Transcript Immunological Monitoring

HLA Ab, Donor Reactivity
and Risk of Rejection
and Graft Loss
Ronald H. Kerman, PhD
The University of Texas Medical School ~ Houston, TX
Division of Immunology and Organ Transplantation
Allograft Rejection
Time:
Mediated by:
Hyperacute
0-48 hrs
Abs
Accelerated
5-7 days
Abs/cells
Early/delayed
Cells/Abs
>60 days
Abs/cells/?
Type:
Acute
Chronic
Responsibilities of the
Histocompatibility Laboratory
To identify clinically relevant
recipient IgG HLA antibodies
Positive crossmatches, due to Abs or
other factors not impacting on graft
outcome, should not influence the donorrecipient pairing for transplantation.
Detection of Recipient Sensitization
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 Immunoglobulin Reactivity
• NIH-CDC
• AHG-CDC
• Flow cytometry
Membrane-dependent assays
Complement-dependent Cytotoxicity NIH Assay
Complement-dependent Cytotoxicity NIH Assay
Complement-dependent Cytotoxicity NIH Assay
Anti-human Globulin (Enhancement) Assay
Anti-human Globulin (Enhancement) Assay
Anti-human Globulin (Enhancement) Assay
Flow Cytometry Assay
NIH - CDC
Negative
AHG – CDC
Negative
Now measuring binding of IgG (absent C’)
Cadaveric Renal Allograft Survival Among
1o CsA-Pred Recipients at 12 months
NIH
Neg.
AHG
Neg.
Pos.
n=166
n=151
n=15
81%
82%
67%
(134/166)
(124/151)
(10/15)
P<0.01
Kerman et al, Transplantation; 51:316, 1991
Cadaveric Renal Allograft Survival Among
1o CsA-Pred Recipients at 12 months
AHG
DTE-AHG
Pos.
Neg.
Pos.
n=15
n=12
n=3
67%
83%
0%
(10/12)
(0/3)
(10/15)
P<0.01
Kerman et al, Transplantation; 51:316, 1991
Cadaveric Renal Allograft Survival Among
1o CsA-Pred Recipients at 12 months
FCXM
DTE/AHG XM
Neg.
n=166
81%
Neg.
Pos.
n=130
n=36
81%
81%
Kerman et al, Transplantation; 51:316, 1991
Neg-NIH Extended XM: FCXM Study
T-FCXM
T-FCXM
Pos.
Neg.
n=148
n=693
75%
82%
P<0.01
Ogura et al, Transplantation; 56:294, 1993
Could Ron Kerman have been
wrong about his crossmatch
results and interpretation?
IgG FCXM: Renal Allograft Study
% Rejection
Frequency of Rejection in a Single Center
50
45
40
35
30
25
20
15
10
5
0
P=NS
Negative
(n=56)
Positive
(n=41)
Kerman et al, Transplantation; 68:1855, 1999
Could Ron Kerman have
been wrong about his
crossmatch results and
interpretation?
I don’t think so!
The Cell Surface Is a Jungle
HLA
Membrane-dependent Assays
• NIH-CDC
•AHG-CDC
• Flow cytometry
Detection of membrane receptors
may not be related to HLA!
Membrane-independent Assays
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)
PRA by Different Methodologies
Type:
Positive
Negative
CDC
102
162
AHG-CDC
116
148
ELISA
127
137
Flow
139
125
Gebel & Bray, Transplantation; 69:1370, 2000
AHG-PRA vs Rejection
493 Consecutive CAD Recipients
AHG-PRA
Rejection
<10%
10%
YES
134
100
NO
159
100
P=NS
ELISA-PRA and Rejection
ELISA-PRA
Rejection
<10%
10%
YES
38
117
NO
168
63
P<0.001
Correlation Between % ELISA-PRA
and Graft Survival
ELISA-PRA
Graft Survival
<10%
>10%
(months)
(n=312)
(n=181)
12
85%
74%
P<0.01
24
82%
70%
P<0.01
36
81%
67%
P<0.01
Sensitivity and sensitization,
defining the unsensitized patient
Gebel & Bray, Transplantation; 69:1370, 2000
Application of membrane-independent
assays to identify HLA antibodies
Correlation of Pre-transplant Abs Detected by Flow
PRA with Biopsy-documented Cardiac Rejection
Tambur et al, Transplantation; 70:1055, 2000
IgG FCXM: Renal Allograft Study
Frequency of Rejection in a Single Center
% Rejection
Were positive crossmatches due
to HLA Abs?
50
45
40
35
30
25
20
15
10
5
0
P=NS
Negative
(n=56)
Positive
(n=41)
Kerman et al, Transplantation; 68:1855, 1999
Immunosuppressive Menu:
• Neoral - CsA
• Steroids
• Prograf - FK506
• Cellcept - MMF
• Rapamycin - Sirolimus
• Thymoglobulin
• OKT3, anti-IL-2R, FTY720
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.
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.)
Results:
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)
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
Flow PRA-1
Rejection
<5%
5%
NO
76
23
YES
21
27
X2=15.7; P<0.001
Flow PRA
Day of 1st
Rejection
Pos.
Neg.
0%
57 ± 34
2
8
No grafts lost
(+) FCXM vs non-HLA Ab
FCXM
Flow PRA
Day of 1st
Rejection
13 ± 9%
55 ± 31
No grafts lost.
FCXM
Pos.
-
Neg.
30
Flow PRA
Day of 1st
Rejection
Pos.
Neg.
28 ± 9%
32 ± 15
12
13
FCXM
(+) HLA Ab and (-) FCXM: rejection, no grafts lost.
(+) HLA Ab and (+) FCXM: rejection, 58% (7/12)
grafts lost.
Flow PRA
Day of 1st
Rejection
Pos.
Neg.
48 ± 31%
17 ± 12
8
7
FCXM
(+) HLA Ab and (-) FCXM: rejection, no grafts lost.
(+) HLA Ab and (+) FCXM: rejection, 63% (5/8)
lost to AMR.
% PRA
N
% Rejection
Day of
Rejection
0
75
5% (4/75)
57 ± 34
13 ± 9
32
13% (4/32)
55 ± 31
28 ± 9
25
100%
32 ± 9
48 ± 31 15
100%
17 ± 12
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.
We have performed heart
transplantation following a
negative AHG-XM.
We evaluated the clinical
relevance of FCXM for heart
recipients.
FCXM Results: Heart Recipient
IgG FCXM
IgG FCXM
Neg.
Pos.
1YGS 86%
68%
P<0.02
Of the 22 IgG FCXM-Pos. Recipients:
7 grafts were lost
15 grafts were successful
WHY?
We Flow PRA Tested the IgG
FCXM-Pos. Sera
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
FCXM (-)
FCXM (+)
Flow PRA I/II Flow PRA I/II
Graft
51%
51%
Survival
12 mo. 100% (13/13) 55% (5/9)
Rejection
0-12 mo.
31% (4/13)
Both comparisons p<0.01
89% (8/9)
HLA Ab and Donor Specific Reactivity
Rank Order of Risk
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 Reactivity
Rank 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)
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!
Applications
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
3. Transplantation of highly sensitized and/or
positive crossmatch recipients.
4. Long term monitoring of the presence
of HLA antibody and graft outcome.
“I have never let my
schooling interfere
with my education.”
-Mark Twain