single hla antigen bead data interpretation: normalized ratios peter stastny
DESCRIPTION
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. - PowerPoint PPT PresentationTRANSCRIPT
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Single HLA Antigen Bead DataInterpretation: Normalized Ratios
Peter Stastny
Transplantation Immunology DivisionDepartments of Internal Medicine and Pathology
UT Southwestern Medical CenterDallas, Texas, USA
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Determine a Cut-off for each of the Single
Antigen Beads
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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.
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Antigen Density on the Beads
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ANTIGEN DENSITY
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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)
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The Reactions of Normal Human Sera
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HLA Class I Beads
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HLA Class II Beads
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Mean plus 3 SD
99.6 %
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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
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Variation in Ranges of Binding of IgG from Normal Human Sera to SA Class II Beads
NHS Mean plus 3SD
2X (Mean Plus 3SD)M
FI
SA Beads HLA Class II
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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.
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METHOD
• Analyze two consecutive sera from 30 patients using normalized ratios and fixed 1000 NMFI cutoff and compared the results.
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NR vs NMFI: Number of positive beads
875
588
1039+
18.7%
+ 15.6%
Num
ber o
f pos
itive
be
ads
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Patients with additional specificities identified by normalized ratios
21/30 patient with additional specificities
14/30 patient with additional specificities
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Analysis of consecutive sera for the 21 discrepant patients for class I antibodies
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Analysis of consecutive sera for the 14 discrepant patients for class II antibodies
64.3%
35.7%
57.1%
42.8%
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Antibodies detected as positive with normalized ratio but negative with normalized MFI can cause
a positive crossmatch
T CELL FLOW CYTOMETRY XM
DSANormalized
RatioNormalized
MFIA0203 >2x 478A0201 >2x 413
Cw0304 3x 450
MESF Delta MESFNegative ctrl 2062 -
Patient 5738 3676Positive ctrl 35455 33393
Positive Cutoff: Delta MESF of 2250
Negative ctrlPatient
Positive ctrl
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Antibodies detected as positive with normalized ratio but negative with normalized MFI can cause
a positive crossmatch
B CELL FLOW CYTOMETRY XM
DSANormalized
RatioNormalized
MFICw07 3X 720.5
MESF Delta MESFNegative ctrl 1022 -
Patient 5430 4408Positive ctrl 70013 68991
Positive Cutoff: Delta MESF of 2169
Negative ctrlPatient
Positive ctrl
Cw07 is a regraft antigen.
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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.
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An early batch from Source A
Class I Beads sorted by MFI
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Antigen Density with W6/32 Source B
Class I Beads sorted by MFI
Trim
med
Mea
n Fl
uore
scen
ce
SA Beads for HLA Class I
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Antigen Density with W6/32 A more recent batch from Source A
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Normalized Ratios
• MFI/(Mean +3SD) • Weak positive: 2X to 10X• Strong positive: greater than
10X
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Analysis of Results obtained with Single Antigen Beads
• Antigen density correctionMonoclonal antibodies for HLA class Iand class II antigens
• Ig binding from normal seraMean + 3SD
• Normalized ratiosMFI/(Mean +3SD for each bead)
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IgG Antibodies against Donor HLA Antigens
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Primary Kidney Allografts
Effect of DSA detected with SA beads when T-cell flow-cytometry crossmatch
was negative.
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Heart Transplants
Association of anti-donor HLA antibodies with transplant-related coronary artery disease.
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Donor-Specific Antibodies
Effect of antibodies against HLA antigens not present in the graft
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From: Stastny et al, Antibodies against donor HLA and the outcome of cardiac allograftsin 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
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