RENAL TRANSPLANTATION INTO HIGH RISK, HIGHLY SENSITIZED RECIPIENTS: A SINGLE CENTER

EXPERIENCE

Randy Hennigar PhD, MDDirector, Nephropathology and Electron

MicroscopyEmory 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

Author # Bxs/Pts Indication C4d+ (% Pt)

Feucht 1993 93/93 Renal dysfunction 46%

Lederer 2001 310/218 Renal dysfunction 46% primary

72% regraft

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%

Regele 2002 213/213 Renal dysfunction 34%

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 Various Renal Tx Populations

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(%) USA average(%)

African-American 63 36 White 32 39 Hispanic/Latino 2 16 Asian 3 8 Other <1 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 52 30

White 45 49 Hispanic/Latino 1 14 Asian 2 6 Other 0 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.

88%75%

12%

25%

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

PRA<20% PRA>20%

UNOS

EUH

Deceased Donor Renal Transplants (1999 – 2004)

Emory University Hospital: Peak PRA Prior to Deceased Donor Renal Tx (2004)

Peak PRA EUH USA

0-9% 51% 64%

10-79% 32% 22%

80+ % 18% 11%

Unknown 0% 4%

From: The Scientific Registry of Transplant Recipients

03 mos

11 22 33YearsYears

5050

100100

% G

raft

Sur

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l%

Gra

ft S

urvi

val

6060

7070

8080

9090UNOSN = 20791UNOSN = 20791

Emory N = >500Emory N = >500

94949393

9999 9797

9090

8181

Cadaveric Renal Allograft Survival (1998 – 2003)

UNOS/SRTR 2003

Evolution of HLA Antibody DetectionCytotoxicity Enhanced Cytotoxicity Flow Cytometry Cytotoxicity Enhanced Cytotoxicity Flow Cytometry

Ly

Ly

C1

Dye

Membrane AttackComplex

Ly

Anti-HLA Antibody

Ly

Ly

Ly

C1

Ly

Membrane AttackComplex

Dye

Anti-Human Globulin

Flow Cytometer

Ly

Ly

Ly

CD19(B cell)

CD3(T cell)

or

FluorescenatedAnti-Human Globulin

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 noncomplement-

fixing 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 StudyFrequency of rejection in a single center

IgG FCXM:Renal Allograft StudyFrequency of rejection in a single center

0

10

20

30

40

50

FCXM PositiveFCXM Negative

n =

41

n =

41

n =

56

n =

56

% r

ejec

tion

Kerman et al Transplantation 68:1855-1858, 1999

IgG

81% 81% vsvs 83%83%

1 yr survival1 yr survival

FCXMs ARE IRRELEVANT!FCXMs ARE IRRELEVANT!

44%40%

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 EVALUATIONMETHODS FOR ANTIBODY EVALUATION

Complement-dependent

Cytotoxicity (CDC):

- Direct CDC (Standard

CDC) - Modifications

WashesExtended Incubation

Anti-human globulin

(AHG-CDC)

DTT / DTE

Flow Cytometry (cells):

- T cell / B cell

- Pronase

Complement-dependent

Cytotoxicity (CDC):

- Direct CDC (Standard

CDC) - Modifications

WashesExtended 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

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

Antigen Non-SpecificAntigen Non-Specific Antigen SpecificAntigen Specific

Flow MicroparticlesFlow MicroparticlesFlow MicroparticlesFlow Microparticles

One Lambdawww.onelambda.com

Solid Phase, Antigen-Specific Assays

B cells + EBV

Purified HLA Antigens

Microparticles

Extract and Purify HLA Antigens

ELISA

Flow Cytometry

Class I or II Phenotypeor Individual Molecule

Microparticles ELISA

90%

Coated with 30 HLA Ior 30 HLA II antigens

Table 6. Flow PRA versus AHG-CDC PRA (n = 203)

Flow PRA-Negative Flow PRA-Positive

AHG-CDC PRA >10% 2 7AHG-CDC PRA <10% 160 34

CDCCDC 102102 162 162CDCCDC 102102 162 162

PRA ANALYSIS BY DIFFERING PRA ANALYSIS BY DIFFERING METHODLOGIESMETHODLOGIES

PRA ANALYSIS BY DIFFERING PRA ANALYSIS BY DIFFERING METHODLOGIESMETHODLOGIES

POSITIVEPOSITIVE NEGATIVENEGATIVEPOSITIVEPOSITIVE NEGATIVENEGATIVE

AHG-CDCAHG-CDC 116 116 (+13%)(+13%) 148 148AHG-CDCAHG-CDC 116 116 (+13%)(+13%) 148 148

ELISAELISA 127 127 (+10%)(+10%) 137 137ELISAELISA 127 127 (+10%)(+10%) 137 137

FlowPRAFlowPRA 139 139 (+10%)(+10%) 125 125FlowPRAFlowPRA 139 139 (+10%)(+10%) 125 125

Gebel and Bray, Transplantation 69:1370-1374, 2000.Gebel and Bray, Transplantation 69:1370-1374, 2000.

Positive FCXM are associated with graft loss when FlowPRA detects high levels of HLA antibodies

Positive FCXM are associated with graft loss when FlowPRA detects high levels of HLA antibodies

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Bray RA, Nickerson PW, Kerman RH, Gebel HM. Immunol Res. 29:41, 2004

Renal Transplantation (DD) into High vs. Low PRA Patients with Negative FCXM

Renal Transplantation (DD) into High vs. Low PRA Patients with Negative FCXM

Submitted for publication

0.00.10.20.30.40.50.60.70.80.91.0

Surv

ivin

g

0 1 2 3 4 5 6

Follow-up (years)

P > 0.05

HighLowCutpoint = 30%

N = 120

N = 372N= 492

Antibody Paradigms - 2005Antibody Paradigms - 2005

Antibody Negative Crossmatch NegativeAntibody Negative Crossmatch Negative

Antibody Negative Crossmatch PositiveAntibody Negative Crossmatch Positive

Antibody Positive Crossmatch NegativeAntibody Positive Crossmatch Negative

Antibody Positive Crossmatch PositiveAntibody Positive Crossmatch Positive

Screening CrossmatchScreening Crossmatch

Low Risk

High Risk

PRAPRA•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.

•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 CLASS II DONOR SPECIFIC ANTIBODIES ARE PATHOGENIC IN PRIMARY RENAL ALLOGRAFTS PATHOGENIC IN PRIMARY RENAL ALLOGRAFTS

Nickerson et al AJT: 4(8) 257, 2004Nickerson et al AJT: 4(8) 257, 2004

Impact of Donor Reactive HLA AntibodiesImpact of Donor Reactive HLA Antibodies

Rejection Time toRejection Time to Ab mediated Time to Ab mediated Time to First Month Rejection Graft Loss Graft Loss First Month Rejection Graft Loss Graft Loss

Rejection Time toRejection Time to Ab mediated Time to Ab mediated Time to First Month Rejection Graft Loss Graft Loss First Month Rejection Graft Loss Graft Loss Donor Reactive Class I 14/15 (93%) 6 (1-17) 4 (27%) 4 (1-14)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)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%) NAHLA Ab (non-donor) 3/21 (14%) 13 (13-19) 0 (0%) NA

Donor Reactive Class I 14/15 (93%) 6 (1-17) 4 (27%) 4 (1-14)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)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%) NAHLA Ab (non-donor) 3/21 (14%) 13 (13-19) 0 (0%) NA

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 77% of positive B cell crossmatches ARE NOT DUE to HLA antibodiesARE NOT DUE to HLA antibodies!!

ApproachesApproaches

PharmacologicalPharmacological BiologicalBiological

Desensitization IVIG PP / IVIG Rituxan

Desensitization IVIG PP / IVIG Rituxan

Identical Sibling

Xenotransplantation

Acceptable Mismatch - Detailed Antibody Analysis - Comprehensive PRA - Virtual Crossmatch

Identical Sibling

Xenotransplantation

Acceptable Mismatch - Detailed Antibody Analysis - Comprehensive PRA - Virtual Crossmatch

Transplant across a + crossmatch anticipating Immunosuppression

Transplant across a + crossmatch anticipating Immunosuppression

Putative Recipient:

A1, A30; B7, B8 ; DR11, 15

Antibodies - A2, 23, 24, 68

Potential Donor:

A25, A33; B42, B18; DR12, DR13

Putative Recipient:

A1, A30; B7, B8 ; DR11, 15

Antibodies - A2, 23, 24, 68

Potential Donor:

A25, A33; B42, B18; DR12, DR13

Acceptable MismatchesAcceptable Mismatches

Strategic ApproachesStrategic 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.

- 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 EveryoneDesensitization Protocols Aren’t For Everyone

- High Titer HLA Antibodies >512

- Refractory Specificities DR52, DR53

- Fragile Patients

- Restricted to Living Donors

- $$$$$$$$$$$$s

- 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