HLA Ab, Donor Reactivity

and Risk of Rejection

and Graft Loss

HLA Ab, Donor Reactivity

and Risk of Rejection

and Graft LossRonald H. Kerman, PhD

The University of Texas Medical School ~ Houston, TX

Division of Immunology and Organ Transplantation

Ronald H. Kerman, PhD

The University of Texas Medical School ~ Houston, TX

Division of Immunology and Organ Transplantation

Allograft RejectionAllograft RejectionType:

Hyperacute

Accelerated

Acute

Chronic

Type:

Hyperacute

Accelerated

Acute

Chronic

Time:

0-48 hrs

5-7 days

Early/delayed

>60 days

Time:

0-48 hrs

5-7 days

Early/delayed

>60 days

Mediated by:

Abs

Abs/cells

Cells/Abs

Abs/cells/?

Mediated by:

Abs

Abs/cells

Cells/Abs

Abs/cells/?

To identify clinically relevant

recipient IgG HLA antibodies

To identify clinically relevant

recipient IgG HLA antibodies

Responsibilities of the Histocompatibility LaboratoryResponsibilities of the Histocompatibility Laboratory

Positive crossmatches, due to Abs or other factors

not impacting on graft outcome, should not influence

the donor-recipient pairing for transplantation.

Positive crossmatches, due to Abs or other factors

not impacting on graft outcome, should not influence

the donor-recipient pairing for transplantation.

Screen sera for reactivity vs target cells by cytotoxicity/fluorescence readouts.

Use the most informative sera when performing the recipient vs donor

crossmatch (historically most reactive, current and pretransplant sera).

Screen sera for reactivity vs target cells by cytotoxicity/fluorescence readouts.

Use the most informative sera when performing the recipient vs donor

crossmatch (historically most reactive, current and pretransplant sera).

Detection of Recipient SensitizationDetection of Recipient Sensitization

• NIH-CDC

• AHG-CDC

• Flow cytometry

Membrane-dependent assays

• NIH-CDC

• AHG-CDC

• Flow cytometry

Membrane-dependent assays

Detection of Immunoglobulin ReactivityDetection of Immunoglobulin Reactivity

Complement-dependent Cytotoxicity NIH AssayComplement-dependent Cytotoxicity NIH Assay

Complement-dependent Cytotoxicity NIH AssayComplement-dependent Cytotoxicity NIH Assay

Complement-dependent Cytotoxicity NIH AssayComplement-dependent Cytotoxicity NIH Assay

Anti-human Globulin (Enhancement) AssayAnti-human Globulin (Enhancement) Assay

Anti-human Globulin (Enhancement) AssayAnti-human Globulin (Enhancement) Assay

Anti-human Globulin (Enhancement) AssayAnti-human Globulin (Enhancement) Assay

Flow Cytometry AssayNIH - CDC NegativeAHG – CDC NegativeNow measuring binding of IgG (absent C’)

Flow Cytometry AssayNIH - CDC NegativeAHG – CDC NegativeNow measuring binding of IgG (absent C’)

Cadaveric Renal Allograft Survival Among 1o CsA-Pred Recipients at 12 months

Cadaveric Renal Allograft Survival Among 1o CsA-Pred Recipients at 12 months

NIH

Neg.

n=166

81% (134/166)

NIH

Neg.

n=166

81% (134/166)

Kerman et al, Transplantation; 51:316, 1991Kerman et al, Transplantation; 51:316, 1991

AHG

Neg. Pos.

n=151 n=15

82% 67% (124/151) (10/15)

AHG

Neg. Pos.

n=151 n=15

82% 67% (124/151) (10/15)

P<0.01P<0.01

Cadaveric Renal Allograft Survival Among 1o CsA-Pred Recipients at 12 months

Cadaveric Renal Allograft Survival Among 1o CsA-Pred Recipients at 12 months

AHG

Pos.

n=15

67% (10/15)

AHG

Pos.

n=15

67% (10/15)

Kerman et al, Transplantation; 51:316, 1991Kerman et al, Transplantation; 51:316, 1991

DTE-AHG

Neg. Pos.

n=12 n=3

83% 0% (10/12) (0/3)

DTE-AHG

Neg. Pos.

n=12 n=3

83% 0% (10/12) (0/3)

P<0.01P<0.01

Cadaveric Renal Allograft Survival Among 1o CsA-Pred Recipients at 12 months

Cadaveric Renal Allograft Survival Among 1o CsA-Pred Recipients at 12 months

DTE/AHG XM

Neg.

n=166

81%

DTE/AHG XM

Neg.

n=166

81%

Kerman et al, Transplantation; 51:316, 1991Kerman et al, Transplantation; 51:316, 1991

FCXM

Neg. Pos.

n=130 n=36

81% 81%

FCXM

Neg. Pos.

n=130 n=36

81% 81%

Neg-NIH Extended XM: FCXM StudyNeg-NIH Extended XM: FCXM Study

Ogura et al, Transplantation; 56:294, 1993Ogura et al, Transplantation; 56:294, 1993

T-FCXM

Pos.

n=148

75%

T-FCXM

Pos.

n=148

75%

T-FCXM

Neg.

n=693

82%

T-FCXM

Neg.

n=693

82%

P<0.01P<0.01

Could Ron Kerman have been wrong

about his crossmatch results and

interpretation?

Could Ron Kerman have been wrong

about his crossmatch results and

interpretation?

Kerman et al, Transplantation; 68:1855, 1999Kerman et al, Transplantation; 68:1855, 1999

0055

101015152020252530303535404045455050

Negative(n=56)

Negative(n=56)

Positive(n=41)

Positive(n=41)

P=NS P=NS

% R

eje

ctio

n%

Reje

ctio

n

IgG FCXM: Renal Allograft StudyFrequency of Rejection in a Single CenterIgG FCXM: Renal Allograft StudyFrequency of Rejection in a Single Center

Could Ron Kerman have been

wrong about his crossmatch

results and interpretation?

Could Ron Kerman have been

wrong about his crossmatch

results and interpretation?

I don’t think so!I don’t think so!

The Cell Surface Is a JungleThe Cell Surface Is a Jungle

HLAHLA

• NIH-CDC

•AHG-CDC

• Flow cytometry

• NIH-CDC

•AHG-CDC

• Flow cytometry

Membrane-dependent AssaysMembrane-dependent Assays

Detection of membrane receptors may not be related to HLA!

Detection of membrane receptors may not be related to HLA!

ELISA-determined IgG HLA Abs vs MHC-I (pooled platelets)

ELISA-determined IgG HLA Abs vs MHC-I/II (PBL cultures)

Flow bead PRA-determined IgG HLA vs I/II (soluble HLA I/II

antigens on microbeads measured by cytometry)

ELISA-determined IgG HLA Abs vs MHC-I (pooled platelets)

ELISA-determined IgG HLA Abs vs MHC-I/II (PBL cultures)

Flow bead PRA-determined IgG HLA vs I/II (soluble HLA I/II

antigens on microbeads measured by cytometry)

Membrane-independent Assays Membrane-independent Assays

PRA by Different MethodologiesPRA by Different MethodologiesType:

CDC

AHG-CDC

ELISA

Flow

Type:

CDC

AHG-CDC

ELISA

Flow

Positive

102

116

127

139

Positive

102

116

127

139

Negative

162

148

137

125

Negative

162

148

137

125

Gebel & Bray, Transplantation; 69:1370, 2000Gebel & Bray, Transplantation; 69:1370, 2000

AHG-PRA vs Rejection 493 Consecutive CAD Recipients

AHG-PRA vs Rejection 493 Consecutive CAD Recipients

Rejection

YES

NO

Rejection

YES

NO

AHG-PRAAHG-PRA

P=NSP=NS

<10%

134

159

<10%

134

159

10%

100

100

10%

100

100

ELISA-PRA and RejectionELISA-PRA and Rejection

Rejection

YES

NO

Rejection

YES

NO

ELISA-PRAELISA-PRA

P<0.001P<0.001

<10%

38

168

<10%

38

168

10%

117

63

10%

117

63

Correlation Between % ELISA-PRA and Graft SurvivalCorrelation Between % ELISA-PRA and Graft Survival

Graft Survival (months)

12

24

36

Graft Survival (months)

12

24

36

ELISA-PRAELISA-PRA

P<0.01P<0.01

<10% (n=312)

85%

82%

81%

<10% (n=312)

85%

82%

81%

>10% (n=181)

74%

70%

67%

>10% (n=181)

74%

70%

67%

P<0.01P<0.01

P<0.01P<0.01

Sensitivity and sensitization, defining the unsensitized

patient

Application of membrane-independent assays to identify HLA antibodies

Sensitivity and sensitization, defining the unsensitized

patient

Application of membrane-independent assays to identify HLA antibodies

Gebel & Bray, Transplantation; 69:1370, 2000Gebel & Bray, Transplantation; 69:1370, 2000

Tambur et al, Transplantation; 70:1055, 2000Tambur et al, Transplantation; 70:1055, 2000

Correlation of Pre-transplant Abs Detected by Flow PRA with Biopsy-documented Cardiac Rejection

Correlation of Pre-transplant Abs Detected by Flow PRA with Biopsy-documented Cardiac Rejection

Kerman et al, Transplantation; 68:1855, 1999Kerman et al, Transplantation; 68:1855, 1999

0055

101015152020252530303535404045455050

Negative(n=56)

Negative(n=56)

Positive(n=41)

Positive(n=41)

P=NSP=NS

% R

eje

ctio

n%

Reje

ctio

n

IgG FCXM: Renal Allograft StudyFrequency of Rejection in a Single CenterIgG FCXM: Renal Allograft StudyFrequency of Rejection in a Single Center

Were positive crossmatches due to HLA Abs?Were positive crossmatches due to HLA Abs?

• Neoral - CsA

• Steroids

• Prograf - FK506

• Cellcept - MMF

• Rapamycin - Sirolimus

• Thymoglobulin

• OKT3, anti-IL-2R, FTY720

• Neoral - CsA

• Steroids

• Prograf - FK506

• Cellcept - MMF

• Rapamycin - Sirolimus

• Thymoglobulin

• OKT3, anti-IL-2R, FTY720

Immunosuppressive Menu:Immunosuppressive Menu:

If new immunosuppressive therapies reduce

the incidence of acute rejection, are pre-Tx

HLA antibodies clinically relevant?

If new immunosuppressive therapies reduce

the incidence of acute rejection, are pre-Tx

HLA antibodies clinically relevant?

RAPA-CsA-Pred treated primary recipients of CAD renal allografts experience fewer acute rejections vs CsA-Pred recipients.

We therefore tested their pre-Tx sera for the presence of HLA Abs and correlated the results to the occurrence of rejection during the first 12 months post-transplant.

RAPA-CsA-Pred treated primary recipients of CAD renal allografts experience fewer acute rejections vs CsA-Pred recipients.

We therefore tested their pre-Tx sera for the presence of HLA Abs and correlated the results to the occurrence of rejection during the first 12 months post-transplant.

147 RAPA-CsA-Pred recipients were studied

48 patients were chosen specifically because they had a rejection episode.

99 patients were chosen because they had not experienced a rejection episode during the

first year post-transplant.

147 RAPA-CsA-Pred recipients were studied

48 patients were chosen specifically because they had a rejection episode.

99 patients were chosen because they had not experienced a rejection episode during the

first year post-transplant.

PRA Testing

Anti-human globulin (AHG)

ELISA (One Lambda, Inc. LAT)

Flow PRA (One Lambda, Inc.)

PRA Testing

Anti-human globulin (AHG)

ELISA (One Lambda, Inc. LAT)

Flow PRA (One Lambda, Inc.)

AHG-PRA detected 18 reactive sera

ELISA-PRA detected 25 reactive sera (11 vs HLA class I, 3 vs II, 11 vs I/II)

Flow PRA detected 59 reactive sera (31 vs HLA class I, 9 vs II, 19 vs I/II)

AHG-PRA detected 18 reactive sera

ELISA-PRA detected 25 reactive sera (11 vs HLA class I, 3 vs II, 11 vs I/II)

Flow PRA detected 59 reactive sera (31 vs HLA class I, 9 vs II, 19 vs I/II)

Results:Results:

There was no significant correlation between

AHG-PRA, ELISA-detected HLA Abs, and Flow

PRA HLA class II Abs and rejection.

There was no significant correlation between

AHG-PRA, ELISA-detected HLA Abs, and Flow

PRA HLA class II Abs and rejection.• AHG vs Rejection P=NS

• LAT-I vs Rejection P=NS

• LAT-II vs Rejection P=NS

• LAT-I/II vs Rejection P=NS

• F-II vs Rejection P=NS

• AHG vs Rejection P=NS

• LAT-I vs Rejection P=NS

• LAT-II vs Rejection P=NS

• LAT-I/II vs Rejection P=NS

• F-II vs Rejection P=NS

Rejection

NO

YES

Rejection

NO

YES

Flow PRA-1Flow PRA-1

X2=15.7; P<0.001X2=15.7; P<0.001

<5%

76

21

<5%

76

21

5%

23

27

5%

23

27

Flow PRA

0%

Flow PRA

0%

No grafts lost

(+) FCXM vs non-HLA Ab

No grafts lost

(+) FCXM vs non-HLA Ab

Day of 1st Rejection

57 ± 34

Day of 1st Rejection

57 ± 34

FCXMPos. Neg.

2 8

FCXMPos. Neg.

2 8

Flow PRA

13 ± 9%

Flow PRA

13 ± 9%

No grafts lost.No grafts lost.

Day of 1st Rejection

55 ± 31

Day of 1st Rejection

55 ± 31

FCXMPos. Neg.

- 30

FCXMPos. Neg.

- 30

Flow PRA

28 ± 9%

Flow PRA

28 ± 9%

(+) HLA Ab and (-) FCXM: rejection, no grafts lost.

(+) HLA Ab and (+) FCXM: rejection, 58% (7/12) grafts lost.

(+) HLA Ab and (-) FCXM: rejection, no grafts lost.

(+) HLA Ab and (+) FCXM: rejection, 58% (7/12) grafts lost.

Day of 1st Rejection

32 ± 15

Day of 1st Rejection

32 ± 15

FCXMPos. Neg.

12 13

FCXMPos. Neg.

12 13

Flow PRA

48 ± 31%

Flow PRA

48 ± 31%

(+) HLA Ab and (-) FCXM: rejection, no grafts lost.(+) HLA Ab and (+) FCXM: rejection, 63% (5/8)

lost to AMR.

(+) HLA Ab and (-) FCXM: rejection, no grafts lost.(+) HLA Ab and (+) FCXM: rejection, 63% (5/8)

lost to AMR.

Day of 1st Rejection

17 ± 12

Day of 1st Rejection

17 ± 12

FCXMPos. Neg.

8 7

FCXMPos. Neg.

8 7

% PRA

0

13 ± 9

28 ± 9

48 ± 31

% PRA

0

13 ± 9

28 ± 9

48 ± 31

% Rejection

5% (4/75)

13% (4/32)

100%

100%

% Rejection

5% (4/75)

13% (4/32)

100%

100%

Day of Rejection

57 ± 34

55 ± 31

32 ± 9

17 ± 12

Day of Rejection

57 ± 34

55 ± 31

32 ± 9

17 ± 12

N

75

32

25

15

N

75

32

25

15

1. Assays that measure binding of immunoglobulin to targets may not represent HLA Ab reactivity.

2. The AHG-XM protects RAPA-CsA-Pred recipients from hyperacute rejection.

3. The Flow PRA assay detects clinically relevant HLA Abs associated with rejection and/or graft loss.

1. Assays that measure binding of immunoglobulin to targets may not represent HLA Ab reactivity.

2. The AHG-XM protects RAPA-CsA-Pred recipients from hyperacute rejection.

3. The Flow PRA assay detects clinically relevant HLA Abs associated with rejection and/or graft loss.

4. How many antibodies are present may be clinically relevant.

5. The antibody titer may also be important.

6. Patients with pre-Tx (+) HLA Abs and

(+) donor reactivity (+ FCXM) are at risk

for graft rejection and loss.

4. How many antibodies are present may be clinically relevant.

5. The antibody titer may also be important.

6. Patients with pre-Tx (+) HLA Abs and

(+) donor reactivity (+ FCXM) are at risk

for graft rejection and loss.

We have performed heart transplantation following

a negative AHG-XM.

We evaluated the clinical relevance of FCXM for

heart recipients.

We have performed heart transplantation following

a negative AHG-XM.

We evaluated the clinical relevance of FCXM for

heart recipients.

IgG FCXMIgG FCXM

Neg.Neg.

1YGS1YGS 86% 86%

IgG FCXMIgG FCXM

Neg.Neg.

1YGS1YGS 86% 86%

Of the 22 IgG FCXM-Pos. Recipients:

7 grafts were lost

15 grafts were successful

WHY?

Of the 22 IgG FCXM-Pos. Recipients:

7 grafts were lost

15 grafts were successful

WHY?

FCXM Results: Heart RecipientFCXM Results: Heart Recipient

IgG FCXM

Pos.

68%

IgG FCXM

Pos.

68%

P<0.02P<0.02

5 sera tested from lost grafts

All 5 sera were Flow PRA reactive vs MHC I (Flow PRAs of 36%, 52%, 68%, 50% and 49%)

11 sera tested from successful recipients

All 11 sera were Flow PRA non-reactive

5 sera tested from lost grafts

All 5 sera were Flow PRA reactive vs MHC I (Flow PRAs of 36%, 52%, 68%, 50% and 49%)

11 sera tested from successful recipients

All 11 sera were Flow PRA non-reactive

We Flow PRA Tested the IgG FCXM-Pos. SeraWe Flow PRA Tested the IgG FCXM-Pos. Sera

Graft Survival

12 mo.

Graft Survival

12 mo.

FCXM (+)

Flow PRA I/II51%

55% (5/9)

FCXM (+)

Flow PRA I/II51%

55% (5/9)

Rejection 0-12 mo.

Rejection 0-12 mo.

89% (8/9)89% (8/9)31% (4/13)31% (4/13)

FCXM (-)

Flow PRA I/II51%

100% (13/13)

FCXM (-)

Flow PRA I/II51%

100% (13/13)

Both comparisons p<0.01Both comparisons p<0.01

1. HLA Ab negative, FCXM negative (at risk for reversible, cellular rejection)

2. HLA Ab negative, FCXM positive (non-HLA allo-Ab - at risk for reversible, cellular rejection)

1. HLA Ab negative, FCXM negative (at risk for reversible, cellular rejection)

2. HLA Ab negative, FCXM positive (non-HLA allo-Ab - at risk for reversible, cellular rejection)

HLA Ab and Donor Specific ReactivityRank Order of Risk

HLA Ab and Donor Specific ReactivityRank Order of Risk

3. HLA Ab positive, FCXM negative (at risk for reversible, cellular, +/- HLA Ab, rejection)

4. HLA Ab positive, FCXM positive (at risk for humoral/cellular rejection and graft loss)

3. HLA Ab positive, FCXM negative (at risk for reversible, cellular, +/- HLA Ab, rejection)

4. HLA Ab positive, FCXM positive (at risk for humoral/cellular rejection and graft loss)

HLA Ab and Donor Specific ReactivityRank Order of Risk

HLA Ab and Donor Specific ReactivityRank Order of Risk

To transplant or not to transplant, that is the question! Whether it is nobler in the minds of transplant surgeons to treat with thymoglobulin, OKT3, Plasmapheresis, IVIg, or the kitchen sink!

To transplant or not to transplant, that is the question! Whether it is nobler in the minds of transplant surgeons to treat with thymoglobulin, OKT3, Plasmapheresis, IVIg, or the kitchen sink!

1. Pre-transplant identification of

immunologically

high risk patients. Consideration of

induction

and/or maintenance

immunosuppression.

2. Clarify the role of HLA antibody in

rejection

episodes (including the role of C4d ).

1. Pre-transplant identification of

immunologically

high risk patients. Consideration of

induction

and/or maintenance

immunosuppression.

2. Clarify the role of HLA antibody in

rejection

episodes (including the role of C4d ).

Applications Applications

3. Transplantation of highly sensitized and/or positive crossmatch recipients.

4. Long term monitoring of the presence of HLA antibody and graft outcome.

3. Transplantation of highly sensitized and/or positive crossmatch recipients.

4. Long term monitoring of the presence of HLA antibody and graft outcome.

Applications Applications

“I have never let my schooling

interfere with my education.”

-Mark Twain

“I have never let my schooling

interfere with my education.”

-Mark Twain