hla ab, donor reactivity and risk of rejection and graft loss hla ab, donor reactivity and risk of...
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HLA Ab, Donor Reactivity
and Risk of Rejection
and Graft Loss
HLA Ab, Donor Reactivity
and Risk of Rejection
and Graft LossRonald H. Kerman, PhD
The University of Texas Medical School ~ Houston, TX
Division of Immunology and Organ Transplantation
Ronald H. Kerman, PhD
The University of Texas Medical School ~ Houston, TX
Division of Immunology and Organ Transplantation
Allograft RejectionAllograft RejectionType:
Hyperacute
Accelerated
Acute
Chronic
Type:
Hyperacute
Accelerated
Acute
Chronic
Time:
0-48 hrs
5-7 days
Early/delayed
>60 days
Time:
0-48 hrs
5-7 days
Early/delayed
>60 days
Mediated by:
Abs
Abs/cells
Cells/Abs
Abs/cells/?
Mediated by:
Abs
Abs/cells
Cells/Abs
Abs/cells/?
To identify clinically relevant
recipient IgG HLA antibodies
To identify clinically relevant
recipient IgG HLA antibodies
Responsibilities of the Histocompatibility LaboratoryResponsibilities of the Histocompatibility Laboratory
Positive crossmatches, due to Abs or other factors
not impacting on graft outcome, should not influence
the donor-recipient pairing for transplantation.
Positive crossmatches, due to Abs or other factors
not impacting on graft outcome, should not influence
the donor-recipient pairing for transplantation.
Screen sera for reactivity vs target cells by cytotoxicity/fluorescence readouts.
Use the most informative sera when performing the recipient vs donor
crossmatch (historically most reactive, current and pretransplant sera).
Screen sera for reactivity vs target cells by cytotoxicity/fluorescence readouts.
Use the most informative sera when performing the recipient vs donor
crossmatch (historically most reactive, current and pretransplant sera).
Detection of Recipient SensitizationDetection of Recipient Sensitization
• NIH-CDC
• AHG-CDC
• Flow cytometry
Membrane-dependent assays
• NIH-CDC
• AHG-CDC
• Flow cytometry
Membrane-dependent assays
Detection of Immunoglobulin ReactivityDetection of Immunoglobulin Reactivity
Flow Cytometry AssayNIH - CDC NegativeAHG – CDC NegativeNow measuring binding of IgG (absent C’)
Flow Cytometry AssayNIH - CDC NegativeAHG – CDC NegativeNow measuring binding of IgG (absent C’)
Cadaveric Renal Allograft Survival Among 1o CsA-Pred Recipients at 12 months
Cadaveric Renal Allograft Survival Among 1o CsA-Pred Recipients at 12 months
NIH
Neg.
n=166
81% (134/166)
NIH
Neg.
n=166
81% (134/166)
Kerman et al, Transplantation; 51:316, 1991Kerman et al, Transplantation; 51:316, 1991
AHG
Neg. Pos.
n=151 n=15
82% 67% (124/151) (10/15)
AHG
Neg. Pos.
n=151 n=15
82% 67% (124/151) (10/15)
P<0.01P<0.01
Cadaveric Renal Allograft Survival Among 1o CsA-Pred Recipients at 12 months
Cadaveric Renal Allograft Survival Among 1o CsA-Pred Recipients at 12 months
AHG
Pos.
n=15
67% (10/15)
AHG
Pos.
n=15
67% (10/15)
Kerman et al, Transplantation; 51:316, 1991Kerman et al, Transplantation; 51:316, 1991
DTE-AHG
Neg. Pos.
n=12 n=3
83% 0% (10/12) (0/3)
DTE-AHG
Neg. Pos.
n=12 n=3
83% 0% (10/12) (0/3)
P<0.01P<0.01
Cadaveric Renal Allograft Survival Among 1o CsA-Pred Recipients at 12 months
Cadaveric Renal Allograft Survival Among 1o CsA-Pred Recipients at 12 months
DTE/AHG XM
Neg.
n=166
81%
DTE/AHG XM
Neg.
n=166
81%
Kerman et al, Transplantation; 51:316, 1991Kerman et al, Transplantation; 51:316, 1991
FCXM
Neg. Pos.
n=130 n=36
81% 81%
FCXM
Neg. Pos.
n=130 n=36
81% 81%
Neg-NIH Extended XM: FCXM StudyNeg-NIH Extended XM: FCXM Study
Ogura et al, Transplantation; 56:294, 1993Ogura et al, Transplantation; 56:294, 1993
T-FCXM
Pos.
n=148
75%
T-FCXM
Pos.
n=148
75%
T-FCXM
Neg.
n=693
82%
T-FCXM
Neg.
n=693
82%
P<0.01P<0.01
Could Ron Kerman have been wrong
about his crossmatch results and
interpretation?
Could Ron Kerman have been wrong
about his crossmatch results and
interpretation?
Kerman et al, Transplantation; 68:1855, 1999Kerman et al, Transplantation; 68:1855, 1999
0055
101015152020252530303535404045455050
Negative(n=56)
Negative(n=56)
Positive(n=41)
Positive(n=41)
P=NS P=NS
% R
eje
ctio
n%
Reje
ctio
n
IgG FCXM: Renal Allograft StudyFrequency of Rejection in a Single CenterIgG FCXM: Renal Allograft StudyFrequency of Rejection in a Single Center
Could Ron Kerman have been
wrong about his crossmatch
results and interpretation?
Could Ron Kerman have been
wrong about his crossmatch
results and interpretation?
I don’t think so!I don’t think so!
• NIH-CDC
•AHG-CDC
• Flow cytometry
• NIH-CDC
•AHG-CDC
• Flow cytometry
Membrane-dependent AssaysMembrane-dependent Assays
Detection of membrane receptors may not be related to HLA!
Detection of membrane receptors may not be related to HLA!
ELISA-determined IgG HLA Abs vs MHC-I (pooled platelets)
ELISA-determined IgG HLA Abs vs MHC-I/II (PBL cultures)
Flow bead PRA-determined IgG HLA vs I/II (soluble HLA I/II
antigens on microbeads measured by cytometry)
ELISA-determined IgG HLA Abs vs MHC-I (pooled platelets)
ELISA-determined IgG HLA Abs vs MHC-I/II (PBL cultures)
Flow bead PRA-determined IgG HLA vs I/II (soluble HLA I/II
antigens on microbeads measured by cytometry)
Membrane-independent Assays Membrane-independent Assays
PRA by Different MethodologiesPRA by Different MethodologiesType:
CDC
AHG-CDC
ELISA
Flow
Type:
CDC
AHG-CDC
ELISA
Flow
Positive
102
116
127
139
Positive
102
116
127
139
Negative
162
148
137
125
Negative
162
148
137
125
Gebel & Bray, Transplantation; 69:1370, 2000Gebel & Bray, Transplantation; 69:1370, 2000
AHG-PRA vs Rejection 493 Consecutive CAD Recipients
AHG-PRA vs Rejection 493 Consecutive CAD Recipients
Rejection
YES
NO
Rejection
YES
NO
AHG-PRAAHG-PRA
P=NSP=NS
<10%
134
159
<10%
134
159
10%
100
100
10%
100
100
ELISA-PRA and RejectionELISA-PRA and Rejection
Rejection
YES
NO
Rejection
YES
NO
ELISA-PRAELISA-PRA
P<0.001P<0.001
<10%
38
168
<10%
38
168
10%
117
63
10%
117
63
Correlation Between % ELISA-PRA and Graft SurvivalCorrelation Between % ELISA-PRA and Graft Survival
Graft Survival (months)
12
24
36
Graft Survival (months)
12
24
36
ELISA-PRAELISA-PRA
P<0.01P<0.01
<10% (n=312)
85%
82%
81%
<10% (n=312)
85%
82%
81%
>10% (n=181)
74%
70%
67%
>10% (n=181)
74%
70%
67%
P<0.01P<0.01
P<0.01P<0.01
Sensitivity and sensitization, defining the unsensitized
patient
Application of membrane-independent assays to identify HLA antibodies
Sensitivity and sensitization, defining the unsensitized
patient
Application of membrane-independent assays to identify HLA antibodies
Gebel & Bray, Transplantation; 69:1370, 2000Gebel & Bray, Transplantation; 69:1370, 2000
Tambur et al, Transplantation; 70:1055, 2000Tambur et al, Transplantation; 70:1055, 2000
Correlation of Pre-transplant Abs Detected by Flow PRA with Biopsy-documented Cardiac Rejection
Correlation of Pre-transplant Abs Detected by Flow PRA with Biopsy-documented Cardiac Rejection
Kerman et al, Transplantation; 68:1855, 1999Kerman et al, Transplantation; 68:1855, 1999
0055
101015152020252530303535404045455050
Negative(n=56)
Negative(n=56)
Positive(n=41)
Positive(n=41)
P=NSP=NS
% R
eje
ctio
n%
Reje
ctio
n
IgG FCXM: Renal Allograft StudyFrequency of Rejection in a Single CenterIgG FCXM: Renal Allograft StudyFrequency of Rejection in a Single Center
Were positive crossmatches due to HLA Abs?Were positive crossmatches due to HLA Abs?
• Neoral - CsA
• Steroids
• Prograf - FK506
• Cellcept - MMF
• Rapamycin - Sirolimus
• Thymoglobulin
• OKT3, anti-IL-2R, FTY720
• Neoral - CsA
• Steroids
• Prograf - FK506
• Cellcept - MMF
• Rapamycin - Sirolimus
• Thymoglobulin
• OKT3, anti-IL-2R, FTY720
Immunosuppressive Menu:Immunosuppressive Menu:
If new immunosuppressive therapies reduce
the incidence of acute rejection, are pre-Tx
HLA antibodies clinically relevant?
If new immunosuppressive therapies reduce
the incidence of acute rejection, are pre-Tx
HLA antibodies clinically relevant?
RAPA-CsA-Pred treated primary recipients of CAD renal allografts experience fewer acute rejections vs CsA-Pred recipients.
We therefore tested their pre-Tx sera for the presence of HLA Abs and correlated the results to the occurrence of rejection during the first 12 months post-transplant.
RAPA-CsA-Pred treated primary recipients of CAD renal allografts experience fewer acute rejections vs CsA-Pred recipients.
We therefore tested their pre-Tx sera for the presence of HLA Abs and correlated the results to the occurrence of rejection during the first 12 months post-transplant.
147 RAPA-CsA-Pred recipients were studied
48 patients were chosen specifically because they had a rejection episode.
99 patients were chosen because they had not experienced a rejection episode during the
first year post-transplant.
147 RAPA-CsA-Pred recipients were studied
48 patients were chosen specifically because they had a rejection episode.
99 patients were chosen because they had not experienced a rejection episode during the
first year post-transplant.
PRA Testing
Anti-human globulin (AHG)
ELISA (One Lambda, Inc. LAT)
Flow PRA (One Lambda, Inc.)
PRA Testing
Anti-human globulin (AHG)
ELISA (One Lambda, Inc. LAT)
Flow PRA (One Lambda, Inc.)
AHG-PRA detected 18 reactive sera
ELISA-PRA detected 25 reactive sera (11 vs HLA class I, 3 vs II, 11 vs I/II)
Flow PRA detected 59 reactive sera (31 vs HLA class I, 9 vs II, 19 vs I/II)
AHG-PRA detected 18 reactive sera
ELISA-PRA detected 25 reactive sera (11 vs HLA class I, 3 vs II, 11 vs I/II)
Flow PRA detected 59 reactive sera (31 vs HLA class I, 9 vs II, 19 vs I/II)
Results:Results:
There was no significant correlation between
AHG-PRA, ELISA-detected HLA Abs, and Flow
PRA HLA class II Abs and rejection.
There was no significant correlation between
AHG-PRA, ELISA-detected HLA Abs, and Flow
PRA HLA class II Abs and rejection.• AHG vs Rejection P=NS
• LAT-I vs Rejection P=NS
• LAT-II vs Rejection P=NS
• LAT-I/II vs Rejection P=NS
• F-II vs Rejection P=NS
• AHG vs Rejection P=NS
• LAT-I vs Rejection P=NS
• LAT-II vs Rejection P=NS
• LAT-I/II vs Rejection P=NS
• F-II vs Rejection P=NS
Rejection
NO
YES
Rejection
NO
YES
Flow PRA-1Flow PRA-1
X2=15.7; P<0.001X2=15.7; P<0.001
<5%
76
21
<5%
76
21
5%
23
27
5%
23
27
Flow PRA
0%
Flow PRA
0%
No grafts lost
(+) FCXM vs non-HLA Ab
No grafts lost
(+) FCXM vs non-HLA Ab
Day of 1st Rejection
57 ± 34
Day of 1st Rejection
57 ± 34
FCXMPos. Neg.
2 8
FCXMPos. Neg.
2 8
Flow PRA
13 ± 9%
Flow PRA
13 ± 9%
No grafts lost.No grafts lost.
Day of 1st Rejection
55 ± 31
Day of 1st Rejection
55 ± 31
FCXMPos. Neg.
- 30
FCXMPos. Neg.
- 30
Flow PRA
28 ± 9%
Flow PRA
28 ± 9%
(+) HLA Ab and (-) FCXM: rejection, no grafts lost.
(+) HLA Ab and (+) FCXM: rejection, 58% (7/12) grafts lost.
(+) HLA Ab and (-) FCXM: rejection, no grafts lost.
(+) HLA Ab and (+) FCXM: rejection, 58% (7/12) grafts lost.
Day of 1st Rejection
32 ± 15
Day of 1st Rejection
32 ± 15
FCXMPos. Neg.
12 13
FCXMPos. Neg.
12 13
Flow PRA
48 ± 31%
Flow PRA
48 ± 31%
(+) HLA Ab and (-) FCXM: rejection, no grafts lost.(+) HLA Ab and (+) FCXM: rejection, 63% (5/8)
lost to AMR.
(+) HLA Ab and (-) FCXM: rejection, no grafts lost.(+) HLA Ab and (+) FCXM: rejection, 63% (5/8)
lost to AMR.
Day of 1st Rejection
17 ± 12
Day of 1st Rejection
17 ± 12
FCXMPos. Neg.
8 7
FCXMPos. Neg.
8 7
% PRA
0
13 ± 9
28 ± 9
48 ± 31
% PRA
0
13 ± 9
28 ± 9
48 ± 31
% Rejection
5% (4/75)
13% (4/32)
100%
100%
% Rejection
5% (4/75)
13% (4/32)
100%
100%
Day of Rejection
57 ± 34
55 ± 31
32 ± 9
17 ± 12
Day of Rejection
57 ± 34
55 ± 31
32 ± 9
17 ± 12
N
75
32
25
15
N
75
32
25
15
1. Assays that measure binding of immunoglobulin to targets may not represent HLA Ab reactivity.
2. The AHG-XM protects RAPA-CsA-Pred recipients from hyperacute rejection.
3. The Flow PRA assay detects clinically relevant HLA Abs associated with rejection and/or graft loss.
1. Assays that measure binding of immunoglobulin to targets may not represent HLA Ab reactivity.
2. The AHG-XM protects RAPA-CsA-Pred recipients from hyperacute rejection.
3. The Flow PRA assay detects clinically relevant HLA Abs associated with rejection and/or graft loss.
4. How many antibodies are present may be clinically relevant.
5. The antibody titer may also be important.
6. Patients with pre-Tx (+) HLA Abs and
(+) donor reactivity (+ FCXM) are at risk
for graft rejection and loss.
4. How many antibodies are present may be clinically relevant.
5. The antibody titer may also be important.
6. Patients with pre-Tx (+) HLA Abs and
(+) donor reactivity (+ FCXM) are at risk
for graft rejection and loss.
We have performed heart transplantation following
a negative AHG-XM.
We evaluated the clinical relevance of FCXM for
heart recipients.
We have performed heart transplantation following
a negative AHG-XM.
We evaluated the clinical relevance of FCXM for
heart recipients.
IgG FCXMIgG FCXM
Neg.Neg.
1YGS1YGS 86% 86%
IgG FCXMIgG FCXM
Neg.Neg.
1YGS1YGS 86% 86%
Of the 22 IgG FCXM-Pos. Recipients:
7 grafts were lost
15 grafts were successful
WHY?
Of the 22 IgG FCXM-Pos. Recipients:
7 grafts were lost
15 grafts were successful
WHY?
FCXM Results: Heart RecipientFCXM Results: Heart Recipient
IgG FCXM
Pos.
68%
IgG FCXM
Pos.
68%
P<0.02P<0.02
5 sera tested from lost grafts
All 5 sera were Flow PRA reactive vs MHC I (Flow PRAs of 36%, 52%, 68%, 50% and 49%)
11 sera tested from successful recipients
All 11 sera were Flow PRA non-reactive
5 sera tested from lost grafts
All 5 sera were Flow PRA reactive vs MHC I (Flow PRAs of 36%, 52%, 68%, 50% and 49%)
11 sera tested from successful recipients
All 11 sera were Flow PRA non-reactive
We Flow PRA Tested the IgG FCXM-Pos. SeraWe Flow PRA Tested the IgG FCXM-Pos. Sera
Graft Survival
12 mo.
Graft Survival
12 mo.
FCXM (+)
Flow PRA I/II51%
55% (5/9)
FCXM (+)
Flow PRA I/II51%
55% (5/9)
Rejection 0-12 mo.
Rejection 0-12 mo.
89% (8/9)89% (8/9)31% (4/13)31% (4/13)
FCXM (-)
Flow PRA I/II51%
100% (13/13)
FCXM (-)
Flow PRA I/II51%
100% (13/13)
Both comparisons p<0.01Both comparisons p<0.01
1. HLA Ab negative, FCXM negative (at risk for reversible, cellular rejection)
2. HLA Ab negative, FCXM positive (non-HLA allo-Ab - at risk for reversible, cellular rejection)
1. HLA Ab negative, FCXM negative (at risk for reversible, cellular rejection)
2. HLA Ab negative, FCXM positive (non-HLA allo-Ab - at risk for reversible, cellular rejection)
HLA Ab and Donor Specific ReactivityRank Order of Risk
HLA Ab and Donor Specific ReactivityRank Order of Risk
3. HLA Ab positive, FCXM negative (at risk for reversible, cellular, +/- HLA Ab, rejection)
4. HLA Ab positive, FCXM positive (at risk for humoral/cellular rejection and graft loss)
3. HLA Ab positive, FCXM negative (at risk for reversible, cellular, +/- HLA Ab, rejection)
4. HLA Ab positive, FCXM positive (at risk for humoral/cellular rejection and graft loss)
HLA Ab and Donor Specific ReactivityRank Order of Risk
HLA Ab and Donor Specific ReactivityRank Order of Risk
To transplant or not to transplant, that is the question! Whether it is nobler in the minds of transplant surgeons to treat with thymoglobulin, OKT3, Plasmapheresis, IVIg, or the kitchen sink!
To transplant or not to transplant, that is the question! Whether it is nobler in the minds of transplant surgeons to treat with thymoglobulin, OKT3, Plasmapheresis, IVIg, or the kitchen sink!
1. Pre-transplant identification of
immunologically
high risk patients. Consideration of
induction
and/or maintenance
immunosuppression.
2. Clarify the role of HLA antibody in
rejection
episodes (including the role of C4d ).
1. Pre-transplant identification of
immunologically
high risk patients. Consideration of
induction
and/or maintenance
immunosuppression.
2. Clarify the role of HLA antibody in
rejection
episodes (including the role of C4d ).
Applications Applications
3. Transplantation of highly sensitized and/or positive crossmatch recipients.
4. Long term monitoring of the presence of HLA antibody and graft outcome.
3. Transplantation of highly sensitized and/or positive crossmatch recipients.
4. Long term monitoring of the presence of HLA antibody and graft outcome.
Applications Applications