Shawn T. Leonard MT(ASCP) Blood Centers of the Pacific

1. Antibody Exclusion “Crossing-Out” Interpreting Panel Results Shawn T. Leonard MT(ASCP) Blood Centers of the Pacific

2. Antibody Identification Involves YOU as the Expert! • Performing selected tests • Determining the strength & pattern of agglutination • Interpreting results • Selecting suitable blood for the patient

3. Objectives • Define Antibody Exclusion • List advantages of crossing out in Ab Identification • Describe limitations of the cross-out process • Identify helpful patient and reagent information • Discuss AABB Standards on assigning Ab specificity • Recognize homozygous vs. heterozygous expression • Apply recommended rules for crossing out

4. “Antibody Exclusion” – Immunohematology definition Systematic process by which panel results are interpreted to eliminate antibody specificities on the basis of non-reactivity of patient plasma with red cell samples that express the antigen. -AABB Technical Manual 16th edition

5. “Antibody Exclusion” – Immunohematology definition Commonly known as… Crossing-Out

6. Advantages of Crossing-Out Allows accurate ID of antibodies Eliminates additional antibody specificities Provides higher predictive value Supports initial hypothesis Determines additional testing Decreases delay in patient care

7. Crossing Out • A first approach • May not lead directly to an answer • A provisional step • Converging evidence ???

8. Helpful Information • Patient’s phenotype • Ethnicity of the patient • Ethnicity of panel cell donor • Age of the panel cell

9. Recommended rules for Crossing-Out Cross-Out protocol varies by institution

10. What about Ruling-In? To increase the probability of correct identification a p-value of <0.05is observed which gives a 95% confidence interval.

11. AABB IRL Standards “Assign specificity after demonstrating reactivity with at least two antigen-positive reagent red cell samples and non-reactivity with at least two antigen-negative reagent red cell samples.” Standards for Immunohematology Reference Laboratories-6th Edition

12. Recommended rules for Crossing-Out

13. Rule #1 It is best to rule out an antibody specificity on a cell with a double-dose expression • Homozygous alleles - Jka/Jka(Double-Dose) • express Jk(a+b-) • Heterozygous alleles - Jka/Jkb(Single-Dose) • express Jk(a+b+)

14. Rule #1 • It is best to rule out an antibody specificity on a cell with a double-dose expression • Exceptions… • K1 and P1 • C or E with Anti-D

15. Rule #2 • It is better to rule out specificities with two unique cells rather than one • By chance, one may not react as well as expected due to: • Variant antigen structure • Lack of antigen recognition by antibody • Weakened antigen expression • Antigen degradation • Tech error

16. Rule #3 “ Reverse of Rule-out ”- for the antibody that shows extreme variability or reacts in more than one phase of testing • Anti-P1 • Anti-Lea • Anti-Leb

17. Rule #4 Antibody Exclusion alone does not define which antibodies are or are not present • Additional Techniques : • Selected Cells • Chemical Treatment • Proteolytic Enzymes - Papain, Ficin, Bromelin • Sulfhydryl Reagents - DTT & AET • Neutralization • P1 & Lewis

18. When All Else Fails If antibodies to common blood group antigens cannot be ruled out, it may be necessary to consult a Blood Bank Reference Laboratory or provide antigen negative units.

19. Example Panels On the following example panels, try crossing out each blood group antigen with at least one homozygous representation.

20. (Panel A) Example #1

21. Example #2 (Panel A)

22. (Panel B) Example #2

23. (Panel A) Example #3

24. Example #4

25. Example #5

26. Example #5 (RT & P1 Neut.)

27. Example #6

28. Example #7

29. Example #7 (Chemicals)