Handouts. Program-Handouts.aspx. 2 All Content Immucor, Inc.

Transcription

1

2 Handouts Program-Handouts.aspx 2 All Content Immucor, Inc.

3 2019 Transplant Webinars Link to register: 3 All Content Immucor, Inc.

4 Link to register: 4 All Content Immucor, Inc.

5 learn.immucor.com 5 All Content Immucor, Inc.

6 Continuing Education PACE, Florida and California DHS 1.0 Contact Hours Each attendee must register to receive CE at: Registration deadline is March 29, 2019 Certificates will be sent via only to those who have registered April 12, All Content Immucor, Inc.

7 Questions? You are all muted Q&A following session - Type in questions 7 All Content Immucor, Inc.

8 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. 8 All Content Immucor, Inc.

9 HLA DEPOT: WHAT S IN YOUR TOOLBOX? Anne Halpin & Esmé Dijke University of Alberta Alberta Transplant Institute Edmonton, Alberta

10 DISCLOSURE This presentation may contain Canadian content

11 EVOLUTION IN TOOLS In the beginning, there were sera, complement, and isolation of cells in straws. And it was good. Pinshow Straw Co

12 EVOLUTION IN TOOLS But as with all great tools, even better tools follow.

13 EVOLUTION IN TOOLS And now these tools are part of the standard of care in the HLA laboratories Flow cytometry for crossmatch (FCXM) Luminex technology (for typing and antibody detection)

14 EVOLUTION OF HLA TYPING TOOLS Serology Typing Molecular Typing SSO SSP rsso qpcr Sequencing SBT NGS

15 EVOLUTION OF CROSSMATCH TOOLS Serology XM ELISA XM Flow Cytometry Crossmatch Flow crossmatch still has many possible sources of variability Pronase use Cell isolation method Halifaster XM is widely used across Canada (courtesy of Dr. Rob Liwski*) Additional solid phase XM tools are also available *Liwski et al Hum Immunol Jan;79(1):28-38

16 SINGLE ANTIGEN BEAD TOOLS Single antigen beads have revolutionized our ability to detect HLA antibodies, such as: Ability to define HLA specificities in high PRA patients Increased sensitivity as compared to CDC Class II antibodies are much more easily characterized BUT Non-specific reactivity can be observed PRA can no longer be defined in the same way

17 CALCULATED PANEL REACTIVE ANTIBODY (CPRA) The use of single antigen beads allow us to to define antibody specificity in a new way BUT created a need to re-define PRA values UNET created a cpra tool based on antigen frequencies in the donor pool The Canadian Transplant Registry (CTR) created a similar tool

18 CPRA UNET The UNOS/UNET cpra calculator has evolved to include many alleles cpra is derived from U.S. deceased donor antigen frequencies

19 CPRA CANADIAN TRANSPLANT REGISTRY Unacceptable antibody is captured in a different field cpra is calculated based on Canadian deceased donor antigen frequencies

20 EPITOPE TOOLS Epitopes were initially defined by serological methods Rodey CREG HLA Matchmaker was first described in 2001 Reflections on HLA Epitope-Based Matching for Transplantation Rene J. Duquesnoy, Front. Immunol., 28 November 2016 Today there are many epitope tools available such as HLA Matchmaker Epitope Registry PIRCHE

21 HOW DO WE USE THESE EPITOPE TOOLS? Epitopes can be used for: HLA matching Transplant risk assessment Investigating antibody patterns

22 ADDITIONAL TOOLS Tools to investigate allele and haplotype frequencies Haplostat Allele frequencies IMGT

23 HOW DO WE USE THESE TOOLS? CASE STUDY EXAMPLES FROM OUR TRANSPLANT CENTRE

24 CASE 1 48-year old male with chronic renal failure Received first kidney transplant in summer 2015 Patient typing: A29 B72 Cw12 DR8 DR52 DQ7 DP04:02 A30 B39 DQ4 DP02:01 Donor typing: A24 B18 Cw17 DR17 DR52 DQ7 DP02:01 A25 B44 Cw11 DR11 DQ2 Pre-transplant cpra = 48%, no DSA detected by SAB HLA antibodies in cpra calculation include B37 and DR53 Both pre-transplant T and B cell flow crossmatch were negative

25 CASE 1 3 ½ years post-transplant patient developed consistent positive urinalysis for protein and hemoglobin (creatinine is stable) HLA antibody testing was performed at the time of biopsy for cause as per local centre protocol No Class I antibody was detected Questionable Class II antibody detected required further investigation

26 CASE 1 Luminex SAB Class II (OLI*): strong reactivity to 1 DQ6 bead: DQB1*06:01 Is this a real antibody? *OLI: One Lambda Thermo Fisher

27 CASE 1 Luminex SAB Class II (LC*): also strong reactivity to DQB1*06:01 Patient has allele-specific reactivity to DQB1*06:01 (not DSA) non-antibody verified epitope that is specific to DQB1*06:01 = 3P 4 *LC: LIFECODES Immucor

28 CASE 1 48-year old male with chronic renal failure Received first kidney transplant in summer 2015 Patient typing: A29 B72 Cw12 DR8 DR52 DQ7 DP04:02 A30 B39 DQ4 DP02:01 Donor typing: A24 B18 Cw17 DR17 DR52 DQ7 DP02:01 A25 B44 Cw11 DR11 DQ2 3 ½ years post-transplant patient developed consistent positive urinalysis for protein and hemoglobin (creatinine is stable) No DSA detected (and biopsy showed no evidence of rejection)

29 CASE 2 56-year old female with coronary artery disease left ventricular dysfunction Listed for heart transplantation Sensitizing events: multiple pregnancies, multiple transfusions Patient typing: A2 B7 Cw10 DR4 DR53 DQ6 DP04:01 A31 B60 Cw15 DR15 DR51 DQ7 Work up for listing: questionable Class I antibody was detected required further investigation No Class II antibody was detected

30 CASE 2 Luminex SAB Class I (OLI*): strong reactivity to Cw5 Is this a real antibody? *OLI: One Lambda Thermo Fisher

31 CASE 2 Luminex SAB Class I (LC*): strong reactivity to Cw5 and Cw8 C*05:01 and C*08:02: share an eplet 138K *LC: LIFECODES Immucor

32 CASE 2 Surrogate crossmatch: Donor typing: A2 B44 Cw5 DR13 DR52 DQ6 DP04:01 A3 B62 Cw9 DR8 DQ4 T-cell flow crossmatch: B-cell flow crossmatch: positive positive Patient has strong reactivity to Cw5 (listed as unacceptable antigen)

33 CASE 2 56-year old female with coronary artery disease left ventricular dysfunction Listed for heart transplantation Sensitizing events: multiple pregnancies, multiple transfusions Patient typing: A2 B7 Cw10 DR4 DR53 DQ6 DP04:01 A31 B60 Cw15 DR15 DR51 DQ7 Donor typing: A2 B18 Cw2 DR15 DR51 DQ6 DP04:01 A25 B27 Cw12 DP23 Both T and B cell crossmatch were negative Transplanted in summer 2018

34 CASE 3 37-year old female with cystic fibrosis listed for lung transplantation Sensitizing events: none known at time of listing Non-specific patterns of reactivity with weak antibodies to DQ7, DQ8, DQ9 was observed in single antigen bead assay Patient typing: A2 B51 Cw7 DR14 DR52 DQ5 DP04:02 B58 Cw16 DR1 DP02:01 Donor typing: A1 B8 Cw7 DR17 DR52 DQ7 DP04:01 A26 B56 Cw1 DR11 DQ2 DP10 Both T and B cell flow crossmatch were NEGATIVE with this previously screened serum sample BUT were POSITIVE with the immediate pre-transplant serum sample

35 Luminex SAB Class I (OLI*): strong reactivity to multiple HLA-A and B alleles CASE 3 Donor-specific antibody Self antibody? Including to A2: A*02:01, A*02:03 and A*02:06 Patient typing: A*02:02 and A*02:05 *OLI: One Lambda Thermo Fisher

36 Luminex SAB Class I (LC*): also strong reactivity to multiple HLA-A and B alleles Including to A*02:01 and A*02:03 But NOT to A*02:05 and A*02:02 CASE 3 Reactivity is allele-specific non-antibody verified epitope = 95V *LC: LIFECODES Immucor

37 CASE 3 Luminex SAB Class II (OLI*): strong reactivity to DQ7, DQ8, DQ9; moderate reactivity to DQ2 Donor-specific antibody *OLI: One Lambda Thermo Fisher

38 CASE 3 37-year old female with cystic fibrosis Listed for lung transplantation Sensitizing events: none known at time of listing Patient typing: A2 B51 Cw7 DR14 DR52 DQ5 DP04:02 B58 Cw16 DR1 DP02:01 Donor typing: A1 B8 Cw7 DR17 DR52 DQ7 DP04:01 A26 B56 Cw1 DR11 DQ2 DP10 Multiple DSA were observed in immediate pre-transplant sample Post transplant monitoring was performed

39 CASE 3 Antibody tracking over time: DSA levels declined over time Patient is doing well in this early post-transplant phase

40 SUMMARY OF CASES- WHAT WAS IN OUR TOOLBOX To investigate these cases we used: Multiple vendor kits for antibody analysis Epitope analysis Surrogate flow crossmatch

41 SUMMARY We have many tools in our HLA toolbox Our earliest tools were limited We now have more tools at our disposal They can be used in combination

42 IS OUR TOOLBOX COMPLETE? The fields of histocompatibility and transplantation are most consistent in their ability to be constantly evolving Our toolbox likely will have more tools in the future! Star Wars: Episode IV A New Hope

43 THANK YOU! QUESTIONS?

44 Questions? You are all muted Q&A following session - Type in questions 44 All Content Immucor, Inc.

45 2019 Advanced Track Webinars Link to register: 45 All Content Immucor, Inc.

46 Questions? You are all muted Q&A following session - Type in questions 46 All Content Immucor, Inc.

47 Continuing Education PACE, Florida and California DHS 1.0 Contact Hours Each attendee must register to receive CE at: Registration deadline is March 29, 2019 Certificates will be sent via only to those who have registered April 12, All Content Immucor, Inc.

48 We like you! Like us on social media! 48 All Content Immucor, Inc.

49 Thank you! 49 All Content Immucor, Inc.