Transcript Document

RENAL TRANSPLANTATION INTO
HIGH RISK, HIGHLY SENSITIZED
RECIPIENTS: A SINGLE CENTER
EXPERIENCE
Randy Hennigar PhD, MD
Director, Nephropathology and Electron
Microscopy
Emory University Hospital
Atlanta ,GA
Incidence of C4d in Renal Transplant
Population: Emory University
Hospital (EUH)
• Objective: To gain more information about the
role of antibody mediated rejection in the renal
transplant population @ EUH.
• Method: From Nov 2003 to Mar 2005, a total
of 313 consecutive biopsies (252 tx patients)
were screened for C4d deposition. Bxs were
performed for renal dysfunction.
Immunoperoxidase Staining for C4d
Incidence of C4d in Various Renal Tx Populations
Author
# Bxs/Pts
Indication
C4d+ (% Pt)
Feucht 1993
93/93
Renal dysfunction
Lederer 2001
310/218
Renal dysfunction
Regele 2001
102/61
Renal dysfunction
51%
Bohmig 2002
113/58
Renal dysfunction
28%
Nickeleit 2002
398/265
Renal dysfunction
35%
Herzenberg 2002
126/93
Rejection
37%
Mauiyyedi 2002
67/67
Renal dysfunction
30%
213/213
Renal dysfunction
34%
Regele 2002
46%
46% primary
72% regraft
Sund 2003
37/37
Protocol
30%
Koo 2004
96/48
Protocol
13%
Modified from Bohmig & Regele, Transpl Int 16:773, 2003
Incidence of C4d in Renal Transplant
Population @ EUH
• Results: 23 of 252 pts (9%) were positive, using
the criteria of Nickeleit and Mihatsch (Nephrol
Dial Transpl 18: 2232-2239, 2003).
• Conclusion: The incidence of C4d deposition
(and presumably antibody-mediated rejection)
among the kidney transplant population at
EUH appears less prevalent than that reported
in the literature.
Emory University Hospital:
Renal Transplant Center Activity (2004)
Deceased donor txs = 111 (74%)
Living donor txs = 39 (26%)
Total =
150
Tx rate among waitlist pts = 0.3
From: The Scientific Registry of Transplant Recipients
Emory University Hospital:
Transplant Recipient Characteristics (2004)
Ethnicity/race of waitlist pts (end of 2004):
EUH(%)
African-American
White
Hispanic/Latino
Asian
Other
63
32
2
3
<1
USA average(%)
36
39
16
8
1
From: The Scientific Registry of Transplant Recipients
Emory University Hospital:
Transplant Recipient Characteristics (2004)
Ethnicity/race of tx patients (deceased donors):
EUH(%)
African-American
White
Hispanic/Latino
Asian
Other
52
45
1
2
0
USA average(%)
30
49
14
6
2
From: The Scientific Registry of Transplant Patients
Panel Reactive Antibodies (PRA)
• A screening mechanism to determine the HLA antibody
profile of potential transplant recipients.
• Periodic screening (monthly/quarterly) of recipient
sera with a panel of HLA-typed cells.
• Sensitization of the recipient is expressed as the
percentage of serum reactivity with the total panel.
Typically, high PRA is indicative of a highly sensitized
recipient- one who is at risk for early graft loss.
Deceased Donor Renal Transplants (1999 – 2004)
100%
UNOS
EUH
90%
80%
70%
60%
88%
75%
50%
40%
30%
20%
25%
10%
12%
0%
PRA<20%
PRA>20%
Emory University Hospital:
Peak PRA Prior to Deceased Donor Renal Tx (2004)
Peak PRA
0-9%
10-79%
80+ %
Unknown
EUH
USA
51%
32%
18%
0%
64%
22%
11%
4%
From: The Scientific Registry of Transplant Recipients
% Graft Survival
Cadaveric Renal Allograft Survival (1998 – 2003)
100
99
90
94
UNOS
97
Emory N = >500
90
93
N = 20791
80
81
70
60
50
0
3 mos
UNOS/SRTR 2003
1
Years
2
3
Evolution of HLA Antibody Detection
Cytotoxicity
Enhanced Cytotoxicity
Flow Cytometry
Anti-HLA Antibody
Ly
Ly
Ly
Anti-Human Globulin
Fluorescenated
Anti-Human Globulin
Ly
C1
Ly
Ly
Ly
Membrane Attack
Complex
Membrane Attack
Complex
Dye
Ly
Ly
Dye
Ly
CD19 or
(B cell)
CD3
(T cell)
Flow Cytometer
Bray et al Immunol Res. 29:41, 2004
From: Gebel et al. Am J Transpl 3:1488-1500, 2003
From: Gebel et al. Am J Transpl 3:1488-1500, 2003
Impact of HLA Antibodies Detected Only
by Flow Cytometric Crossmatch (Regrafts)
Gebel et al. Am J Transpl 3:1488-1500, 2003
In 2002, of the >150 labs participating in the
ASHI-CAP class I crossmatch surveys (MX1-A,
B, C), only 68–70% reported AHG augmented
CDC and 47–52% flow-based crossmatches.
From: Gebel et al. Am J Transpl 3:1488-1500, 2003
Perceived Pitfalls of Flow Cytometry
Crossmatching (FCXM)
• Too sensitive
– Detection of low titer and noncomplementfixing antibodies of little or no clinical
relevance
• Would inappropriately deny a patient
access to transplantion
• Does not reliably predict poor clinical
outcomes
IgG FCXM:Renal Allograft Study
Frequency of rejection in a single center
50
44%
40%
% rejection
40
30
n=
20
10
41
n=
81% vs 83%
1 yr survival
56
FCXM Positive
FCXM Negative
FCXMs ARE IRRELEVANT!
0
IgG
Kerman et al Transplantation 68:1855-1858, 1999
In 2002, of the >150 labs participating in the
ASHI-CAP class I crossmatch surveys (MX1-A,
B, C), only 68–70% reported AHG augmented
CDC and 47–52% flow-based crossmatches.
Panel Reactive Antibodies (PRA)
• A screening mechanism to determine the HLA antibody
profile of potential transplant recipients.
• Periodic screening (monthly/quarterly) of recipient
sera with a panel of HLA typed cells.
• Sensitization of the recipient is expressed as the
percentage of serum reactivity with the total panel.
Typically, high PRA is indicative of a highly sensitized
recipient- one who is at risk for early graft loss.
• Historically, PRA has been antigen-nonspecific.
METHODS FOR ANTIBODY EVALUATION
Antigen Non-Specific
Antigen Specific
Complement-dependent
Cytotoxicity (CDC):
- Direct CDC (Standard CDC)
- Modifications
Washes
Extended Incubation
Anti-human globulin
(AHG-CDC)
DTT / DTE
ELISA
- Yes / No
- PRA % (I & II)
- Specificity (I & II)
Flow Cytometry (cells):
- T cell / B cell
- Pronase
Multi-plex
- Suspension Arrays
- Protein Chips
“FlowPRA”
Flow cytometry using
microparticles (“beads”)
- PRA % (I and II )
- Specificity (I & II)
Flow Microparticles
One Lambda
www.onelambda.com
Solid Phase, Antigen-Specific Assays
Extract and Purify
HLA Antigens
B cells + EBV
Class I or II Phenotype
or Individual Molecule
Flow Cytometry
Microparticles
Purified HLA Antigens
ELISA
Microparticles
ELISA
Coated with 30 HLA I
or 30 HLA II antigens
90%
Table 6. Flow PRA versus AHG-CDC PRA (n = 203)
Flow PRA-Negative
AHG-CDC PRA >10%
AHG-CDC PRA <10%
2
160
Flow PRA-Positive
7
34
PRA ANALYSIS BY DIFFERING
METHODLOGIES
POSITIVE
NEGATIVE
CDC
102
162
AHG-CDC
116
(+13%)
148
ELISA
127
(+10%)
137
FlowPRA
139
(+10%)
125
Gebel and Bray, Transplantation 69:1370-1374, 2000.
Positive FCXM are associated with graft loss when
FlowPRA detects high levels of HLA antibodies
% Graft
Survival
100
90
80
70
60
50
40
30
20
10
0
30
8
7
20
Fl
ow
PR
A
PR
A
-/F
C
XM
3+
30
Fl
%
ow
/F
PR
C
XM
A
>3
+
0%
/F
C
XM
+
XM
Fl
ow
>3
0%
/F
C
XM
Fl
ow
PR
A
330
%
/F
C
/F
C
XM
-
-
12
PR
A
PR
AFl
ow
Fl
ow
20
Bray RA, Nickerson PW, Kerman RH, Gebel HM. Immunol Res. 29:41, 2004
Surviving
Renal Transplantation (DD) into High vs.
Low PRA Patients with Negative FCXM
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
P > 0.05
N = 372
N= 492
N = 120
High
Low
Cutpoint = 30%
0
1
2
3
4
Follow-up (years)
Submitted for publication
5
6
Antibody Paradigms - 2005
Screening
Crossmatch
Low Risk
Antibody Negative
Crossmatch Negative
Antibody Negative
Crossmatch Positive
Antibody Positive
Crossmatch Negative
High Risk
Antibody Positive
Crossmatch Positive
PRA
•PRA can be a qualitative and/or quantitative
assessment of alloimmunization in transplant
patients.
•Optimally, PRA testing should identify the
specificity of an antibody and provide the
“transplantability” index of a patient.
•More succinctly, PRA testing should correlate
with the final crossmatch.
CLASS II DONOR SPECIFIC ANTIBODIES ARE
PATHOGENIC IN PRIMARY RENAL ALLOGRAFTS
Nickerson et al AJT: 4(8) 257, 2004
Impact of Donor Reactive HLA Antibodies
Rejection
First Month
Time to
Rejection
Ab mediated
Graft Loss
Time to
Graft Loss
Donor Reactive Class I
14/15 (93%)
6 (1-17)
4 (27%)
4 (1-14)
Donor Reactive Class II
8/10 (80%)
5 (2-7)
3 (30%)
5 (2-9)
HLA Ab (non-donor)
3/21 (14%)
13 (13-19)
0 (0%)
NA
BCM+ class II, n=14
BCM+ autoAb, n=10
77% of positive B cell crossmatches
ARE NOT DUE to HLA antibodies!
BCM+ Ab UNKNOWN, n=38
BCM-,n=930
Le Bas-Bernardet,et al Transplantation 75:477,2003
Approaches
Pharmacological
Desensitization
IVIG
PP / IVIG
Rituxan
Transplant across a +
crossmatch anticipating
Immunosuppression
Biological
Identical Sibling
Xenotransplantation
Acceptable Mismatch
- Detailed Antibody Analysis
- Comprehensive PRA
- Virtual Crossmatch
Acceptable Mismatches
Putative Recipient:
A1, A30; B7, B8 ; DR11, 15
Antibodies - A2, 23, 24, 68
Potential Donor:
A25, A33; B42, B18; DR12, DR13
Strategic Approaches
- Based on recognition that matching is not for
everyone- 85% of DD Txs are mismatched.
- Focus on appropriate mismatching rather
than looking for an HLA “match”.
- Requires detailed evaluation of the
patient’s HLA antibodies.
- Shifts emphasis to antibody evaluation
and away from crossmatching to identify
acceptable mismatches.
Desensitization Protocols Aren’t For Everyone
- High Titer HLA Antibodies
>512
- Refractory Specificities
DR52, DR53
- Fragile Patients
- Restricted to Living Donors
- $$$$$$$$$$$$s
Recommendations to define the ‘non-sensitized’ patient:
• Validate patient history for the lack of sensitizing
events.
• Confirm that a patient is nonsensitized using a solid
phase assay documented to be more sensitive than
CDC assays.
Recommendations to evaluate the ‘sensitized’ patient:
• To optimize detection of low titer HLA antibodies,
monitoring should be performed using sensitive
solid-phase assays.
• Monitoring should include evaluation for both
antibodies to class I and class II HLA antigens.
• A crossmatch test must be performed before
transplantation using, as a minimum, an enhanced CDC
technique.
• The final crossmatch technique should be of equal
sensitivity to the solid-phase assay used to screen for the
presence of HLA antibody.
• A B-cell crossmatch should be included in the final
crossmatch.
• Peak sera should be included in the final crossmatch.
• Auto-crossmatches should be utilized to aid in the
interpretation of allo-crossmatches.
END OF LECTURE