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
Feucht 1993 Lederer 2001
# Bxs/Pts
93/93 310/218
Indication
Renal dysfunction Renal dysfunction
C4d+ (% Pt)
46% 46% primary 72% regraft
Regele 2001
Bohmig 2002 Nickeleit 2002
102/61
113/58 398/265
Renal dysfunction
Renal dysfunction Renal dysfunction
51%
28% 35%
Herzenberg 2002
Mauiyyedi 2002 Regele 2002 Sund 2003
126/93
67/67 213/213 37/37
Rejection
Renal dysfunction Renal dysfunction Protocol
37%
30% 34% 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 = Living donor txs = Total = 111 (74%) 39 (26%) 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(%) USA average(%)
African-American White Hispanic/Latino Asian Other
63 32 2 3 <1
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(%) USA average(%)
African-American White Hispanic/Latino Asian Other
52 45 1 2 0
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% 90% 80% 70% 60% 50% 40% 30% 20% 25% 10% 0%
PRA<20% PRA>20%
UNOS EUH
88% 75%
12%
Emory University Hospital:
Peak PRA Prior to Deceased Donor Renal Tx (2004)
Peak PRA 0-9% 10-79% 80+ % Unknown
EUH
USA 64% 22% 11% 4%
51% 32% 18% 0%
From: The Scientific Registry of Transplant Recipients
Cadaveric Renal Allograft Survival (1998 – 2003)
100
99
97
Emory N = >500
93
% Graft Survival
90 80
70
94
UNOS
90
N = 20791
81
60 50
3 mos
0
1
UNOS/SRTR 2003
Years
2
3
Evolution of HLA Antibody Detection
Cytotoxicity
Anti-HLA Antibody
Enhanced Cytotoxicity
Flow Cytometry
Ly
Ly
Ly
Anti-Human Globulin
C1
Ly
Fluorescenated Anti-Human Globulin
Ly
Ly
Ly
Membrane Attack Complex
Dye
Ly
Membrane Attack Complex
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 40
44% 40%
% rejection
30
n=
20 10 0
n=
81% vs 83%
1 yr survival
41
56
FCXM Positive FCXM Negative
FCXMs ARE IRRELEVANT!
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
Complement-dependent Cytotoxicity (CDC): - Direct CDC (Standard CDC) - Modifications Washes Extended Incubation Anti-human globulin (AHG-CDC) DTT / DTE Flow Cytometry (cells): - T cell / B cell - Pronase
Antigen Specific
ELISA - Yes / No - PRA % (I & II) - Specificity (I & II)
“FlowPRA” Flow cytometry using microparticles (“beads”) - PRA % (I and II ) - Specificity (I & II) Multi-plex - Suspension Arrays - Protein Chips
Flow Microparticles
One Lambda www.onelambda.com
Solid Phase, Antigen-Specific Assays
Extract and Purify HLA Antigens Class I or II Phenotype or Individual Molecule
B cells + EBV
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 CDC AHG-CDC ELISA FlowPRA 102 116 127 139
(+13%) (+10%) (+10%)
NEGATIVE 162 148 137 125
Gebel and Bray, Transplantation 69:1370-1374, 2000.
Positive FCXM are associated with graft loss when FlowPRA detects high levels of HLA antibodies
100 90 80 70 60 50 40 30 20 10 0
/F C
% Graft Survival
8
30
20
7
12
20
Bray RA, Nickerson PW, Kerman RH, Gebel HM. Immunol Res. 29:41, 2004
Fl ow
3+ 30 Fl % ow /F PR C XM A >3 + 0% /F C XM +
-
XM -
XM
XM
Fl ow PR A -/F C
330 % /F C
Fl ow
PR A
PR A
>3 0% /F C
PR A-
Fl ow
Fl ow
PR A
XM
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 0
P > 0.05 N = 372
N = 120
N= 492
Surviving
Cutpoint = 30%
High Low
1
Submitted for publication
2 3 4 Follow-up (y ears)
5
6
Antibody Paradigms - 2005
Screening
Low Risk
Crossmatch
Crossmatch Negative
Crossmatch Positive
Antibody Negative
Antibody Negative
Antibody Positive
High Risk
Crossmatch Negative
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 Donor Reactive Class I Donor Reactive Class II 14/15 (93%) 8/10 (80%)
Time to Rejection 6 (1-17) 5 (2-7)
Ab mediated Graft Loss 4 (27%) 3 (30%)
Time to Graft Loss 4 (1-14) 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