1. 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

2. 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 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.

3. Immunoperoxidase Staining for C4d

4. Incidence of C4d in Various Renal Tx Populations
Author
# Bxs/Pts
Indication
C4d+ (% Pt)
Feucht 1993
93/93
Renal dysfunction
46%
Lederer 2001
310/218
46% primary
72% regraft
Regele 2001
102/61
51%
Bohmig 2002
113/58
28%
Nickeleit 2002
398/265
35%
Herzenberg 2002
126/93
Rejection
37%
Mauiyyedi 2002
67/67
30%
Regele 2002
213/213
34%
Sund 2003
37/37
Protocol
Koo 2004
96/48
13%
Modified from Bohmig & Regele, Transpl Int 16:773, 2003

5. 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: , 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.

6. ????

7. Emory University Hospital: Renal Transplant Center Activity (2004)
Deceased donor txs = 111 (74%)
Living donor txs = 39 (26%)
Total =
Tx rate among waitlist pts = 0.3
From: The Scientific Registry of Transplant Recipients

8. 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 <
From: The Scientific Registry of Transplant Recipients

9. Emory University Hospital: Transplant Recipient Characteristics (2004)
Ethnicity/race of tx patients (deceased donors):
EUH(%) USA average(%)
African-American
White
Hispanic/Latino
Asian
Other
From: The Scientific Registry of Transplant Patients

10. 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.

11. Deceased Donor Renal Transplants (1999 – 2004)

12. Emory University Hospital: Peak PRA Prior to Deceased Donor Renal Tx (2004)
EUH
USA
0-9%
51%
64%
10-79%
32%
22%
80+ %
18%
11%
Unknown 0% 4%
From: The Scientific Registry of Transplant Recipients

13. Cadaveric Renal Allograft Survival (1998 – 2003)
100 97
Emory N = >500 90 94 93 90
UNOS
N = 20791 80 81
% Graft Survival 70 60 50
3 mos 1 2 3
Years
UNOS/SRTR 2003

16. Evolution of HLA Antibody Detection
Cytotoxicity Enhanced Cytotoxicity Flow Cytometry Ly C1
Dye
Membrane Attack
Complex
Anti-HLA Antibody Ly C1
Membrane Attack
Complex
Dye
Anti-Human Globulin
Flow Cytometer Ly
CD19
(B cell)
CD3
(T cell) or
Fluorescenated
Anti-Human Globulin
Bray et al Immunol Res. 29:41, 2004

17. From: Gebel et al. Am J Transpl 3:1488-1500, 2003

18. From: Gebel et al. Am J Transpl 3:1488-1500, 2003

19. Impact of HLA Antibodies Detected Only by Flow Cytometric Crossmatch (Regrafts) Gebel et al. Am J Transpl 3: , 2003

20. 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.

21. From: Gebel et al. Am J Transpl 3:1488-1500, 2003

22. Perceived Pitfalls of Flow Cytometry Crossmatching (FCXM)
Too sensitive
Detection of low titer and noncomplement-fixing antibodies of little or no clinical relevance
Would inappropriately deny a patient access to transplantion
Does not reliably predict poor clinical outcomes

23. Kerman et al Transplantation 68:1855-1858, 1999
IgG FCXM:Renal Allograft Study Frequency of rejection in a single center
44%
40%
n = 41
n = 56
81% vs 83%
1 yr survival
% rejection
FCXMs ARE IRRELEVANT!
IgG
Kerman et al Transplantation 68: , 1999

24. 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.

25. 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.

26. 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
Flow Cytometry (cells):
- T cell / B cell
- Pronase
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

27. Flow Microparticles
One Lambda

28. 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

29. Coated with 30 HLA I or 30 HLA II antigens
Microparticles
ELISA
Coated with 30 HLA I
or 30 HLA II antigens
90%

30. Table 6. Flow PRA versus AHG-CDC PRA (n = 203)
Flow PRA-Negative Flow PRA-Positive
AHG-CDC PRA >10%
AHG-CDC PRA <10%

31. PRA ANALYSIS BY DIFFERING METHODLOGIES
POSITIVE NEGATIVE
CDC
AHG-CDC 116 (+13%)
ELISA 127 (+10%)
FlowPRA (+10%)
Gebel and Bray, Transplantation 69: , 2000.

32. Positive FCXM are associated with graft loss when FlowPRA detects high levels of HLA antibodies
% Graft Survival 8 30 20 7 12 20
Bray RA, Nickerson PW, Kerman RH, Gebel HM. Immunol Res. 29:41, 2004

33. Renal Transplantation (DD) into High vs
Renal Transplantation (DD) into High vs. Low PRA Patients with Negative FCXM
P > 0.05
Cutpoint = 30%
N = 372
N= 492
N = 120
Submitted for publication

34. 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

35. 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.

36. Impact of Donor Reactive HLA Antibodies
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 First Month Rejection Graft Loss Graft Loss
Donor Reactive Class I /15 (93%) (1-17) (27%) (1-14)
Donor Reactive Class II /10 (80%) (2-7) (30%) (2-9)
HLA Ab (non-donor) /21 (14%) (13-19) (0%) NA

37. Le Bas-Bernardet,et al Transplantation 75:477,2003
BCM+ class II, n=14
BCM+ autoAb, n=10
BCM+ Ab UNKNOWN, n=38
BCM-,n=930
Le Bas-Bernardet,et al Transplantation 75:477,2003
77% of positive B cell crossmatches
ARE NOT DUE to HLA antibodies!

38. Approaches Pharmacological Biological Desensitization
IVIG
PP / IVIG
Rituxan
Identical Sibling
Xenotransplantation
Acceptable Mismatch
- Detailed Antibody Analysis
- Comprehensive PRA
- Virtual Crossmatch
Transplant across a + crossmatch anticipating
Immunosuppression

39. Acceptable Mismatches
Putative Recipient:
A1, A30; B7, B8 ; DR11, 15
Antibodies - A2, 23, 24, 68
Potential Donor:
A25, A33; B42, B18; DR12, DR13

40. 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.

41. Desensitization Protocols Aren’t For Everyone
- High Titer HLA Antibodies
>512
- Refractory Specificities
DR52, DR53
- Fragile Patients
- Restricted to Living Donors
- $$$$$$$$$$$$s

42. 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.

43. 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.

44. END OF LECTURE