Transcript AbSorber

XM-ONE® - A complementary approach
to assessing risk in Solid Organ
Transplantation
”Insight into the role of non-HLA antibodies and Rejections”
Content
 AMR, DSA and C4d
 Pre transplant assessment of risk
in Organ Transplant Patients
Risk Assessment using XM-ONE®
Antibody Detection Methods
 HLA
- CDC
- ELISA
- Flow PRA
- Solid Phase Assay (Luminex)
- Flow XM (using T- and/or B cells)
 Non-HLA
-ELISA (MICA / MICB)
-Solid Phase Assay (MICA)
-XM-ONE (Anti Endothelial Cell Antibodies)
Pre Tx antibodies influence graft failure
Reference:
Gerhard Opelz for the
Collaborative
Transplant Study*
Lancet 2005; 365: 1570–76
HLA-identical siblings also reject!
The PRA activity in HLA
identical siblings is
associated with poorer graft
survival
Patients cannot form
antibodies against their own
HLA antigens; therefore they
cannot form anti-HLA against
lymphocytes of an HLAidentical sibling donor.
Reference:
Gerhard Opelz for the
Collaborative
Transplant Study*
Lancet 2005; 365: 1570–76
These findings further
supports the role of non-HLA
antibodies
Development of antibody awareness and
diagnostics
1968:
1985:
1997:
2005:
2009:
Post tx HLA ab’s
associated with
poor survival
(Morris and coworkers)
First publications
regarding endothelial
alloantigens and
transplantation
First cases
published on AECA
and rejection
(Heart tx, Kidney
Tx)
The Collaborative
Transplant Study
HLA identicals
have PRA (G Opelz)
XM-ONE
Multicenter
study (M
Breimer)
non-HLA
HLA
1969:
1993:
2002:
2006:
2008:
HLA crossmatching
(Patel & Terasaki)
First Olerup
SSP HLA
typing test
on market
Introducing
the LUMINEX100
XM-ONE CE
marked in
Europe
XM-ONE
cleared by
FDA in US
Banff Criteria for AMR
•C4d+
•Presence of circulating DSA
•Acute tissue injury
Solez et al, Am J
Transpl: 2008; 8:
753–760
Donor Specific Antibodies are considered to
be a risk factor in organ transplantation
• The presence of donor-specific antibodies is associated with
an increased risk of graft loss (Lachmann, N et al, Transplantation. 2009 May
27;87(10):1505-13)
• The presence of preformed DSA is strongly associated with
increased graft loss in kidney transplants, related to an
increased risk of AMR (Lefaucheur C et al, Humoral Immunity in Kidney Transplantation.
Contrib Nephrol. Basel, Karger, 2009, vol 162, pp 1-12 )
• High baseline DSA patients have high rates of AMR and poor
long-term allograft survival highlighting the need for
improved therapy for these candidates (J. M. Gloor et al, Am J transpl
American Journal of Transplantation, 2010, Vol 10 (3):582–589)
• Pre-transplant Donor-Specific Antibodies Detected by Single-Antigen
Bead Flow Cytometry Are Associated With Inferior Kidney Transplant
Outcomes (Singh, N et al, Transplantation: 27 November 2010 - Vol 90 (10):1079-1084)
C4d – the footprint of DSA
• ”C4d is a fragment of complement component C4
released during activation of the classical complement
pathway by antigen-antibody complexes”
Chakravarti DN,
Campbell RD,
Porter RR:
Molecular
Immunology 24:
1187–1197,
1987
C4d is associated with Graft Failure
Mauiyyedi, S et al,
J Am Soc Nephrol
13: 779–787, 2002
C4d deposition correlates to DSA
Mauiyyedi, S et al,
J Am Soc Nephrol
13: 779–787, 2002
Antibody Detection Methods
 HLA
- CDC
- ELISA
- Flow PRA
- Solid Phase Assay (Luminex)
- Flow XM (using T- and/or B cells)
Rejections in HLA DSA
negative patients in Heart,
Lung or Kidney
Transplantation
AMR in Heart Tx
•985 biopsies from 107 heart transplant
recipients were evaluated
•C4d positive staining was found in 36
patients (34%)
•HLA DSA identified by LUMINEX was
present in 14 patients (13%) at the time
of rejection
•AMR was diagnosed in 8 patients (7%)
according to ISHLT recommendations
Ref: Fedrigo et al,
Transplantation,
Vol 90 (7)
Oct 15, 2010
No Graft Dysfunction = asAMR,
Graft Dysfunction = symptomatic
AMR
107 HTx
Control
(n=71)
C4d+, DSA-,
no GD
C4d+, DSA+,
no GD
C4d+, DSA+,
GD
(n=22)
(n=6)
(n=8)
Questions:
C4d positive staining can occur without DSA?
C4d positive staining can only occur in the presence of
AB’s?
The above AB’s were not detected by LUMINEX:
due to lack of sensitivity?
since the AB’s responsible are non-HLA AB’s?
Ref: Fedrigo et al,
Transplantation,
Vol 90 (7)
Oct 15, 2010
Magro et al:
Am J of Transpl
2003; 3:
1264–1272
Early (<7 days) AMR in KTx
Amico et al:
Transplantation
2008;85:
1557–1563
Amico et al:
Transplantation
2008;85:
1557–1563
AMR and DSA
• Patients receive an allograft
based on a negative complementdependent cytotoxicity (CDC) crossmatch
• At time of transplantation many patients display
donor-specific antibodies (DSA) by sensitive methods
(solid-phase assays, FCXM)
• Study comparing different crossmatch techniques
(PRA, SPA, FCXM) in detecting DSA correlated to
“AMR” (defined according to Banff minus DSA)
Ref: Vlad et al;
Human Imm 70
(2009) 589–594
Demographics
n
Patients
325
Primary Graft
260
Secondary Graft
AMR*
PRA
Pos DSA SPA
Pos DSA FCXM (T/B)
65 (20%)
29 (9%)
129 (40%)
27 (8%)
47 (14%)
*AMR was diagnosed if C4d+ and morphologic tissue injury
Ref: Vlad et al;
Human Imm 70
(2009) 589–594
AMR* occur in 9% of CDC negative patients
*AMR=Banff without DSA
100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
Ref: Vlad et al;
Human Imm 70
(2009) 589–594
PRA<10%
(1.5%)
FCXM(4.3%)
DSA(6.2%)
Negative
Positive
PRA >10%
(7.4%)
FCXM+
(4.6%)
DSA+
(2.8%)
PRA > or < 10%
DSA SPA
FCXM
Graft Survival
Negative in test (SPA, FCXM)
but ”AMR” diagnosed
Discussion
• ”Although the Luminex SPA method is excellent for
identifying anti-HLA antibodies, non-HLA antibodies
represent a “blind-spot” for this type of testing.
Essentially, the SPA will always return a false-negative
result if the target is not an HLA antigen”
• ”Cell-based methods are less susceptible to this type
of problem, because the donor cells used for testing
emulate the comprehensive antigenic makeup of the
prospective graft with much greater fidelity”
Ref: Vlad et al;
Human Imm 70
(2009) 589–594
Background summary
• Assessing risk of post transplant complications
• DSA, Donor Specific Antibodies, are associated with poorer
outcome
• C4d – ”the footprint of DSA” – can occur without detected
HLA DSA
• PRA does not correlate with HLA DSA – evidence of non-HLA
DSA
• Would assessing non-HLA DSA improve the risk
assessment?
XM-ONE® :
Detects antibodies against cell bound antigens
HLA I
non-HLA
Tie-2-r
HLA II
Peripheral blood endothelial cell precursor
XM-ONE identifies more than HLA
HLA I
non-HLA
HLA DSA positive (n=37)
XM-ONE
pos
35
XM-ONE
neg
2
HLA DSA negative (n=98)
45
53
HLA II
Reference:
AbSorber, Data on file
The sensitivity of XM-ONE as compared to the Flow PRA Single
Antigen bead assay is 35 (positive in XM-ONE) / 37 (positive in
Flow PRA), 95%.
Expression of surface markers on Tie2
isolated cells from PBMC
Tie2 isolated
Ref:
AbSorber , Data on File
MHC I
Yes
MHC II
Yes
CD11b
Yes
CD14
Yes
CD31
Yes
CD32
Yes
CD34
Low
CD68
Low
CD133
Yes
CD144
Low
VEGFR1
Low
VEGFR2
Low
Green indicates a higher expresion when
compared to total non-isolated cells
Organ Donor
Recipient
Collect blood in
BD CPT™ tubes
Collect blood in
serum tube
Centrifuge to
isolate PBMC
Centrifuge to
obtain sera
Retrieve PBMCs into 50 ml tube
and wash cells
Incubate PBMCs with
magnetic beads
to isolate EPC
Anti-Tie-2
Incubate isolated EPCs with
recipient sera and control sera.
Wash three times
Incubate with secondary
FITC conjugated antibodies
against IgG and IgM
Analyse by Flow
Cytometry
Reading XM-ONE® by flow cytometry
Picture shows CE marked version;
for US no CD3 and CD19 antibodies are included
XM-ONE Background
• Developed to detect antibodies causing Hyper Acute
Rejections (HAR) in patients at Karolinska Institute,
Sweden
• Multicenter study designed to assess risk of HAR from
pre transplant testing for non-HLA DSA (8% XM-ONE
positivity, 25% HAR)
• Study sample size was calculated to be 280 patients
• Ethics Committee (EC) decided to have interim
analysis for every 100 patients enrolled
XM-ONE® prospective multicenter study
Ref: Breimer et al;
Transplantation
87(4):549-556,
February 27, 2009.
Baylor University Med Center, Dallas, TX, Johns Hopkins University, Baltimore, MD, Ohio
State University Med Center, Columbus, OH, Massachusetts General Hospital, Boston, MA,
USA
Karolinska Institute, Huddinge Hospital, Stockholm, Sahlgrenska University Hospital,
Gothenburg, Sweden
Study Design
Recruitment
Patient Information
Lymphocyte
crossmatch (LXM)
Endothelial Cell
crossmatch
(XM-ONE)
n=147
Clinical follow-up
> 3 month / rejection
Ref: Breimer et al;
Transplantation
87(4):549-556,
February 27, 2009.

Decisions about transplantation and immunosuppressive treatment
based on results from LXM and solid phase assay

If a rejection episode occured responsible staff was informed about
XM-ONE results before making decisions about immunosuppressive
treatment
Patient Demographics (N=147 )
Gender
Male
59%
Female
41%
Recipient Race
Caucasians
123 (84%)
Afro-Americans
12 (8%)
Hispanics
2 (1%)
Other
10 (7%)
Donors
LD
83%
DD
17%
Pregnancy
47%
Blood transfusion
28%
Previous transplant
14%
Sensitizing events
Ref: Breimer et al;
Transplantation
87(4):549-556,
February 27, 2009.
XM-ONE® result correlates to rejections
Total
(n=29)
XM-ONE
positive
(n=35)
XM-ONE
negative
(n=112)
12%
(13/112)
TOTAL
(n=147)
20%
(29/147)
46%
(16/35)
XM-ONE® result correlates to rejections,
PRA levels does not
PRA>10% with PRA<10% with
ARE (n=9)
ARE (n=20)
XM-ONE
positive
(n=35)
XM-ONE
negative
(n=112)
TOTAL
(n=147)
Total
(n=29)
46%
(6/13)
45%
(10/22)
46%
(16/35)
15%
(3/20)
11%
(10/92)
12%
(13/112)
27%
(9/33)
18%
(20/114)
20%
(29/147)
XM-ONE® assesses risk for acute rejections in
kidney transplant recipients
XM-ONE
positive
(n=35)
XM-ONE
negative
(n=112)
Total with
PRA>10% with PRA<10%
(n=29)
ARE (n=9)
ARE
(n=20)
XM-ONE
positive
46%*
46%
45%
(n=35)
(16/35)
(6/13)
(10/22)
XM-ONE
negative
12%
15%
11%
(n=112)
(13/112)
(3/20)
(10/92)
TOTAL
(n=147)
TOTAL
27%
(n=147)
(9/33)
20%
18%
(29/147)
(20/114)
Total
(n=29)
46%
*p<0.0005
(16/35)
12%
(13/112)
20%
(29/147)
XM-ONE® positive crossmatch correlates
to acute rejections
100%
80%
60%
19
118
99
40%
16
20%
29
13
0%
All patients
Rejection
Ref: Breimer et al;
Transplantation
87(4):549-556,
February 27, 2009.
XM-ONE +
No rejection
XM-ONE -
XM-ONE® positive patients experienced
rejections early after transplantation
Ref: Breimer et al;
Transplantation
87(4):549-556,
February 27, 2009.
All C4d positive rejections had non-HLA
DSA as defined by XM-ONE positivity
XM-ONE® positive XM-ONE® negative
(35)
(112)
Ref: Breimer et al;
Transplantation
87(4):549-556,
February 27, 2009.
C4d positive at
first rejection
episode
6
(17%)
0
(0%)
C4d negative at
first rejection
episode
10
(29%)
13
(12%)
Creatinine levels were significantly
higher in XM-ONE® positive patients
1.77
mg/dL
1.73
mg/dL
1.39
mg/dL
p < 0,05
Ref: Breimer et al;
Transplantation
87(4):549-556,
February 27, 2009.
1.43
mg/dL
p < 0,05
Study Conclusions
 XM-ONE® positive patients experience significantly more
rejections than XM-ONE® negative patients
 XM-ONE® positive patients experienced earlier and more severe
rejections than XM-ONE® negative patients
 XM-ONE® positive patients have higher creatinine values at 3
and 6 months after transplantation
Ref: Breimer et al;
Transplantation
87(4):549-556,
February 27, 2009.
Enhancing the risk assessment
 Factors influencing graft survival
 HLA as well as non-HLA antibodies are associated with impaired graft half-life
 Patients experiencing acute rejections have shorter graft survival
 S-creatinine is recognized as a surrogate marker for graft half-life
 XM-ONE® identifies patients at risk*
 Detects patients at increased risk of rejections and reduced kidney function
 XM-ONE® provides valuable information on the expected immune respons on
the transplanted organ
*Referenced to Breimer et al, Transplantation 2009
Karolinska Cases
 A girl transplanted in 1993 *
- Abrupt graft failure (x3) due to Hyperacute Rejection
A boy transplanted in 1998
- Abrupt graft failure (x2) due to Hyperacute Rejection
Endothelial Antibodies detected i both
patients (through UVEC)
 Both patients have retrospectively been
confirmed as XM-ONE® positive against
the first donor
*Reference:
Sumitran-Karuppan S et al,
Transplant Immunology
1997;5:321-327
A French case
• Patient transplanted with a kidney from a living related donor
• No HLA antibodies detected in crossmatches or solid phase screens
• The kidney was lost a few days after transplantation
• Pretransplant sera as well as rejection sera was sent to
Karolinska for XM-ONE® tests
• Pretransplant sera contained donor specific IgM antibodies. At the
time of rejection the patient had class switched to IgG
• Data were presented at the recent EFI meeting
A US Case
• A patient with Negative PRA and Negative lymphocyte
crossmatches experienced Graft loss due to antibody
mediated rejection
• The clinic contacted OSUMC that was participating in
the XM-ONE® prospective study and XM-ONE tests
were performed
• The pretransplant sera was positive in XM-ONE tests
against the donor but negative against several
independent blood donors
• Presented at the ATC meeting, Jon von Visger et al
ABOi Case Study
Johns Hopkins University Hospital
• Day 0
• Non sensitized, AB0 incompatible KTx (AB to 0)
• Anti-A titer: 256 to 8 on day of transplant
• XM-ONE®, strongly positive
• Immediate graft function
• Day 3 post-tx
• S-cr rose to 2.6 mg/dl, urine output fell
• Torsion of kidney, repaired and regain of allograft function
• Biopsy: g2, i0, t0, v0, ptc3,
• C4d3, Suggested of AMR
• No rise in anti-A titer
• Day 6 post-tx,
• 2 x pp
Ref: A Jackson,
ATC Workshop,
May 1, 2010
• XM-ONE® negative
Discharged with S-cr =1.3 mg/dl
Case, Karolinska Hospital,
Stockholm, Sweden
• 22 -year-old female
• FSGS since childhood and
started hemodialysis in 1999
• First KTx 2000;
• the graft failed within hours
• Negative CDC and FXM (the
rejection was believed to be
caused by non-HLA Abs)
• 2004:
• transplantation was again
considered, the mother
was evaluated as donor
Ref: Holgerson, J
et at, Clin Transpl.
2006:535-8
• Blood group-incompatible
(A1 RhD-positive and the
donor A1B RhD-positive)
• Anti-donor RBC titers: 1:16 for
both IgM and IgG.
• HLA:
• HLA typed using PCR-SSP (no
repeated HLA mismatches)
• No anti-HLA class I or II Abs (FCbased FlowPRA, conventional Tand B-lymphocyte CDCs, Tlymphocyte FXM, all negative
• Non HLA, XM-ONE®
• IgM +, IgG -
Immunosuppressive protocol for AB0i + IA
Disease Course
Ref: Holgerson, J
et at, Clin Transpl.
2006:535-8
Disease Course
Decision on biopsy on XM-ONE result
Ref: Holgerson, J
et at, Clin Transpl.
2006:535-8
Post Tx
• Day 9 post-transplant:
• S-cr from 72 to 91 μmol/L.
• XM-ONE® switched to be positive for IgG (pre-Tx IgM +)
• anti-A1B titers low (IgM 2, IgG 1).
• Ultrasonography: slight increase in the R.I. index to 0.6–0.7
• T-lymphocyte XM negative
• Immunoadsorptions were re-initiated.
• Day 10: Kidney biopsy showed acute vascular rejection
(Banff type IIA-B) with a humoral component (C4d
positive).
• Day 14: S-Cr 349 μmol/L.
Ref: Holgerson, J
et at, Clin Transpl.
2006:535-8
Following rejection treatment (0.5 g Solu-Medrol on three
consecutive days) and repeated immunoadsorptions, the
patient’s kidney function was normalized
Summary
• Acute rejection occur in HLA negative patients
• The multicenter study published in 2009 showed that, in
these HLA negative patients, 24% are positive to non-HLA
ab (as identified by XM-ONE®)
• As shown by Breimer et al, XM-ONE® positivity is a risk
factor for early rejection and subsequent impaired
renal function
• In patients being HLA negative in the standard assays
Would you like to know more?
Immunosuppression from Karolinska Case
(slide 51)
•Rituxmab (375 mg per m2 bodysurface) 2 months
pre-operative and on the day of operation;
•Repeated (n = 5) protein A immunoadsorption
•IvIg (25 g) after the last immunoadsorption
•Conventional immunosuppressive regimen
• Prograf, CellCept, Prednisolon regimen starting >1 week
pre-operative
Ref: Holgerson, J
et at, Clin Transpl.
2006:535-8