1. Single HLA Antigen Bead Data Interpretation: Normalized Ratios Peter Stastny Transplantation Immunology Division Departments of Internal Medicine and Pathology UT Southwestern Medical Center Dallas, Texas, USA

2. Determine a Cut-off for each of the Single Antigen Beads

3. NORMALIZED RATIOS Normalized ratios are a method of expressing the results of SA assays developed at UT Southwestern. Normalized ratios : [(Raw MFI x ag density corr. factor) - NC)]/NHS Mean+3SD Each bead has a different cutoff value. NR of >2x are considered positive. Above 10x strong positive.

4. Antigen Density on the Beads

6. ANTIGEN DENSITY

8. Monoclonal antibodies used for antigen density correction HLA Class I: w6/32 HLA Class II – HLA-DR: L243 – HLA-DQ: SPVL3 (Beckman) – HLA-DP: SPM421 (Abcam)

9. The Reactions of Normal Human Sera

10. HLA Class I Beads

11. HLA Class II Beads

12. Mean plus 3 SD 99.6 %

13. NHS Mean plus 3SD Variation in Ranges of Binding of IgG from Normal Human Sera to SA Class I Beads 2X (Mean Plus 3SD) MFI SA Beads HLA Class I Cut-off at 1000 MFI Cut-off at 500 MFI

14. Variation in Ranges of Binding of IgG from Normal Human Sera to SA Class II Beads NHS Mean plus 3SD 2X (Mean Plus 3SD) MFI SA Beads HLA Class II

15. NORMALIZED MFI No correction for antigen density. No individual cutoff based on NHS panel. Fix 1000 NMFI cutoff. Normalized MFI are obtained by subtracting negative ctrl from raw MFI.

16. METHOD Analyze two consecutive sera from 30 patients using normalized ratios and fixed 1000 NMFI cutoff and compared the results.

17. NR vs NMFI: Number of positive beads 875 588 1039 + 18.7% + 15.6% Number of positive beads

18. Patients with additional specificities identified by normalized ratios 21/30 patient with additional specificities 14/30 patient with additional specificities

19. Analysis of consecutive sera for the 21 discrepant patients for class I antibodies

20. Analysis of consecutive sera for the 14 discrepant patients for class II antibodies 64.3% 35.7% 57.1% 42.8%

21. Antibodies detected as positive with normalized ratio but negative with normalized MFI can cause a positive crossmatch T CELL FLOW CYTOMETRY XM DSA Normalized Ratio Normalized MFI A0203>2x478 A0201>2x413 Cw03043x450 MESFDelta MESF Negative ctrl2062- Patient57383676 Positive ctrl3545533393 Positive Cutoff: Delta MESF of 2250 Negative ctrl Patient Positive ctrl

22. Antibodies detected as positive with normalized ratio but negative with normalized MFI can cause a positive crossmatch B CELL FLOW CYTOMETRY XM DSA Normalized Ratio Normalized MFI Cw073X720.5 MESFDelta MESF Negative ctrl1022- Patient54304408 Positive ctrl7001368991 Positive Cutoff: Delta MESF of 2169 Negative ctrl Patient Positive ctrl Cw 07 is a regraft antigen.

23. CONCLUSIONS Normalized ratios generally identified more weak positive specificities than normalized MFI. Normalized ratios yielded more reproducible results when comparing sequential serum samples than normalized MFI. Normalized ratios has a different cutoff for each bead. Specificities that were identified with normalized ratios but not with normalized MFI could yield a positive crossmatch. Methods used to analyze SA tests can impact the antibody specificities identified.

24. An early batch from Source A Class I Beads sorted by MFI

25. Antigen Density with W6/32 Source B Class I Beads sorted by MFI Trimmed Mean Fluorescence SA Beads for HLA Class I

26. Antigen Density with W6/32 A more recent batch from Source A

27. Normalized Ratios MFI/(Mean +3SD) Weak positive: 2X to 10X Strong positive: greater than 10X

30. Analysis of Results obtained with Single Antigen Beads Antigen density correction Monoclonal antibodies for HLA class I and class II antigens Ig binding from normal sera Mean + 3SD Normalized ratios MFI/(Mean +3SD for each bead)

31. IgG Antibodies against Donor HLA Antigens

32. Primary Kidney Allografts Effect of DSA detected with SA beads when T-cell flow-cytometry crossmatch was negative.

34. Heart Transplants Association of anti-donor HLA antibodies with transplant-related coronary artery disease.

36. Donor-Specific Antibodies Effect of antibodies against HLA antigens not present in the graft

37. From: Stastny et al, Antibodies against donor HLA and the outcome of cardiac allografts in adults and children, Transplantation 84: 738, 2007. Antibodies against HLA antigens not expressed in the graft did not harm the heart transplant in 5 ½ years