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
Single HLA Antigen Bead DataInterpretation: Normalized Ratios
Peter Stastny
Transplantation Immunology DivisionDepartments of Internal Medicine and Pathology
UT Southwestern Medical CenterDallas, Texas, USA
Determine a Cut-off for each of the Single
Antigen Beads
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.
Antigen Density on the Beads
ANTIGEN DENSITY
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)
The Reactions of Normal Human Sera
HLA Class I Beads
HLA Class II Beads
Mean plus 3 SD
99.6 %
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
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
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.
METHOD
• Analyze two consecutive sera from 30 patients using normalized ratios and fixed 1000 NMFI cutoff and compared the results.
NR vs NMFI: Number of positive beads
875
588
1039+
18.7%
+ 15.6%
Num
ber o
f pos
itive
be
ads
Patients with additional specificities identified by normalized ratios
21/30 patient with additional specificities
14/30 patient with additional specificities
Analysis of consecutive sera for the 21 discrepant patients for class I antibodies
Analysis of consecutive sera for the 14 discrepant patients for class II antibodies
64.3%
35.7%
57.1%
42.8%
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
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.
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.
An early batch from Source A
Class I Beads sorted by MFI
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
Antigen Density with W6/32 A more recent batch from Source A
Normalized Ratios
• MFI/(Mean +3SD) • Weak positive: 2X to 10X• Strong positive: greater than
10X
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)
IgG Antibodies against Donor HLA Antigens
Primary Kidney Allografts
Effect of DSA detected with SA beads when T-cell flow-cytometry crossmatch
was negative.
Heart Transplants
Association of anti-donor HLA antibodies with transplant-related coronary artery disease.
Donor-Specific Antibodies
Effect of antibodies against HLA antigens not present in the graft
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