1. Marcelo Pando UNOS Region 5 Collaborative March, 2017
Histocompatibility Evaluation for Sensitized Patients: Maximizing the Patient’s Benefit.
Marcelo Pando
UNOS Region 5 Collaborative
March, 2017

2. Disclosure
Relevant Financial Relationship
None
Off Label Usage

3. Learning Objectives Understand the complexity of the HLA system.
Discuss some of the methodologies we use to detect antibodies against HLA.
Understand the current HLA practice in solid organ transplantation.
Discuss some educational cases.

4. The Human Leukocyte Antigen
In Medicine:
In Nature:
Organ Transplantation
Ag presentation
DIVERSITY!
Disease association
Pregnancy
HLA
Interaction with KIR
Drug hypersensitivity
Mate selection!

5. Then…
HLA in Transplantation.

6. The First Crossmatch

7. Crossmatch by complement-dependent cytotoxicity (CDC):
Donor cells + CFDA
Patient serum
Complement
EtBr
carboxy- fluorescein diacetate

8. Crossmatch by Complement-Dependent Cytotoxicity (CDC):
NEGATIVE
POSITIVE

9. The HLA System: Antigens and alleles
HLA-A
Serology: A2
Molecular: A*02:01
Alleles of A2: A*02:01, A*02:02, A*02:03, A*02:04…… A*02:336
carboxy- fluorescein diacetate

10. HLA Epitopes
 AA Pos.                    10         20         30         40         50         60         70         80         90        100  A*01:01:01:01      GSHSMRYFFT SVSRPGRGEP RFIAVGYVDD TQFVRFDSDA ASQKMEPRAP WIEQEGPEYW DQETRNMKAH SQTDRANLGT LRGYYNQSED GSHTIQIMYG  A*02:01:01:01       R G---KV H-VD A ----V-R---    AA Pos.                   110        120        130        140        150        160        170        180        190        200  A*01:01:01:01      CDVGPDGRFL RGYRQDAYDG KDYIALNEDL RSWTAADMAA QITKRKWEAV HAAEQRRVYL EGRCVDGLRR YLENGKETLQ RTDPPKTHMT HHPISDHEAT  A*02:01:01:01      ----S-W H-Y K T--H----A -V---L-A-- --T--EW A AV    AA Pos.                   210        220        230        240        250        260        270        280        290        300  A*01:01:01:01      LRCWALGFYP AEITLTWQRD GEDQTQDTEL VETRPAGDGT FQKWAAVVVP SGEEQRYTCH VQHEGLPKPL TLRWELSSQP TIPIVGIIAG LVLLGAVITG  A*02:01:01:01      ------S Q P F    AA Pos.                   310        320        330        340  A*01:01:01:01      AVVAAVMWRR KSSDRKGGSY TQAASSDSAQ GSDVSLTACK V  A*02:01:01:01       S
 AA Pos    10         20         30         40         50  A*01      GSHSMRYFFT SVSRPGRGEP RFIAVGYVDD TQFVRFDSDA ASQKMEPRAP  A*02       R    AA Pos.   110        120        130        140        150  A*01      CDVGPDGRFL RGYRQDAYDG KDYIALNEDL RSWTAADMAA QITKRKWEAV  A*02      ----S-W H-Y K T--H----A    AA Pos.   210        220        230        240        250  A*01      LRCWALGFYP AEITLTWQRD GEDQTQDTEL VETRPAGDGT FQKWAAVVVP  A*02      ------S    AA Pos.   310        320        330        340  A*01      AVVAAVMWRR KSSDRKGGSY TQAASSDSAQ GSDVSLTACK V  A*02       S 1 2 3 4 6 5 β1 α1 α2 β2
Epitopes are defined by the differences of the self antigen and the immunogenic antigen.

11. HLA Epitopes: The Bw4/Bw6 Case
AA Pos
B*07:02: GSHSMRYFYT SVSRPGRGEP RFISVGYVDD TQFVRFDSDA ASPREEPRAP WIEQEGPEYW DRNTQIYKAQ AQTDRESLRN LRGYYNQSEA GSHTLQSMYG
B*13:01: AM T T---MA E---S-TN T--Y--N--T ALR II-R---
B*13:02: AM T T---MA E---S-TN T--Y--N--T ALR W-T---
B*15:13: AM A MA S-TN T--Y--N--I ALR II-R---
B*15:16: F AM A MA E-RNM--S ---Y--N--I ALR W-R---
B*44:02:01: AM T L T---K E---S-TN T--Y--N--T ALR II-R---
B*14:01: C-TN T W---
B*14:02: A C-TN T W---
B*15:01:01: AM A MA E---S-TN T--Y R---
B*15:02: AM A MA S-TN T--Y II-R---
B*15:03: AM E---S-TN T--Y R---
B*15:10: AM C-TN T--Y R---
B*15:11: AM A MA TN T--Y R---
B*15:11: * AM A MA TN T--Y R---
B*15:11: * AM A MA TN T--Y R---

12. The Dogma of AMR Assumptions: Antibody to the Donor
Antibody Mediated Rejection
Assumptions:
Antibody is specific to the donor
The test detects clinical relevant antibodies
There is enough antibody to trigger IR

13. The Dogma of AMR
DSA
AMR

14. Tests for Antibody Screening

15. Luminex: Two different targets
PRA Beads
Phenotype: A2, A3; B7, B44; Cw4, Cw7
Less sensitive
More stable
SAB
SA: A2
High sensitivity
Less stable = denature Ags = False Positives

16. SAB

17. Phenotype Beads (PRA Beads)
AECI294
AECI294

18. Calculated Panel of Reactive Antibodies (cPRA)
the percentage of donors that will be excluded
probability of having a positive crossmatch
likelihood of getting an incompatible organ
100-cPRA = probability of getting a compatible organ

19. Examples of cPRA Patient with 20% cPRA 20% of donors will be excluded
20% of crossmatches will be positive
80% probability of getting a compatible organ

20. Maximizing Successful Offers
The CPRA game
Likelihood of Compatible Donor

21. Allele Specific Antibody
DRB3*02
Single Antigen Beads o con un unico antigeno de HLA

22. CPRA
Single Antigen Beads o con un unico antigeno de HLA

23. Single Antigen Bead (SAB) test results:
Single Antigen Beads o con un unico antigeno de HLA

24. Case 1: 100% cPRA patient

25. Case 2: 100% cPRA patient

26. Unacceptable antigens
Case 1:
A1 A2 A3 A11 A23 A24 A25 A26 A29 A36 A34 A43 A66 A68 A69 A80
B13 B27 B2708 B44 B58 B60 B61 B7 B73 B76 B8 B81 B57
DR4
DQ7 DQ8 DQ9
Case 2:
A1 A3 A11 A2403 A25 A26 A29 A30 A32 A33 A34 A36 A43 A66 A68 A69 A74 A80
B7 B8 B13 B27 B2708 B42 B45 B49 B50 B56 B57 B58 B62 B63 B67 B71 B72 B73 B75 B76 B77 B81
Cw1 Cw5 Cw6 Cw7 Cw8 Cw12 Cw14 Cw16 Cw17 Cw18
DR1 DR4 DR7 DR8 DR10 DR103 DR11 DRB1*13:03 DRB1*14:02 DR15 DR16
DRw51 DRw52
DQB1*06:03 DQ2 DQ5 DQ7 DQA1*04:01
DPB1*01:01 DPB1*04:01 DPB1*05:01 DPB1*19:01

27. cPRA
Case 1:
A1 A2 A3 A11 A23 A24 A25 A26 A29 A36 A34 A43 A66 A68 A69 A80
B13 B27 B2708 B44 B58 B60 B61 B7 B73 B76 B8 B81 B57
DR4
DQ7 DQ8 DQ9
Case 2:
A1 A3 A11 A2403 A25 A26 A29 A30 A32 A33 A34 A36 A43 A66 A68 A69 A74 A80
B7 B8 B13 B27 B2708 B42 B45 B49 B50 B56 B57 B58 B62 B63 B67 B71 B72 B73 B75 B76 B77 B81
Cw1 Cw5 Cw6 Cw7 Cw8 Cw12 Cw14 Cw16 Cw17 Cw18
DR1 DR4 DR7 DR8 DR10 DR103 DR11 DRB1*13:03 DRB1*14:02 DR15 DR16
DRw51 DRw52
DQB1*06:03 DQ2 DQ5 DQ7 DQA1*04:01
DPB1*01:01 DPB1*04:01 DPB1*05:01 DPB1*19:01
100% %
100% %

28. Probability of 1: 203 donors
cPRA
Case 1:
Case 2:
100% %
Probability of 1: 203 donors
100% %
Probability of 1: 34,483 donors

29. Maximizing Successful Offers
Unacceptable Ags
A2: 48% CPRA

30. Summary
An accurate definition of a antibody profile can give more opportunities to waiting list patients.
Determining the list of HLA antigens to avoid is patient specific and depends on several factors.
Highly sensitized patients, particularly the 100% cPRA, needs especial assessment to maximize their opportunities.
These patients, although a minority in the waiting list, have enormous differences in the opportunity of getting a compatible organ.

31. Thanks!