Post on 24-Feb-2016
description
181811
111111
811888
111111
888111
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118181
111111
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181888
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888811
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811111
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118111
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PRA = 36% (21/58) Anti-A11 and B44
181811
111111
811888
111111
888111
111111
118181
111111
111111
111111
181888
111111
888811
111111
811111
111111
118111
111111
111181
111111
PRA = 36% (21/58) Anti-A11 and B44
888888
111111
888888
111111
888888
111111
888888
111111
888888
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888888
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888888
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111111
888888
111111
811181
111111
PRA = 95% (55/58) Specificity?
T cell B cell
Flow Cytometry Crossmatch
T cell B cell
FITC-a-IgG
Flow Cytometry Crossmatch
T cell B cell
Flow Cytometry Crossmatch
FITC-a-IgGFITC-a-IgG
T cell B cell
Anti-CD3Anti-CD19
Flow Cytometry Crossmatch
FITC-a-IgGFITC-a-IgG
T cell B cellAnti-CD3 Anti-CD19
Flow Cytometry Crossmatch
Detect fluorescent labels by flow cytometry
FITC-a-IgGFITC-a-IgG
Flow Crossmatch Implementedin Halifax, June 2010
Negative
Weakpositive
Strongpositive
T cell X-match B cell X-match
Flow Cytometry Crossmatch
Gating strategy
FITC-a-IgG FITC-a-IgG
•Retrospective flow cytometry crossmatch study
•249 patients transplanted (June 1992 and June 2000) with negative CDC-AHG crossmatch
Karpinski et al. JASN 2001
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
Tells the instrument which bead is being examined
Tells the instrument how much antibody is bound to the bead
2 lasers
1 2 3 4 8 109765
HLA antibody detection by Luminex assay
1 2 3 4 8 109765
A1 A2 A3 A11 A23 A24 A25 A26 A29 A30
HLA antibody detection by Luminex assay
1
2
3
4
810
9
7
65
A1
A2
A3
A11
A23 A24
A25
A26
A29
A30
2
A2
1
A1
3
A3
4
A11 5
A23
6
A24
7
A25
8
A26
9
A29
10
A30
HLA antibody detection by Luminex assay
1
2
3
4
810
9
7
65
A1
A2
A3
A11
A23 A24
A25
A26
A29
A30
2
A2
1
A1
3
A3
4
A11 5
A23
6
A24
7
A25
8
A26
9
A29
10
A30
HLA antibody detection by Luminex assay
1
2
3
4
810
9
7
65
A1
A2
A3
A11
A23 A24
A25
A26
A29
A30
2
A2
1
A1
3
A3
4
A11 5
A23
6
A24
7
A25
8
A26
9
A29
10
A30
HLA antibody detection by Luminex assay
1
2
3
4
810
9
7
65
A1
A2
A3
A11
A23 A24
A25
A26
A29
A30
2
A2
1
A1
3
A3
4
A11 5
A23
6
A24
7
A25
8
A26
9
A29
10
A30
HLA antibody detection by Luminex assay
1
2
3
4
810
9
7
65
A1
A2
A3
A11
A23 A24
A25
A26
A29
A30
2
A2
1
A1
3
A3
4
A11 5
A23
6
A24
7
A25
8
A26
9
A29
10
A30
PE-a-IgG
HLA antibody detection by Luminex assay
1
2
3
4
810
9
7
65
A1
A2
A3
A11
A23 A24
A25
A26
A29
A30
2
A2
1
A1
3
A3
4
A11 5
A23
6
A24
7
A25
8
A26
9
A29
10
A30
HLA antibody detection by Luminex assay
Patient Case
Patient A3,31 B7,60 DR1,14 (52) DQB5,6
HLA Class I antibody analysis
Patient A3,31 B7,60 DR1,14 (52) DQB5,6Donor A1, B8 DR7,17 (53,52) DQB2
HLA Class I antibody analysis
Patient A3,31 B7,60 DR1,14 (52) DQB5,6Donor A1, B8 DR7,17 (53,52) DQB2Unacceptable antigens: A1, A36, B8
HLA Class I antibody analysis
Patient A3,31 B7,60 DR1,14 (52) DQB5,6Donor A1, B8 DR7,17 (53,52) DQB2
HLA Class II antibody analysis
Patient A3,31 B7,60 DR1,14 (52) DQB5,6Donor A1, B8 DR7,17 (53,52) DQB2Unacceptable antigens: DR7, DR53, DQ2
HLA Class II antibody analysis
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
http://optn.transplant.hrsa.gov/ Resources, professional resources, choose cPRA calculator from options
CPRA Calculator
Correlation between virtual and Flow crossmatch
Some allele specific non-DSASome weak DSA
FP 3.1%
FN 14% Non-HLA absFalse 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.
Tambur et al. AJT 2009
A Virtual Crossmatch Protocol Significantly Increases Access of Highly Sensitized Patients to Deceased Donor Kidney Transplantation.
Bingaman et al. Transplantation 2008
FP = 3%
Cost effectiveDecreased TATIncreases access to transplantation of highly sensitized patients
12%
Negative virtual crossmatch predicts negative flow crossmatch
Crossmatches performed since implementation of flow crossmatch (June 2010 – September 2011).
157
4
FP rate = 2.5%
No DSA0
20
40
60
80
100
120
140
160
negativepositive
# of
cro
ssm
atch
es
Virtual Crossmatch
Halifax Lab experience
Renal Transplant Patient Workup• 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
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
VXMHLA typing HLA antibodies identified
Virtual Crossmatch
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
Donor A1,2 B7,8 DR4,17
VXMHLA typing HLA antibodies identified
Virtual Crossmatch
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
Donor A1,2 B7,8 DR4,17
VXMHLA typing HLA antibodies identified
Virtual Crossmatch
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
Donor A1,2 B7,8 DR4,17
VXMHLA typing HLA antibodies identified
Highly Sensitized Patient Case
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PRA = 95% (55/58) Specificity?
Class I specificityA1 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)
Highly sensitized patient, Case 1
Virtual crossmatch in transplantation from live donors
Case 1 • Potential recipient Mother
• Potential donor Son
Recipient HLA typingA3,3 B7,7 Cw7,7 DR4,15 DQ6,7
Donor HLA typingA1,3 B7,8 Cw7,7 DR4,17 DQ2,7
Class I HLA antibody analysis
Recipient HLA typingA3,3 B7,7 Cw7,7 DR4,15 DQ6,7
Donor HLA typingA1,3 B7,8 Cw7,7 DR4,17 DQ2,7
Donor specific antibodies:A1, B8
Class II HLA antibody analysis
Recipient HLA typingA3,3 B7,7 Cw7,7 DR4,15 DQ6,7
Donor HLA typingA1,3 B7,8 Cw7,7 DR4,17 DQ2,7
Donor specific antibodies:DR17, DQ2?
Case 2
Case 2Recipient Sister Brother Mother Father
A 03 03 02 03 03 02 03 02 02 03B 35 49 08 49 35 15(62) 49 15(62) 08 35C 04 07 07 07 04 03(10) 07 03(10) 07 04Bw 6 4 6 4 6 6 4 6 6 6
DRB1 04 04 13 04 04 04 04 04 13 04DRB3/4/5 53 53 52 53 53 53 53 53 52 53DQB1 03(7) 03(8) 06 03(8) 03(7) 03(8) 03(8) 03(8) 06 03(7)DQA1 03 03 01 03 03 03 03 03 01 03
Case 2Recipient Sister Brother Mother Father
A 03 03 02 03 03 02 03 02 02 03B 35 49 08 49 35 15(62) 49 15(62) 08 35C 04 07 07 07 04 03(10) 07 03(10) 07 04Bw 6 4 6 4 6 6 4 6 6 6
DRB1 04 04 13 04 04 04 04 04 13 04DRB3/4/5 53 53 52 53 53 53 53 53 52 53DQB1 03(7) 03(8) 06 03(8) 03(7) 03(8) 03(8) 03(8) 06 03(7)DQA1 03 03 01 03 03 03 03 03 01 03
MM 4/10 3/10 3/10 4/10
Class I specificityB8 B76 B82 Cw5 Patient typing A*03,03 B*35,49 Cw*04,07 DRB1*04,04 DR53, 53 DQ*03(7),03(8)
Class I HLA antibody analysis
Family StudyRecipient Sister Brother Mother Father
A 03 03 02 03 03 02 03 02 02 03
B 35 49 08 49 35 15(62) 49 15(62) 08 35C 04 07 07 07 04 03(10) 07 03(10) 07 04Bw 6 4 6 4 6 6 4 6 6 6
DRB1 04 04 13 04 04 04 04 04 13 04DRB3/4/5 53 53 52 53 53 53 53 53 52 53DQB1 03(7) 03(8) 06 03(8) 03(7) 03(8) 03(8) 03(8) 06 03(7)DQA1 03 03 01 03 03 03 03 03 01 03
MM 4/10 3/10 3/10 4/10
Unacceptable antigensB8 B76 B82 Cw5
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 1Group AHLA-A1,3
Recipient 1Group BNo HLA abs
Donor 2Group OHLA-A2,3
Recipient 2Group AAnti-HLA-A2
XX
2 way exchange
Living Donor Paired Exchange
Donor 1
Recipient 1
Donor 2
Recipient 2
N way exchange
Donor 3
Recipient 3
Donor N
Recipient N
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