP Today
Basic & Applied Concepts in Immunohematology, 2nd edition, 2009
Ortho WIRE,