Transcript Slide 1

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PRA = 36% (21/58)
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Anti-A11 and B44
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PRA = 36% (21/58)
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PRA = 95% (55/58)
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Specificity?
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Flow Cytometry Crossmatch
T cell
B cell
Flow Cytometry Crossmatch
FITC-a-IgG
T cell
B cell
Flow Cytometry Crossmatch
FITC-a-IgG
FITC-a-IgG
T cell
B cell
Flow Cytometry Crossmatch
Anti-CD3
Anti-CD19
FITC-a-IgG
FITC-a-IgG
T cell
B cell
Flow Cytometry Crossmatch
FITC-a-IgG
FITC-a-IgG
Anti-CD3
T cell
Anti-CD19
Detect fluorescent labels by flow cytometry
B cell
Flow Crossmatch Implemented
in Halifax, June 2010
Flow Cytometry Crossmatch
T cell X-match
B cell X-match
Negative
Gating strategy
Weak
positive
Strong
positive
FITC-a-IgG
FITC-a-IgG
Karpinski et al. JASN 2001
•Retrospective flow cytometry crossmatch study
•249 patients transplanted (June 1992 and June 2000) with
negative CDC-AHG crossmatch
Karpinski et al. JASN 2001
Strategies used to avoid/minimize
transplant rejection
• HLA typing and matching of recipient/donor pairs
• Detection of donor specific HLA antibodies.
– Lymphocyte crossmatch
• Complement dependent cytotoxicity (CDC) crossmatch.
• Flow cytometry crossmatch (newer technique, much more sensitive)
– Virtual crossmatch
• Identification of HLA antibodies in recipient serum by solid phase assay
• HLA typing of the donor (and recipient)
• Correlation of recipient HLA antibodies and donor/recipient typing
HLA antibody identification by Luminex
(solid phase) Assay
HLA antigen coated microspheres
2 lasers
Tells the instrument
which bead is being
examined
Tells the instrument how much
antibody is bound to the bead
HLA antibody detection by Luminex assay
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HLA antibody detection by Luminex assay
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A1
A2
A3
A11
A23
A24
A25
A26
A29
A30
HLA antibody detection by Luminex assay
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A26
A29
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A25
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A30
A11
A24
A1
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A26
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A3
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A23
A29
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A23
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A25
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A30
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A11
A24
HLA antibody detection by Luminex assay
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A26
A29
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A25
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A30
A11
A24
A1
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A3
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A23
A29
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A23
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A25
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A30
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A11
A24
HLA antibody detection by Luminex assay
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A26
A29
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A25
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A30
A11
A24
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A23
A29
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A23
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A25
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A30
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HLA antibody detection by Luminex assay
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A26
A29
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A25
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A30
A11
A24
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A23
A29
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A23
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A25
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A30
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HLA antibody detection by Luminex assay
PE-a-IgG
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A26
A29
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A25
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A30
A11
A24
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A23
A29
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A25
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HLA antibody detection by Luminex assay
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A26
A29
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A25
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A11
A24
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A26
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A23
A29
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Patient Case
HLA Class I antibody analysis
Patient A3,31 B7,60 DR1,14 (52)
DQB5,6
HLA Class I antibody analysis
Patient A3,31 B7,60 DR1,14 (52) DQB5,6
Donor A1, B8 DR7,17 (53,52) DQB2
HLA Class I antibody analysis
Patient A3,31 B7,60 DR1,14 (52) DQB5,6
Donor A1, B8 DR7,17 (53,52) DQB2
Unacceptable antigens: A1, A36, B8
HLA Class II antibody analysis
Patient A3,31 B7,60 DR1,14 (52) DQB5,6
Donor A1, B8 DR7,17 (53,52) DQB2
HLA Class II antibody analysis
Patient A3,31 B7,60 DR1,14 (52) DQB5,6
Donor A1, B8 DR7,17 (53,52) DQB2
Unacceptable antigens: DR7, DR53, DQ2
What is the clinical relevance of
donor specific HLA antibodies
detected pre-transplant by solid
phase assay?
Amico et al. Transplantation 2009
Significant increase in biopsy proven AMR in patients with pre-transplant DSA
Lefaucheur et al. JASN 2010
Significant decrease in graft survival in patients with pre-transplant DSA
Class I and Class II DSA confer similar risk.
What about PRA?
(probability of a positive crossmatch)
Calculated PRA
• calculated PRA (cPRA) is based on the
unacceptable HLA antigens listed for a patient
• cPRA is determined using an established
algorithm (Zachary et al) and HLA frequencies
derived from the HLA phenotypes of more than
12,000 donors recently entered into the US OPTN
registry
CPRA Calculator
http://optn.transplant.hrsa.gov/
Resources, professional resources, choose cPRA calculator from options
Tambur et al. AJT 2009
Correlation between virtual and Flow crossmatch
FP 3.1%
FN 14%
Some allele specific non-DSA
Some weak DSA
Non-HLA abs
False pos FCXM
Tambur et al. AJT 2009
• Virtual crossmatch is a good tool to predict HLA
compatibility.
• Caveats:
•
Antibodies against all donor HLA antigens have to be
investigated.
•
Strength of the antibody has to be considered.
•
Non-HLA antibodies.
A Virtual Crossmatch Protocol Significantly Increases Access of Highly
Sensitized Patients to Deceased Donor Kidney Transplantation.
Bingaman et al. Transplantation 2008
FP = 3%
12%
Cost effective
Decreased TAT
Increases access to transplantation of highly sensitized patients
Negative virtual crossmatch predicts negative
flow crossmatch
Crossmatches performed since implementation
of flow crossmatch (June 2010 – September 2011).
157
# of crossmatches
160
FP rate = 2.5%
140
120
negative
positive
100
80
60
4
40
20
0
No DSA
Virtual Crossmatch
Halifax Lab experience
Renal Transplant Patient Workup
•
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•
•
HLA typing, SSO.
Sera collected monthly and after sensitizing event.
Antibody identification by Luminex every 3 months.
Unacceptable antigens and HLA typing are entered into MOTP
database.
• Donor HLA typing performed and entered into MOTP database.
• Smartmatch excludes potential recipients with unacceptable
mismatches.
• Top 5 potential recipients are selected for crossmatch.
• Top 2 recipients with negative crossmatch proceed to Tx
• Day of transplant serum and sera collected at 3 weeks and 3 months
post transplant are also tested.
Virtual Crossmatch
HLA typing
VXM
HLA antibodies identified
Patient 1
A1,3
B8,50
DR4,17
A11,A24,A25,B18,B44,DR12
Patient 2
A2,3
B44,62
DR7,8
A1,A26,A33,B52,DR15
Patient 3
A3,11
B8,18
DR4,15
A2,A31,A66,B7,B52
Patient 4
A1,24
B7,45
DR9,12
A3,A30,B60,B61,DR15,DR16
Patient 5
A23,24
B27,35
DR10,16
A1,B8,B44,DR7
Patient 6
A2,23
B51,55
DR9,17
A3,B60,B61,B62,B63,DR12,DR13
Patient 7
A1,30
B7,60
DR11,13
A2,DR1,DR7,DR8
Patient 8
A3,31
B27,61
DR4,7
A1,A23,A24,B18,B45,DR11,DR12
Patient 9
A2,24
B7,45
DR4,8
B27,B51,DR15,DR16
Patient 10
A2,2
B37,44
DR9,12
B8,B60,B61,DR10
Virtual Crossmatch
Donor
A1,2
B7,8
HLA typing
VXM
DR4,17
HLA antibodies identified
Patient 1
A1,3
B8,50
DR4,17
A11,A24,A25,B18,B44,DR12
Patient 2
A2,3
B44,62
DR7,8
A1,A26,A33,B52,DR15
Patient 3
A3,11
B8,18
DR4,15
A2,A31,A66,B7,B52
Patient 4
A1,24
B7,45
DR9,12
A3,A30,B60,B61,DR15,DR16
Patient 5
A23,24
B27,35
DR10,16
A1,B8,B44,DR7
Patient 6
A2,23
B51,55
DR9,17
A3,B60,B61,B62,B63,DR12,DR13
Patient 7
A1,30
B7,60
DR11,13
A2,DR1,DR7,DR8
Patient 8
A3,31
B27,61
DR4,7
A1,A23,A24,B18,B45,DR11,DR12
Patient 9
A2,24
B7,45
DR4,8
B27,B51,DR15,DR16
Patient 10
A2,2
B37,44
DR9,12
B8,B60,B61,DR10
Virtual Crossmatch
Donor
A1,2
B7,8
HLA typing
VXM
DR4,17
HLA antibodies identified
Patient 1
A1,3
B8,50
DR4,17
Neg
A11,A24,A25,B18,B44,DR12
Patient 2
A2,3
B44,62
DR7,8
Pos
A1,A26,A33,B52,DR15
Patient 3
A3,11
B8,18
DR4,15
Pos
A2,A31,A66,B7,B52
Patient 4
A1,24
B7,45
DR9,12
Neg
A3,A30,B60,B61,DR15,DR16
Patient 5
A23,24
B27,35
DR10,16
Pos
A1,B8,B44,DR7
Patient 6
A2,23
B51,55
DR9,17
Neg
A3,B60,B61,B62,B63,DR12,DR13
Patient 7
A1,30
B7,60
DR11,13
Pos
A2,DR1,DR7,DR8
Patient 8
A3,31
B27,61
DR4,7
Pos
A1,A23,A24,B18,B45,DR11,DR12
Patient 9
A2,24
B7,45
DR4,8
Neg
B27,B51,DR15,DR16
Patient 10
A2,2
B37,44
DR9,12
Neg
B8,B60,B61,DR10
Virtual Crossmatch
Donor
A1,2
B7,8
HLA typing
VXM
DR4,17
HLA antibodies identified
Patient 1
A1,3
B8,50
DR4,17
Neg
A11,A24,A25,B18,B44,DR12
Patient 4
A1,24
B7,45
DR9,12
Neg
A3,A30,B60,B61,DR15,DR16
Patient 6
A2,23
B51,55
DR9,17
Neg
A3,B60,B61,B62,B63,DR12,DR13
Patient 9
A2,24
B7,45
DR4,8
Neg
B27,B51,DR15,DR16
Patient 10
A2,2
B37,44
DR9,12
Neg
B8,B60,B61,DR10
Highly Sensitized Patient
Case
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Specificity?
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Highly sensitized patient, Case 1
Class I specificity
A1 A23 A24 A25 A32 B13 B27 B37 B38 B41 B44 B45 B47 B48 B49 B50 B51 B52 B53 B57 B58 B59
B60 B61 B63 B7 B76 B77 B8 B81 B82
cPRA = 96%
Patient typing A*11,33 B*35,35 Cw*04,04 DRB1*04,13 DR52, 53 DQ*03(7),03(8)
Donor typing A*11,03 B*35,62 Cw*04,10 DRB1*04,11 DR52, 53 DQ*03(7),03(8)
Virtual crossmatch in
transplantation from live donors
Case 1
• Potential recipient
Mother
• Potential donor
Son
Recipient HLA typing
A3,3
B7,7
Cw7,7
DR4,15
Donor HLA typing
A1,3
B7,8
Cw7,7
DR4,17
DQ6,7
DQ2,7
Class I HLA antibody analysis
Donor specific antibodies:
A1, B8
Recipient HLA typing
A3,3
B7,7
Cw7,7
DR4,15
Donor HLA typing
A1,3
B7,8
Cw7,7
DR4,17
DQ6,7
DQ2,7
Class II HLA antibody analysis
Donor specific antibodies:
DR17, DQ2?
Recipient HLA typing
A3,3
B7,7
Cw7,7
DR4,15
Donor HLA typing
A1,3
B7,8
Cw7,7
DR4,17
DQ6,7
DQ2,7
Case 2
Case 2
Recipient
Sister
Brother
Mother
A
03
03
02
03
03
02
B
35
49
08
49
35
C
04
07
07
07
Bw
6
4
6
DRB1
04
04
DRB3/4/5
53
DQB1
DQA1
03
Father
02
02
03
15(62) 49
15(62) 08
35
04
03(10) 07
03(10) 07
04
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6
6
4
6
6
6
13
04
04
04
04
04
13
04
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52
53
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53
03(7)
03(8)
06
03(8)
03(7)
03(8)
03(8)
03(8)
06
03(7)
03
03
01
03
03
03
03
03
01
03
Case 2
Recipient
Sister
A
03
03
02
03
03
02
B
35
49
08
49
35
C
04
07
07
07
Bw
6
4
6
DRB1
04
04
DRB3/4/5
53
DQB1
DQA1
MM
Brother
Mother
02
03
15(62) 49
15(62) 08
35
04
03(10) 07
03(10) 07
04
4
6
6
4
6
6
6
13
04
04
04
04
04
13
04
53
52
53
53
53
53
53
52
53
03(7)
03(8)
06
03(8)
03(7)
03(8)
03(8)
03(8)
06
03(7)
03
03
01
03
03
03
03
03
01
03
4/10
3/10
03
Father
3/10
02
4/10
Class I HLA antibody analysis
Class I specificity
B8 B76 B82
Cw5
Patient typing A*03,03 B*35,49 Cw*04,07 DRB1*04,04 DR53, 53 DQ*03(7),03(8)
Family Study
Recipient
Sister
A
03
03
02
03
03
02
B
35
49
08
49
35
C
04
07
07
07
Bw
6
4
6
DRB1
04
04
DRB3/4/5
53
DQB1
DQA1
MM
Brother
Mother
02
02
03
15(62) 49
15(62)
08
35
04
03(10) 07
03(10) 07
04
4
6
6
4
6
6
6
13
04
04
04
04
04
13
04
53
52
53
53
53
53
53
52
53
03(7)
03(8)
06
03(8)
03(7)
03(8)
03(8)
03(8)
06
03(7)
03
03
01
03
03
03
03
03
01
03
4/10
Unacceptable antigens
B8 B76 B82
Cw5
3/10
03
Father
3/10
4/10
Living Donor Paired Exchange
• National Program for incompatible recipient/donor pairs
(living kidney donation)
• Pairs incompatibility due to:
– Presence of donor specific HLA antibodies
– ABO blood group incompatibility
• Recipient/donor pair information is entered into database
– HLA typing, HLA antibodies, blood group, clinical
parameters.
• Computer program matches incompatible pairs with others
using a virtual crossmatch principle.
• Major impact on rate of kidney transplantation.
Living Donor Paired Exchange
Donor 1
Group A
HLA-A1,3
Donor 2
Group O
HLA-A2,3
X
X
Recipient 1
Group B
No HLA abs
Recipient 2
Group A
Anti-HLA-A2
2 way exchange
Living Donor Paired Exchange
Donor 1
Donor 2
Donor 3
Donor N
Recipient 1
Recipient 2
Recipient 3
Recipient N
N way exchange
Conclusions
• Major improvement in HLA testing over the last few years
• Implementation of state of the art technology and
methodology
• Allows more complete assessment of immunologic risk
• Better clinical outcomes
• Decreased TAT/Decreased cost
• Increased rate of transplantation through participation in
LDPE program
Thank you