6/19/2012. Who is in the room today? What is your level of understanding of Donor Antigens and Candidate Unacceptables in KPD?

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1 6/19/212 KPD Webinar Series: Part 3 Demystifying the OPTN Kidney Paired Donation Pilot Program Unacceptable HLA antigens: The key to finding a compatible donor for your patient M. Sue Leffell, Ph.D. J. Michael Cecka, Ph.D. Facilitator: Ruthanne L Hanto RN, MPH Who is in the room today? Histocompatibility Lab Director Other Lab Personnel Physician or Surgeon Transplant Coordinator Mixed group Other What is your level of understanding of Donor Antigens and Candidate Unacceptables in KPD? 1 Clueless 2 Novice 3 Basic understanding 4 Good at some stuff 5 Expert ask me anything 1

2 6/19/212 Goal Provide an understanding of the importance of defining unacceptable antigens for successful KPD matches. Provide an understanding of the importance of donor antigen reporting in KPD paired donors This is an Exchange There are 4 Matches in this Exchange If the Match between Donor A and Candidate B is declined due to positive crossmatch all other Matches in the Exchange also fall apart OPTN KPDPP Match Offer Refusal Reasons 2% Data are incomplete 4% other matches in the exchange fell through Of the remaining (4%) of refused matches: 33% refused due to positive crossmatch or unacceptable antigens 7% due to candidate involved in a pending exchange 6% due to a variety of other donor and candidate reasons 2

3 6/19/212 Objectives Define appropriate candidate unacceptable antigens for successful KPD transplants. Accurately report donor antigens for successful KPD donation. Demonstrate data entry into the KPD application of UNet Unacceptable HLA Antigens: The key to finding a compatible donor for your patient in KPD Donor Antigens J. Michael Cecka, Ph.D., Professor Emeritus UCLA Immunogenetics Center 8 UNOS Kidney Paired Donation Recommendations Laboratory recommendations Molecular HLA typing HLA-A,B,C,Bw4,Bw6, DRB1-5, DQA, DQB DQA required for donors only Solid-phase tests for antibody (single antigen) Two levels of stringency Unacceptable antigens + All others Update antibody profile whenever Antibodies change on quarterly screens A sensitizing event occurs A patient is inactive >3 mo An unexplained positive crossmatch occurs Histocompatibility Committee review of positive crossmatches in real time. 9 3

4 6/19/212 Virtual Crossmatch Incompatible HLA Type A1, A2, B8, B57, Bw4, Bw6, DR4, DR17, DR52, DR53, DQA1*3, DQA1*5, DQ2, DQ7, DP2, DP6 Compatible Patient Anti-HLA Antibodies A2, A23, A24,A68,A69,B57,B58 Donor 1 HLA Type A1, A3, B7, B13, Bw4, Bw6, DR1, DR7, DR53,DQA1*1, DQA1*2, DQ2, DQ5, DP42 Donor 2 Critical Elements of the virtual crossmatch Accurate and realistic identification of patient antibodies that define unacceptable HLA antigens Accurate and complete identification AND reporting of donor HLA types If the donor antigens don t match the patient s antibody specificities there will be mistakes If the donor HLA type is not complete, there will be mistakes 11 Most virtual crossmatch failures are due to: a. Inability to identify all unacceptable HLA antigens b. Lack of allele-level HLA typing of the donor c. Data entry errors d. Failure to type the donor for HLA-DP e. Sleeping during the webinar 12 4

5 6/19/212 HLA antigens are complex HLA Antigens and Alleles Locus A B C DR DQ Antigens Alleles 965 1, WHO Nomenclature Committee Some HLA antigens are common HLA-A Some HLA antigens have a different distribution HLA-B

6 6/19/212 Some HLA antigens are rare and limited 1 14 HLA-B54 16 HLA Antigens were defined by antibodies: Parent Antigens and Splits HLA-A9 Antigens HLA-B14 HLA-A23 HLA-A24 A*23:1 A*24:2 A*23:2 A*24:3 A*23:3 A*24:4 A*23:4 A*24:6 HLA-B64 HLA-B65 Alleles B*14:1 B*14:2 B*14:3 B*14:4 B*14:5 17 Don t report broad HLA Antigens or allele groups Broad Ag Antigen A9 23,24 A1 25,26,34,66 A ,74 A28 68,69 B12 44,45 B16 38,39 B17 57,58 B21 49,5 B22 54,55,56 Allele Groups Antigen B14 64,65 B15 62,63,71 75,76,77 B4 6,61 Cw3 9,1 DRB1*3 17,18 DQB1*1 5,6 DQB1*3 7,8,9 B*14 B*15 B*4 DRB1*3 DQB1*3 are NOT antigens 18 6

7 6/19/212 Some common uncommon alleles A*11:1 A*11:2 (~1% of 11s) A*3:1 A*3:2 (~5% of 3s) A*66:1 A*66:2 (~2% of 66s) B*27:5 B*27:8 (~1% of 27s) B*15:2 (75) B*15:11 (~5% of 75s) DRB1*4 DRB1*4:5 (1% of 4s) DRB1*12:1 DRB1*12:2 (~5% of 12s) DRB1*15:1 DRB1*15:2 (~1% of 15) 19 The promiscuous habits of HLA-DQ alpha (-) (+) (+) (+) (+) DQ2 DQ2 DQ2 DQ9 DQ4 DQB1*2:1 DQB1*2:1 DQB1*2:2 DQB1*3:3 DQB1*4:1 DQA1*4:1 DQA1*2:1 DQA1*2:1 DQA1*2:1 DQA1*2:1 DQA1*2 associated with DR7 Surrogate CPRA=23% 2 HLA-DP B1 Allele Frequencies % 2% % 2% 51 3% 11 6% 31 1% 11 2% other 5% 41 42% 98% of 12, unrelated HSCT donors and recipients had common HLA-DP alleles (>2%) 75% both common 23% one common 42 12% 21 12% Petersdorf, E Blood 27; 11(13)

8 6/19/212 Based on the following HLA profile, what would you enter into the KPD database? A 2,11 B 12, 35 Bw 4 pos; Bw6 pos Cw 5,4 DR 4, 13 DR51 neg; DR52 neg; DR53 pos DQ 5, 7 DQA 1, 3 DP undetermined 22 Base on the B 12, 35 HLA prolife, you would enter B 12 and B 35 into the KPD database? True False 23 Based on the following HLA profile, what would you enter into the KPD database? A 2,11 B 12, 35 Bw 4 pos; Bw6 pos Cw 5,4 DR 4, 13 DR51 neg; DR52 neg; DR53 pos DQ 5, 7 DQA 1, 3 DP undetermined (Split B12 tob44) (Report DR1 DR4) (Perform DP typing) 24 8

9 6/19/ Summary Make a close friend in the HLA lab preferably the director Check the donor HLA type for accuracy, correctness and completeness prior to activating the pair then have the lab check again Do not enter antigens that are not entered as unacceptable antigens (Parent, alleles) Do not forget Bw4,6 and DR51-53 DQ alpha Type donors for DP even though it is not required Use the preselect option review with the laboratory 26 Unacceptable HLA Antigens: The key to finding a compatible donor for your patient in KPD Unacceptable HLA antigens M. Sue Leffell, Ph.D. Professor of Medicine 27 9

10 6/19/212 Why is the definition of unacceptable antigens so important for KPD? Candidates who come to KPD are usually sensitized (>6% have CPRAs >8) Compatible KPD matches are based on the presence of no or low levels of donor specific antibody (DSA) Unacceptable antigens (UAs): Antigens considered a contraindication to Tx Usually based on antibodies to potential donors Accurate UA definition optimizes planning for compatible exchanges Failure to accurately assess unacceptable antigens (UAs) disrupts exchanges 28 What are the effects of inaccurately entering unacceptable antigens on exchanges? a. There are no effects b. Your individual match is declined but the rest of the exchange can still move forward c. The entire exchange could be terminated 29 Steps for UA Definition Development of center specific criteria for acceptable risk for KPD and/or KPD with desensitization Correlation of solid phase antibody assays (SPI) with crossmatch tests and center s risk criteria Analysis of the antibody specificities for candidates to define corresponding UAs Re-evaluation of antibodies periodically and following any sensitizing event 3 1

11 6/19/212 Center Specific Criteria Dependent upon clinical protocols and the level, if any, of donor specific antibody (DSA) that is acceptable Factors to consider: Are any repeat mismatches acceptable? Are DSA levels that are XM - acceptable? Is desensitization an option? These factors determine the stringency that must be applied to UA definition 31 Correlation of SPI with Crossmatch Results T-CDC XM vs. Phenotype Panel MFI Negative Positive Cutoff: 9, MFI correlation: r =.92 correct prediction: 96% Zachary AA, et al. Hum Imm. 7: 574, 29 T cell FCXM vs. Phenotype Panel Negative Positive Cutoff: 53 MFI, correlation: r =.87 correct prediction: 98% Zachary AA, et al. Hum Imm. 7: 574, 29 11

12 SFI Normalized 6/19/212 T-CDC XM v. Single Antigen Panel Negative Positive Cutoff: 53 MFI correlation r =.68 correct prediction: 84% Zachary AA, et al. Hum Imm. 7: 574, 29 Acceptable Pretransplant DSA Levels After Desensitization Therapy: Luminex SFI vs Flow TXM MCS 1.E+6 7,966 msfi 57,182 msfi 11,244 msfi 1.E+5 Class II DSA > 1 5 AMR 1.E+4 1.E+3 Flow XM Neg Flow XM Pos CDC XM Pos 1.E MCS y = e.157x R 2 =.6888 Reinsmoen et al, Transplantation 86:82, 28 Considerations for SPI: Crossmatch Correlations Correlations must be assay and antigen specific HLA-Cw, DQ, DP concentrations are enhanced compared to HLA-A,B,DR (Zachary, et al. Meth Mol Biol.212; Zachary, Reinsmoen, Cur Opin Organ Transplant. 211) Individual HLA-A,B,DR concentrations may vary Correlations should be on-going to account for: Lot-to-lot variability Changes in phenotypes and/or single antigen panels 36 12

13 6/19/212 Considerations for Individual Candidate s Antibody Analysis Define the level of DSA that represents a contraindication or UA for each candidate Any DSA? DSA+, but virtual CDC XM or FCXM negative? Depends upon your center s criteria Should account for day-to-day assay variability Should consider possible test interference 37 Cut Off MFI Values for Positive Antibody Levels There are no generally accepted cut off MFI values Reported MFI cut offs range from 5 to 3 Often what is considered positive is lower than what is unacceptable Values should be antigen specific E.g., DQ values are higher than for DR UAs 38 High Risk vs. Low Risk UAs High risk when DSA is at a level that is absolute contraindication at your center List as UA Low risk DSA is present but at lower levels and could be positive in combination with other donor antigens or when target antigen is homozygous Try to identify antigen combinations that could reach unacceptable risk level Consider these combinations in evaluating prospective KPD exchanges 39 13

14 MFI 4/12 4/19 4/26 5/3 5/1 5/17 5/24 5/31 6/7 6/14 6/21 6/28 7/5 7/12 7/19 7/26 6/19/212 Interpretation of Results: Positive Control Variation A3 C15 B51 B6 ci Pos con SPI: Test Interference Substances inherent to the serum High IgM levels Immune complexes Antibody to plastic Complement: Ab binding of C1q in high titered sera (Schnaidt M, et al. Transplantation 92: 51, 211) Extrinsic factors Therapeutic Abs: thymoglobulin, eculizumab, high dose IVIg Bortezomib Untreated * * * * Lowest reacting B7: 3578 MFI IgM Depleted 14

15 GTI Score GTI Score 6/19/212 Methods to Deal with Interference and/or prozone Dilution Hypotonic dialysis (Zachary, et al. Human Immunol.29) Dithiothreitol (DTT) (Kosmoliaptsis, et al. Human Immunol.21) Ethylenediaminetetraacetic acid (EDTA) (Schnaidt M, et al. Transplantation.211) 43 Frequency of Antibody Testing Once at initial evaluation is NOT enough Specificity should be confirmed on at least two samples, preferably three to assess antibody trend Regular testing is necessary while the candidate is on the wait list to be sure UAs are current Test after any sensitizing event Transfusions Infection, other inflammatory events A 14. B Baseline Time of Infection Baseline Time of Event Locke JE, et al. Am J Transplant.29;9: renal transplant candidates with proinflammatory event c/in 1 month of aby testing A. 35 with culture+ infection; 97.1% had significant increase in aby B. 3 with proimflamm. events Surgery Myocardial infarction AV line placement 15

16 6/19/212 Recommended Best Practices (Guidelines from the OPTN/UNOS Histocompatibility Committee and a recent national KPD Consensus Conference) UAs should be based on Center specific risk criteria Correlation of antibody assays with risk criteria is essential Antibody definition should be done by at least two methods, one of which should be a solid phase assay Crossmatch confirmation should be cell based Specificity should be confirmed using a single antigen assay for: HLA-A,B,C, DRB1, DRB3-5, DQA1, DQB1, DPB1 Antibody interpretation may require high resolution typing of both recipient and donor alleles 46 Best Practices (con t.) Antibodies and corresponding UAs should be confirmed on at least two samples, updated at least quarterly, after any sensitizing event, and after any unexpected positive crossmatch Both high and low risk UAs should be considered Labs should achieve > 95% accuracy in virtual crossmatch prediction Histocompatibility evaluation should be used for preapproval of exchange matches 47 Effective UA Definition: Recipient DSA Analysis UA Definition Potential Donor Mismatches High Risk Unacceptable Low Risk Possibly Acceptable No Risk Acceptable Center Decision Preliminary - Final XM 48 16

17 6/19/212 Given the following results, what would you list for UAs? HLA Class I Abs Normalized MFI Specificity 16,952 A2 16,582 A69 12,951 A B A1 312 B58 HLA Class II Abs Normalized MFI Specificity 22,25 DR1 19,248 DR51 (B5*2:2) 18,564 DR9 17,438 DR51 (B5*1:1) 14,8 DQ6 12,24 DQ5 49 Specificity A68 with a normalized MFI 12,951would be listed as an UA? True False June 19, Given the following results, what would you list for UAs? HLA Class I Abs Normalized MFI Specificity 16,952 A2 16,582 A69 12,951 A B A1 312 B58 HLA Class II Abs Normalized MFI Specificity 22,25 DR1 19,248 DR51 (B5*2:2) 18,564 DR9 17,438 DR51 (B5*1:1) 14,8 DQ6 12,24 DQ5 Answer: At JHMI, UAs are based on Abs that are CDC XM+: Class I: A2, A69, A68 Class II: DR1, DR51, DR

18 6/19/212 June 19, Only unacceptable antigens that are highly likely to cause a positive crossmatch June 19, June 19,

19 6/19/212 All other antigens to which the candidate has antibodies, not including the unacceptable antigens June 19, June 19, Summary Accurate identification of both donor HLA and candidate unacceptable antibodies is critical to successful matching Transplant centers and HLA labs must work together to identify, enter, and verify appropriate data into the KPD system Declined Matches affect all other Matches in an Exchange 19

20 6/19/212 For more information M. Sue Leffell, Ph.D J. Michael Cecka, Ph.D Ruthanne L Hanto RN, MPH ruthanne.hanto@unos.org 2