P1247676903gubFk

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HLA Ab, Donor Reactivity and Risk of Rejection
and Graft Loss
Ronald H. Kerman, PhD The University of Texas
Medical School Houston, TX Division of
Immunology and Organ Transplantation
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• Allograft Rejection

Type Hyperacute Accelerated Acute Chronic
Mediated by Abs Abs/cells Cells/Abs Abs/cells/?
Time 0-48 hrs 5-7 days Early/delayed gt60 days
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Responsibilities of the Histocompatibility
Laboratory

• To identify clinically relevant recipient IgG HLA
  antibodies

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• Positive crossmatches, due to Abs or other
  factors not impacting on graft outcome, should
  not influence the donor-recipient pairing for
  transplantation.

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Detection of Recipient Sensitization

• 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).

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Detection of Immunoglobulin Reactivity

• NIH-CDC
• AHG-CDC
• Flow cytometry
• Membrane-dependent assays

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Complement-dependent Cytotoxicity NIH Assay
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Complement-dependent Cytotoxicity NIH Assay
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Complement-dependent Cytotoxicity NIH Assay
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Anti-human Globulin (Enhancement) Assay
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Anti-human Globulin (Enhancement) Assay
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Anti-human Globulin (Enhancement) Assay
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Flow Cytometry Assay NIH - CDC Negative AHG
CDC Negative Now measuring binding of IgG
(absent C)
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Cadaveric Renal Allograft Survival Among 1o
CsA-Pred Recipients at 12 months
NIH Neg. n166 81 (134/166)
AHG Neg. Pos. n151 n15 82 67
(124/151) (10/15)
Plt0.01
Kerman et al, Transplantation 51316, 1991
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Cadaveric Renal Allograft Survival Among 1o
CsA-Pred Recipients at 12 months
AHG Pos. n15 67 (10/15)
DTE-AHG Neg. Pos. n12 n3 83 0
(10/12) (0/3)
Plt0.01
Kerman et al, Transplantation 51316, 1991
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Cadaveric Renal Allograft Survival Among 1o
CsA-Pred Recipients at 12 months
DTE/AHG XM Neg. n166 81
FCXM Neg. Pos. n130 n36 81 81

Kerman et al, Transplantation 51316, 1991
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Neg-NIH Extended XM FCXM Study
T-FCXM Pos. n148 75
T-FCXM Neg. n693 82
Plt0.01
Ogura et al, Transplantation 56294, 1993
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• Could Ron Kerman have been wrong about his
  crossmatch results and interpretation?

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IgG FCXM Renal Allograft Study Frequency of
Rejection in a Single Center
Rejection
Kerman et al, Transplantation 681855, 1999
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• Could Ron Kerman have been wrong about his
  crossmatch results and interpretation?

I dont think so!
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The Cell Surface Is a Jungle
HLA
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Membrane-dependent Assays

• NIH-CDC
• AHG-CDC
• Flow cytometry

Detection of membrane receptors may not be
related to HLA!
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Membrane-independent Assays

• 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)

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PRA by Different Methodologies
Type CDC AHG-CDC ELISA Flow
Positive 102 116 127 139
Negative 162 148 137 125
Gebel Bray, Transplantation 691370, 2000
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AHG-PRA vs Rejection 493 Consecutive CAD
Recipients
AHG-PRA
Rejection YES NO
lt10 134 159
?10 100 100
PNS
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ELISA-PRA and Rejection
ELISA-PRA
Rejection YES NO
lt10 38 168
?10 117 63
Plt0.001
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Correlation Between ELISA-PRA and Graft Survival
ELISA-PRA
Graft Survival (months) 12 24 36
lt10 (n312) 85 82 81
gt10 (n181) 74 70 67
Plt0.01
Plt0.01
Plt0.01
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• Sensitivity and sensitization, defining the
  unsensitized patient
• Application of membrane-independent assays to
  identify HLA antibodies

Gebel Bray, Transplantation 691370, 2000
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Correlation of Pre-transplant Abs Detected by
Flow PRA with Biopsy-documented Cardiac Rejection
Tambur et al, Transplantation 701055, 2000
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IgG FCXM Renal Allograft Study Frequency of
Rejection in a Single Center
Were positive crossmatches due to HLA Abs?
Rejection
Kerman et al, Transplantation 681855, 1999
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Immunosuppressive Menu

• Neoral - CsA
• Steroids
• Prograf - FK506
• Cellcept - MMF
• Rapamycin - Sirolimus
• Thymoglobulin
• OKT3, anti-IL-2R, FTY720

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• If new immunosuppressive therapies reduce the
  incidence of acute rejection, are pre-Tx HLA
  antibodies clinically relevant?

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• 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.

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• 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.

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• PRA Testing
• Anti-human globulin (AHG)
• ELISA (One Lambda, Inc. LAT)
• Flow PRA (One Lambda, Inc.)

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Results

• 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)

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• There was no significant correlation between
  AHG-PRA, ELISA-detected HLA Abs, and Flow PRA HLA
  class II Abs and rejection.

• AHG vs Rejection PNS
• LAT-I vs Rejection PNS
• LAT-II vs Rejection PNS
• LAT-I/II vs Rejection PNS
• F-II vs Rejection PNS

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Flow PRA-1
Rejection NO YES
lt5 76 21
?5 23 27
X215.7 Plt0.001
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Day of 1st Rejection 57 34
Flow PRA 0
FCXM Pos. Neg. 2 8
No grafts lost () FCXM vs non-HLA Ab
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Day of 1st Rejection 55 31
Flow PRA 13 9
FCXM Pos. Neg. - 30
No grafts lost.
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Day of 1st Rejection 32 15
Flow PRA 28 9
FCXM Pos. Neg. 12 13
() HLA Ab and (-) FCXM rejection, no grafts
lost. () HLA Ab and () FCXM rejection, 58
(7/12) grafts lost.
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Day of 1st Rejection 17 12
Flow PRA 48 31
FCXM Pos. Neg. 8 7
() HLA Ab and (-) FCXM rejection, no grafts
lost. () HLA Ab and () FCXM rejection, 63
(5/8) lost to AMR.
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Day of Rejection 57 34 55 31 32 9 17
12
PRA 0 13 9 28 9 48 31
Rejection 5 (4/75) 13 (4/32) 100 100
N 75 32 25 15
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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.
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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.
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We have performed heart transplantation following
a negative AHG-XM. We evaluated the clinical
relevance of FCXM for heart recipients.
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FCXM Results Heart Recipient
IgG FCXM Neg. 1YGS 86
IgG FCXM Pos. 68
Plt0.02
Of the 22 IgG FCXM-Pos. Recipients 7 grafts
were lost 15 grafts were successful WHY?
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We Flow PRA Tested the IgG FCXM-Pos. Sera

• 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

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FCXM () Flow PRA I/II ?51 55 (5/9)
FCXM (-) Flow PRA I/II ?51 100 (13/13)
Graft Survival 12 mo.
89 (8/9)
31 (4/13)
Rejection 0-12 mo.
Both comparisons plt0.01
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HLA Ab and Donor Specific Reactivity Rank Order
of Risk
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)
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HLA Ab and Donor Specific Reactivity Rank 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)
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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!
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Applications
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 ).
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Applications
3. Transplantation of highly sensitized and/or
positive crossmatch recipients. 4. Long
term monitoring of the presence
of HLA antibody and graft outcome.
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• I have never let my schooling interfere with my
  education.
• -Mark Twain