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Single HLA Antigen Bead Data Interpretation: Normalized Ratios Peter Stastny

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

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Single HLA Antigen Bead Data Interpretation: Normalized Ratios Peter Stastny

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  1. Single HLA Antigen Bead DataInterpretation: Normalized RatiosPeter 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

  5. ANTIGEN DENSITY

  6. ANTIGEN DENSITY

  7. ANTIGEN DENSITY

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

  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 1039 + 18.7% 875 588 Number of positive beads + 15.6%

  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% 57.1% 42.8% 35.7%

  21. Antibodies detected as positive with normalized ratio but negative with normalized MFI can cause a positive crossmatch Negative ctrl Patient Positive ctrl T CELL FLOW CYTOMETRY XM

  22. Antibodies detected as positive with normalized ratio but negative with normalized MFI can cause a positive crossmatch Negative ctrl Patient Positive ctrl Cw07 is a regraft antigen. B CELL FLOW CYTOMETRY XM

  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

  28. Analysis of Results obtained with Single Antigen Beads • Antigen density correctionMonoclonal antibodies for HLA class I and class II antigens • Ig binding from normal seraMean + 3SD • Normalized ratiosMFI/(Mean +3SD for each bead)

  29. IgG Antibodies against Donor HLA Antigens

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

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

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

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

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