Transcript Study of MICA in 201 African Americans by multiplexed

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
• 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
0
A3001
A1101
A1102
B1801
B62
B75
B2708
A3002
B5001
B5703
Cw10
B5301
B3501
B76
B4901
B2705
A2403
B6701
B5801
A3402
A2501
B4501
A2901
B5601
B64
A0301
B8201
B7301
B63
B7801
Cw10
A3401
A3303
B4701
B0801
B4201
A2902
A3201
B0702
A6801
A0206
A2402
A6802
A2301
A6601
A3101
Cw9
A0101
Cw0801
A6901
A7401
B4601
B5701
A2601
B5101
A6602
B8101
Cw1802
Cw1601
B60
A4301
B77
B65
B3901
B4402
A0201
B3701
Cw0102
A3601
B61
B5401
B1302
B5501
Cw0202
Cw1402
B71
B72
Cw1701
Cw0501
B4101
A0203
B3801
Cw0702
Cw1502
A3301
B5201
A8001
B4403
Cw0602
B4801
B5901
B5102
Cw1203
Cw0401
Bead MFI
12000
One Lambda SA1 Group 4 Lot 001 Antigen Density
10000
8000
6000
4000
2000
16000
0
DP0101
DP1701
DQ0501
DR0404
DQ0303
DR1303
DP0301
DQ0401
DQ0609
DQ0301
DR1602
DQ0603
DQ0301
DR0101
DQ0301
DQ0301
DR0401
DR1501
DR1601
DQ0303
DQ0601
DP0401
DR1201
DP0901
DQ0402
DR0701
DQ0502
DRB40103
DQ0302
DRB50202
DR1001
DQ0201
DR1301
DR0103
DRB40101
DP1001
DR0102
DQ0604
DP1101
DQ0301
DRB50101
DR0301
DR0901
DR1502
DP1801
DQ0402
DR1101
DR0405
DR0403
DR1202
DR1401
DR0801
DP0501
DP0201
DQ0304
DQ0201
DQ0201
DQ0602
DQ0202
DRB30101
DR0902
DRB30202
DQ0401
DR0402
DQ0201
DP1901
DP1401
DQ0303
DQ0602
DQ0302
DR0302
DP1301
DP0402
DP1501
DP2301
DP0202
Bead MFI
ANTIGEN DENSITY
One Lambda SA2 Group 1 Lot 005 Antigen Density
Bead MFI
14000
12000
10000
8000
6000
4000
2000
0
DR0902
DR0401
DP0401
DR0103
DR0404
DP0402
DR0101
DP1801
DR1001
DP2801
DRB40103
DR0403
DR0402
DR0405
DR1303
DR1501
DP0501
DR1502
DR0102
DR1601
DR17
DR0901
DRB50202
DR1301
DRB50101
DP0201
DR1401
DP0901
DQ0603
DRB40101
DR1503
DR1104
DP0301
DR0701
DQ0609
DP0301
DP1401
DP0101
DR0801
DR1602
DQ0601
DR1101
DQ0401
DQ9
DP1801
DP1801
DP2001
DR18
DQ7
DRB30202
DR1202
DR1201
DQ0501
DP1101
DQ9
DQ8
DP1101
DP0101
DP1901
DP1701
DRB30101
DQ7
DP0301
DP2801
DQ7
DQ0201
DQ0604
DQ7
DQ0602
DP0601
DQ0502
DQ0602
DQ7
DQ0201
DP1301
DP1301
DP1501
DQ9
DP1001
DQ8
DQ8
DQ0402
DQ0202
DP2301
DQ8
DQ0201
DQ0401
DP0202
DQ0201
DQ0402
DP1301
DRB30301
Bead MFI
One Lambda SA2 Group 1 Lot 008 Antigen Density
Bead MFI
12000
10000
8000
6000
4000
2000
Monoclonal antibodies used
for antigen density correction
• HLA Class I: w6/32
• HLA Class II
– HLA-DR:
– HLA-DQ:
– HLA-DP:
L243
SPVL3 (Beckman)
SPM421 (Abcam)
The Reactions of Normal
Human Sera
HLA Class I Beads
HLA Class II Beads
Mean plus 3 SD
99.6 %
Variation in Ranges of Binding of IgG from Normal Human
Sera to SA Class I Beads
NHS Mean plus 3SD
MFI
2X (Mean Plus 3SD)
Cut-off at 1000
MFI
Cut-off at 500 MFI
SA Beads HLA Class I
Variation in Ranges of Binding of IgG from Normal
Human Sera to SA Class II Beads
NHS Mean plus
3SD
2X (Mean
Plus 3SD)
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
1200
1039
1000
+
18.7%
Number of positive
beads
875
800
680
600
588
Class I
Class II
+
15.6%
400
200
0
NMFI
Normalized Ratios
Patients with additional specificities
identified by normalized ratios
Class I
Class II
30%
47%
53%
70%
Additional specificies
No additional specificies
21/30 patient with additional
specificities
Additional specificies
No additional specificies
14/30 patient with additional
specificities
Analysis of consecutive sera for the 21
discrepant patients for class I antibodies
18
Number of patients
16
80.9%
14
52.6%
12
47.6%
10
8
6
19.0%
4
2
0
Normalized MFI
Normalized ratio
POS/POS
0
17
POS/NEG
0
4
NEG/NEG
11
0
NEG/POS
10
0
Analysis of consecutive sera for the 14
discrepant patients for class II antibodies
10
9
64.3%
57.1%
Number of patients
8
7
42.8%
6
35.7%
5
4
3
2
1
0
Normalized MFI
Normalized ratio
POS/POS
0
9
POS/NEG
0
5
NEG/NEG
8
0
NEG/POS
6
0
Antibodies detected as positive with normalized
ratio but negative with normalized MFI can cause
a positive crossmatch
Negative ctrl
Patient
Positive ctrl
Normalized
DSA
Ratio
A0203
>2x
A0201
>2x
Cw0304
3x
Negative ctrl
Patient
Positive ctrl
MESF
2062
5738
35455
Normalized
MFI
478
413
450
Delta MESF
3676
33393
Positive Cutoff: Delta MESF of 2250
T CELL FLOW
CYTOMETRY XM
Antibodies detected as positive with normalized
ratio but negative with normalized MFI can cause
a positive crossmatch
Negative ctrl
Patient
DSA
Cw07
Normalized
Ratio
3X
Normalized
MFI
720.5
Positive ctrl
Cw07 is a regraft
antigen.
Negative ctrl
Patient
Positive ctrl
MESF
1022
5430
70013
Delta MESF
4408
68991
Positive Cutoff: Delta MESF of 2169
B CELL FLOW
CYTOMETRY XM
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
Trimmed Mean Fluorescence
Class I Beads sorted by MFI
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 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)
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
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 allografts
in adults and children, Transplantation 84: 738, 2007.