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 =
Living donor txs =
Total = 150

111 (74%)
39 (26%)

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
32 39
Hispanic/Latino
Asian
3 8
Other
<1

63 36
2

16

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)

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

3 mos

UNOS/SRTR 2003

Years

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

C1
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 6870% reported AHG augmented
CDC and 4752% 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
44%

% rejection

40%

n=
41

81% vs 83%
1 yr survival

n=
56

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 6870% reported AHG augmented
CDC and 4752% 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

B cells + EBV

Class I or II Phenotype
or Individual Molecule

Microparticles
Purified HLA Antigens

Flow Cytometry

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.

% Graft
Survival

Positive FCXM are associated with graft loss when

FlowPRA detects high levels of HLA antibodies

30

20

12

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

Cutpoint = 30%

2
3
4
Follow-up (years)
Submitted for publication

High
Low

Antibody Paradigms - 2005

Screening

Crossmatch
Low Risk

Antibody Negative

Crossmatch Negative

Antibody Negative
Antibody Positive

Crossmatch 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

Time to

Ab mediated

Time to

Donor Reactive Class I

First
Month
14/15
(93%)

Rejection
6 (1-17)

Graft
Loss
4 (27%)

Graft
Loss
4 (1-14)

Donor Reactive Class II

8/10 (80%)

5 (2-7)

HLA Ab (non-donor)

3/21 (14%)

13 (13-19)

3 (30%)
0 (0%)

5 (2-9)
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
patients HLA antibodies.
- Shifts emphasis to antibody evaluation
and away from crossmatching to identify
acceptable mismatches.

Desensitization Protocols Arent 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