1 1 8 1 1 8 1 8 1 1 8 1 1 8 1 1 8 1 1 1 8 1 1 8 1 1 1 1 8 PRA = 36% (21/58) Anti-A11 and B44

1 1 8 1 1 8 1 8 1 1 8 1 1 8 1 1 8 1 1 1 8 1 1 8 1 1 1 1 8 PRA = 36% (21/58) Anti-A11 and B44

1 8 1 8 1 8 1 8 1 8 1 8 1 8 1 8 1 8 1 8 1 PRA = 95% (55/58) Specificity?

Flow Cytometry Crossmatch B cell T cell

Flow Cytometry Crossmatch FITC-a-IgG B cell T cell

Flow Cytometry Crossmatch FITC-a-IgG FITC-a-IgG B cell T cell

Flow Cytometry Crossmatch Anti-CD3 Anti-CD19 FITC-a-IgG FITC-a-IgG B cell T cell

Flow Cytometry Crossmatch FITC-a-IgG FITC-a-IgG B cell Anti-CD3 T cell Anti-CD19 Detect fluorescent labels by flow cytometry

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 Tells the instrument which bead is being examined 2 lasers Tells the instrument how much antibody is bound to the bead

HLA antibody detection by Luminex assay 1 2 3 4 5 6 7 8 9 10

HLA antibody detection by Luminex assay 1 2 3 4 5 6 7 8 9 10 A1 A2 A3 A11 A23 A24 A25 A26 A29 A30

HLA antibody detection by Luminex assay 8 9 7 A26 A29 A25 4 10 6 8 10 A11 9 A30 1 5 A24 A26 A30 1 A29 A1 A23 2 A1 5 A2 3 6 A23 7 A3 A24 3 4 2 A25 A3 A11 A2

HLA antibody detection by Luminex assay 8 9 7 A26 A29 A25 4 10 6 8 10 A11 9 A30 1 5 A24 A26 A30 1 A29 A1 A23 2 A1 5 A2 3 6 A23 7 A3 A24 3 4 2 A25 A3 A11 A2

HLA antibody detection by Luminex assay 8 9 7 A26 A29 A25 4 10 6 8 10 A11 9 A30 1 5 A24 A26 A30 1 A29 A1 A23 2 A1 5 A2 3 6 A23 7 A3 A24 3 4 2 A25 A3 A11 A2

HLA antibody detection by Luminex assay 8 9 7 A26 A29 A25 4 10 6 8 10 A11 9 A30 1 5 A24 A26 A30 1 A29 A1 A23 2 A1 5 A2 3 6 A23 7 A3 A24 3 4 2 A25 A3 A11 A2

HLA antibody detection by Luminex assay PE-a-IgG 8 9 7 A26 A29 A25 4 10 6 8 10 A11 9 A30 1 5 A24 A26 A30 1 A29 A1 A23 2 A1 5 A2 3 6 A23 7 A3 A24 3 4 2 A25 A3 A11 A2

HLA antibody detection by Luminex assay 8 9 7 A26 A29 A25 4 10 6 8 10 A11 9 A30 1 5 A24 A26 A30 1 A29 A1 A23 2 A1 5 A2 3 6 A23 7 A3 A24 3 4 2 A25 A3 A11 A2

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% Non-HLA abs False pos FCXM FN 14% Some allele specific non-DSA Some weak DSA

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 FP rate = 2.5% 4

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.

HLA antibodies identified 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 DR4,8 B27,B51,DR15,DR16 Patient 10 A2,2 B37,44 B8,B60,B61,DR10

HLA antibodies identified Virtual Crossmatch Donor A1,2 B7,8 DR4,17 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 DR4,8 B27,B51,DR15,DR16 Patient 10 A2,2 B37,44 B8,B60,B61,DR10

HLA antibodies identified Virtual Crossmatch Donor A1,2 B7,8 DR4,17 HLA typing VXM 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 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 DR4,8 B27,B51,DR15,DR16 Patient 10 A2,2 B37,44 B8,B60,B61,DR10

HLA antibodies identified Virtual Crossmatch Donor A1,2 B7,8 DR4,17 HLA typing VXM 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 A3,A30,B60,B61,DR15,DR16 Patient 6 A2,23 B51,55 DR9,17 A3,B60,B61,B62,B63,DR12,DR13 Patient 9 A2,24 DR4,8 B27,B51,DR15,DR16 Patient 10 A2,2 B37,44 B8,B60,B61,DR10

Highly Sensitized Patient Case

1 8 1 8 1 8 1 8 1 8 1 8 1 8 1 8 1 8 1 8 1 PRA = 95% (55/58) Specificity?

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 DQ6,7 Donor HLA typing A1,3 B7,8 Cw7,7 DR4,17 DQ2,7

Class I HLA antibody analysis Donor specific antibodies: A1, B8 Recipient HLA typing A3,3 B7,7 Cw7,7 DR4,15 DQ6,7 Donor HLA typing A1,3 B7,8 Cw7,7 DR4,17 DQ2,7

Class II HLA antibody analysis Donor specific antibodies: DR17, DQ2? Recipient HLA typing A3,3 B7,7 Cw7,7 DR4,15 DQ6,7 Donor HLA typing A1,3 B7,8 Cw7,7 DR4,17 DQ2,7

Case 2

Case 2 Recipient Sister Brother Mother Father A 03 02 B 35 49 08 15(62) C 04 07 03(10) Bw 6 4 DRB1 13 DRB3/4/5 53 52 DQB1 03(7) 03(8) 06 DQA1 01

Case 2 Recipient Sister Brother Mother Father A 03 02 B 35 49 08 15(62) C 04 07 03(10) Bw 6 4 DRB1 13 DRB3/4/5 53 52 DQB1 03(7) 03(8) 06 DQA1 01 MM 4/10 3/10

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 08 Unacceptable antigens B8 B76 B82 Cw5 Recipient Sister Brother Mother Father A 03 02 B 35 49 08 15(62) C 04 07 03(10) Bw 6 4 DRB1 13 DRB3/4/5 53 52 DQB1 03(7) 03(8) 06 DQA1 01 MM 4/10 3/10 Unacceptable antigens B8 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 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 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