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1 8//07 Transplant Webinar Series: Ep. - Pre-transplant Immunological workup for Solid Organ Transplants Future Webinars Pre-transplant Immunological workup for Bone Marrow Transplants featuring Dr Uma Kanga AIIMS, New Delhi, India 8 January 08 6:30-7:30 IST (:00-:00 GMT) Repeated 08:00-09:00 and :00-5:00 EST Link to register: All Content 05 Immucor, Inc. Handouts Program-Handouts.aspx All Content 05 Immucor, Inc. All Content 05 Immucor, Inc.

2 8//07 Continuing Education ABHI, ASCLS/P.A.C.E., Florida and California Credits.0 Contact Hours Each attendee must register to receive CE credits at: Registration deadline is January 08 Certificates will be sent via only to those who have registered by 6 January 08 All Content 05 Immucor, Inc. All Content 05 Immucor, Inc. Presentation Recording Session will be recorded and posted to Immucor s LEARN site. Access information will be sent to each registrant when the recording becomes available CE credits will be issued to anyone who listens to the recording within one year of the original presentation date (today December 07). All Content 05 Immucor, Inc. All Content 05 Immucor, Inc. Questions? You are all muted Q&A following session - Type in questions All Content 05 Immucor, Inc. All Content 05 Immucor, Inc.

3 8//07 Course content is for information and illustration purposes only. Immucor makes no representation or warranties about the accuracy or reliability of the information presented and this information is not to be used for clinical or maintenance evaluations. The opinions contained in this presentation are those of the presenter and do not necessarily reflect those of Immucor. All Content 05 Immucor, Inc. All Content 05 Immucor, Inc. Pre-transplant Immunological Workup for Solid Organ Transplantation Rajalingam Raja, Ph.D., D (ABHI) Professor of Clinical Surgery & Director of Immunogenetics and Transplantation Laboratory University of California, San Francisco (UCSF) 8 Thanks to Dr. Uma Kanga AIIMS Dr. P. Srinivasan, Jeevan Stem Cell Foundation Dr. Ramona Chopra, Immucor India PVT. LTD., Lisa Waltham, Immucor, UK 9 3

4 8//07 Outline of the Presentation Kidney Transplantation (KTx): USA vs. India Testing performed at UCSF for KTx Protocol used at UCSF for KTx Testing Details HLA Typing, Antibody, and Crossmatch Optimal protocol for KTx in India? 0 Kidney Transplantation USA vs. India. Donor. Cost The cost for a kidney transplantation + first-year immunosuppression: USA - $6,000 India - $5,000 to 5,000 Most kidney Tx in USA are from Deceased Donors Most kidney Tx in India are from Living Donors UCSF Test Menu HLA Typing HLA-A, -B, -C, DRB/3//5, -DQB, -DQA, -DPB, -DPA. Intermediate resolution level using Luminex rsso/real-time PCR/SSP. HLA Antibody Testing Single Antigen Bead (SAB) assay. These tests identify HLA antibody specificities and their relative strengths expressed in Mean Florescence Intensity (MFI). Ambiguous results are re-evaluated by Multi Antigen Bead assay (PRA). Crossmatch (VXM) Virtual Crossmatch (VXM) is an assessment of immunological compatibility based on the patient s HLA antibody profile compared to the donor s HLA antigens. Flow Cytometry Crossmatch (FXM) determines if donor-specific IgG antibodies bind to donor T & B cells. If the patient was treated with Rituximab or anti-thymoglobulin, the B cell flow crossmatch will likely be false positive, and may need Pronase xm.

5 Pre-Tx Post-Tx 8//07 UCSF Kidney Transplantation Protocol Phase Living Donor Tx Deceased Donor Tx Initial HLA typing- Patient & Donors HLA antibody by SAB Initial XM by Virtual XM HLA typing- Patient HLA antibody by SAB - List unacceptable antigens in UNOS Waitlist HLA antibody by SAB - Top of the list patients for every 3 months - Update the unacceptable antigens in UNOS Transplant Final XM by Flow XM - Patient s serum (<30 days old, Antibody tested) HLA typing- Donor Pre-transplant XM by Virtual XM Antibody testing by SAB to identify the donor specific antibodies (DSA) Recipients who had pre-transplant DSA and/or CPRA >80% At wk., mo., and 6 mo. (+biopsy) post-tx; if rejection is suspected due graft dysfunctions 3 HLA Typing Basic protocol for DNA typing of HLA DNA isolation DNA PCR Sequence-specific priming (SSP) Gel-based detection Melting-Curve detection Sequence-specific oligonucleotide (SSO) hybridization Dot-blot Blot-dot Microarray Luminex rsso Sequencing-based typing (SBT) Sanger NGS 5 5

6 8//07 Sequence-Specific Primer (SSP) directed PCR amplification A*00 GCTGCGCGCG TGCTCCCCTG TTGGGGGTGC TATATTGGGC CACGGTAGAG TTGGGCCATG CTAAGCGGTA CGACGCCACG CTAGGCTACT GAGGGTCGGT A* ATA A-C-A CC----T C G-GG--T G---- -C A* ATA C---A CC----T G-GG--T C A* G-G C-C-A C-C---C G C A* G-AG AC---G C-----C G--A T--C --GA---G C B* TT ATG--A C-C-A C-----A A C--C ---G G -----C---- B* TT G G GC----AA-A G G -----C---- B* TT G G GC----AA-A G G -----C---- B* TT ATA C-C-A C-----A C T C--C G -----C---- B* TT ATG--A G CC----TG C G--A T--C AG---G -----C---- Cw* AC ATA C-C-A CC----T C T--C T G---- T----C---- Cw* AC ATA C-C-A CC----C A--C G ---T G---- T----C---- Cw* AC C-G C-C-A C C T--- A G---- T----C---- Cw* AC C-G C-C-A C C--C T----C---- Well Positive Negative No reaction reaction reaction Internal control band Positive typing band Primer band 6 SSP typing of HLA: reactivity pattern 7 Real-time PCR method SYBR GREEN 8 6

7 8//07 Sequence-Specific Oligonucleotide Probe (SSOP) hybridization A*00 GCTGCGCGCG TGCTCCCCTG TTGGGGGTGC TATATTGGGC CACGGTAGAG TTGGGCCATG CTAAGCGGTA CGACGCCACG CTAGGCTACT GAGGGTCGGT A* ATA A-C-A CC----T C G-GG--T G---- -C A* ATA C---A CC----T G-GG--T C A* G-G C-C-A C-C---C G C A* G-AG AC---G C-----C G--A T--C --GA---G C B* TT ATG--A C-C-A C-----A A C--C ---G G -----C---- B* TT G G GC----AA-A G G -----C---- B* TT G G GC----AA-A G G -----C---- B* TT ATA C-C-A C-----A C T C--C G -----C---- B* TT ATG--A G CC----TG C G--A T--C AG---G -----C---- Cw* AC ATA C-C-A CC----T C T--C T G---- T----C---- Cw* AC ATA C-C-A CC----C A--C G ---T G---- T----C---- Cw* AC C-G C-C-A C C T--- A G---- T----C---- Cw* AC C-G C-C-A C C--C T----C---- Dot blotting Probe-A * Hybridization Hybridized blott 9 Luminex Technology Polystyrene Microspheres 0 Luminex Technology A A A A3 A3 500 Color-coded Polystyrene beads using a blend of different fluorescent intensities of two dyes Each bead is conjugated with oligonucleotide probe specific for a HLA allele (s) 7

8 8//07 Cell Luminex: rsso Method Amplified DNA Denatured PCR products DNA * PCR amplification using florochrome-tagged locus-specific primers + A A Hybridization A A3 A3 A A A A3 A3 Detection & Interpretation 3 8

9 8//07 Patient and Donor HLA Typing Report 5 Solid Organ Matching Terminology HLA types Recipient A,A3,B8,B7,DR3,DR5 Donor A,A3,B8,B7,DR3,DR5 Recipient A,A3,B8,B7,DR3,DR5 Donor A,A,B8,B7,DR3,DR5 Recipient A,A3,B8,B7,DR3,DR5 Donor A,A,B8,B8,DR3,DR3 Recipient A,A3,B8,B7,DR3,DR5 Donor A,A3,B8,B7,DR3,DR9 Recipient A,A3,B8,B7,DR3,DR5 Donor A,A,B8,B,DR3,DR9 Matching HLA-identical Zero mismatch Zero mismatch DR mismatch Haploidentical Best match 6. Recipient A,A3,B8,B7,DR3,DR5 Donor A,A,B7,B6,DR,DR9 HLA Disparate (6 antigens mismatch) Worst match 6 Next-Gen Sequencing 7 9

10 8//07 High Resolution HLA Typing Report 8 HLA Antibody Testing 9 HLA is the Challenging Barrier to Transplantation HLA mismatched Allograft Recipient Induction Therapy Maintenance Therapy T NK T B NK B T T NK T B Rejection Lymphocytes Plasma cell HLA antibodies Lymphocytes Depletion Anti-Thymoglobulin T & NK cells Anti-CD3 T cells Anti-CD5 Activated T cells Anti-CD5 mature lymphocytes Anti-CD0 B cells Antibody Depletion Plasmaphoresis Antibody Blocking IVIG Anti-C5a Unacceptable Antigens Immunosuppression Cyclosporine MMF Steroids Transplantation Pregnancy Transfusion 30 0

11 8//07 Consequences of Pre-formed Donor-Specific HLA Antibodies Hyperacute rejection Delayed graft function Accelerated acute rejection Chronic rejection Prolonged waiting times No transplantation 3 HLA Antibody Testing Methods Complement-dependent Cytotoxicity (CDC) Method Solid Phase Methods Mixed Antigen Beads Multi Antigen Beads Single Antigen Beads Positive/Negative PRA and Specificity CPRA, Specificity and Strength (MFI) 3 Multi Antigen PRA Assay 33

12 8//07 Panel Reactive Antibody (PRA) Tray Pos. Cell I.D. A Panel Typing B Cw Tray Pos. Cell I.D. A Panel Typing B Cw A F B E C 56 D D 8 56 C E B F A F A E B D 0 56 C C D B E A F Cell lines in the panel = Cell lines reactive = 0 (PRA =.6%) Specificity = A Masked antigens = B8 3 Single Antigen Bead (SAB) Assay patient s serum Single Antigen beads + A B55 A66 A B55 A66 Detection & Interpretation 35 Antibodies Against A CREG Specificities: A, A68, A69, B57, B58 CPRA: 6% One Antibody 36

13 8//07 Public and Private Epitopes (antigenic determinants) A A68 A69 B57 B58 A A36 Public Epitopes Private Epitopes 37 Antibodies Against A CREG Specificities: A, A68, A69, B57, B58 CPRA: 6% One Antibody CPRA: 6% 38 No antibodies to self-hla are made. Individuals alloimmunized by a specific HLA type can make antibodies to many HLA types. Allograft A, A, B7, B8 Recipient Anti-A A8 A68 A3 A69 B57 A B58 A, A, B7, B8 Cross-REactive groups (CREG) 39 3

14 8//07 Cross-reactive groups (CREG) CREG HLA Specificities CPRA value A A,A3,A,A3,A,A9,A30,A3,A36,A80 78% A A,A3,A,A68,A69,B57,B58 75% A0 A,A5,A6,A3,A33,A3,A3,A66,A68, A69, A7 0% Bw A3,A,A5,A3,Bw 7% B5 B8,B35,B6,B9,B50,B5,B5,B53,B57, B58, B6,B63,B7,B7,B73,B75,B76,B77,B78 63% Bw6 Bw6 85% B7 B7,B8,B3,B7,B,B,B7,B8,B5,B55,B56,B59,B60,B6,B67,B8,B8 59% B8 B8,B8,B38,B39,B59,B6,B65,B67 36% B B3,B37,B,B,B5,B7,B9,B50,B60,B6 8% C Cw,Cw7,Cw8,Cw9,Cw0,Cw,Cw,Cw6,B6,B73 77% C Cw,Cw,Cw5,Cw6,Cw5,Cw7,Cw8 66% DR DR,DR0,DR03 % DR5 DR5,DR5,DR6 9% DR5 DR5,DR,DR,DR3,DR,DR7,DR8 6% DR53 DR53,DR,DR7,DR9 50% DQ DQ5,DQ6 6% DQ DQ 37% DQ3 DQ7,DQ8,DQ9 56% DQ DQ 0% DPc* DP,DP3,DP,DP6,DP9,DP0,DP,DP,DP7,DP8.DP0,DP DPc* DP,DP5,DP3,DP5,DP9,DP *DP-specific antibodies that are shown to occur frequently together in UCSF waitlist population 0 Bw6/Bw Epitope Bw6: B7, B8, B8, B708, B35, B39, B0, B005, B, B, B5, B8, B50, B5, B55, B56, B60, B6, B6, B6, B65, B67, B70, B7, B7, B75, B76, B78, B8, B8 Bw: Bw-T80: B3, B7, B37, B, B7 Bw-I80: B38, B9, B5, B5, B53, B57, B58, B59, B63, B77, A3, A, A5, A3 Bw-: B6, B73 Bw6 Antibodies A B Cw DR DR DQ 3(Bw) (Bw) CPRA=85%

15 8//07 Bw Antibodies A B Cw DR DR DQ 35(Bw6) (Bw6) 5 7 CPRA=6% 3 LABXpress Pipettor HLA Antibody Report Page- of Page- of 5

16 8//07 Antibody Binding Sites on HLA (epitopes) Conformational epitope Peptide epitope Peptide+HLA epitope Linear epitope HLA Antibody Profile in Kidney Waiting List Candidates (n=0) Kidney waitlist candidates n=07 Serum: DTT-treated re-tx females 03 re-tx males 53 st -tx females 77 st -t males 7 HLA class I & II Antibody testing by One lambda Single Antigen Bead Assay Data : 07 x antibodies = 509,07 antibodies with MFI Frequency analysis MFI analysis via Tukey s test Over reactive beads analysis 7 Hierarchy of antibody-provoking HLA types that warrant matching in kidney transplantation st -Transplant Relist or nd Tx females= 5 males= 5 No antibody -3 years HLA antibody testing (One Lambda single antigen bead assay) Allograft failure 8 6

17 8//07 9 HLA Antibodies Produced by Kidney Transplantation 50 HLA Antibodies Produced by Pregnancies 5 7

18 8//07 HLA Antibodies Produced in unsensitized Males 5 Crossmatching 53 Crossmatch (xm) types Methods Goal T cell xm Class I DSA B cell xm Class II DSA CDC xm Cytotoxic Antibodies AHG xm Sensitive CDC xm DTT xm Depletes IgM Flow xm Sensitive xm Pronase xm Removes Fc/background Endothelial cell xm Non-HLA Antibodies Auto xm Auto-Antibodies Virtual xm Most sensitive xm 5 8

19 Strength of T cell crossmatch (MCS) 8//07 Donor Recipient Flow Cytometry Crossmatch T cell B cell Lymphocytes Serum + Rituximab Negative Control HLA antibodies Patient Serum T cell Pronase FcR CD0 B cell T cell MCS > 50 B cell MCS > 0 Measure FITC intensity by flow cytometry Anti-human IgG F(ab ) FITC HLA class I (A,B,C) HLA class II (DR, DQ, DP) 55 Crossmatch - Report 56 Single Class I DSA MFI vs. T cell Crossmatch MCS Strength of single HLA class I DSA (MFI) Cunniffe et al. Human Immunology 06, 77:

20 Strength MCS of B cell crossmatch (MCS) 8//07 Single Class II DSA MFI vs. B cell Crossmatch MCS DPB DRB DQB DQA DRB Strength of single MFI HLA class II DSA (MFI) 000 Cunniffe et al. Human Immunology 06, 77:3. 58 Virtual Flow Cytometry Crossmatch Donor Recipient Median Chanel Shift (MCS) a quantitative readout (Ag+Ab) Detects only IgG antibodies Non-specific reactivity can be reduced by Pronase digestion Lymphocytes + Serum T cell B cell Anti-human IgG F(ab ) FITC Negative Control Measure FITC intensity by flow cytometry Patient Serum T cell MCS > 50 B cell MCS >

21 8//07 A new reality: Virtual crossmatch can be used successfully as pre-transplant compatibility testing for most deceased donor transplants at a high volume center 6 Post-TX DSA Report Section 6 Post-TX DSA Report Section 63

22 8//07 Post-TX DSA Report Section 3 6 If I run a HLA lab in India What would be my protocol for kidney Transplantation? 65 Optimal Kidney Transplant Protocol for India? HLA Identical Living donor KTx HLA typing- Patient & Donors HLA Haploidentical HLA Disparate Flow XM with Pronase Deceased donor KTx HLA typing- Patient Flow XM with Pronase - Wait for compatible Deceased donor HLA antibody by SAB Negative Positive Negative Positive No Transplant HLA antibody by SAB, identify DSA, and assess the risk for rejection No/low Risk High Risk Kidney Transplantation Post-tx Donor-Specific HLA Antibodies (DSA) monitoring Antibody testing by SAB to identify DSA / Donor-specific Flow XM Frequency per history of sensitization; if rejection is suspected due graft dysfunctions - Kidney Exchange for compatible living donor 66

23 8//07 Thank You Continuing Education ABHI, ASCLS/P.A.C.E., Florida and California Credits.0 Contact Hours Each attendee must register to receive CE credits at: Registration deadline is January 08 Certificates will be sent via only to those who have registered by 6 January 08 All Content 05 Immucor, Inc. All Content 05 Immucor, Inc. We like you! Like us on social media! All Content 05 Immucor, Inc. 3

24 8//07 Future Webinars Pre-transplant Immunological workup for Bone Marrow Transplants featuring Dr Uma Kanga AIIMS, New Delhi, India 8 January 08 6:30-7:30 IST (:00-:00 GMT) Repeated 08:00-09:00 and :00-5:00 EST Link to register: All Content 05 Immucor, Inc. Thank you! All Content 05 Immucor, Inc. All Content 05 Immucor, Inc.