1. 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

2. 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

3. 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?

4. Flow Cytometry Crossmatch
B cell
T cell

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

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

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

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

9. Flow Crossmatch Implemented
in Halifax, June 2010

10. Flow Cytometry Crossmatch
T cell X-match
B cell X-match
Negative
Gating strategy
Weak
positive
Strong
positive
FITC-a-IgG
FITC-a-IgG

11. Karpinski et al. JASN 2001
Retrospective flow cytometry crossmatch study
249 patients transplanted (June 1992 and June 2000) with negative CDC-AHG crossmatch

12. Karpinski et al. JASN 2001

13. 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

14. 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

15. HLA antibody detection by Luminex assay1 2 3 4 5 6 7 8 9 10

16. HLA antibody detection by Luminex assay1 2 3 4 5 6 7 8 9 10 A1 A2 A3
A11
A23
A24
A25
A26
A29
A30

17. HLA antibody detection by Luminex assay8 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

18. HLA antibody detection by Luminex assay8 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

19. HLA antibody detection by Luminex assay8 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

20. HLA antibody detection by Luminex assay8 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

21. 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

22. HLA antibody detection by Luminex assay8 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

23. Patient Case

24. HLA Class I antibody analysis
Patient A3,31 B7,60 DR1,14 (52) DQB5,6

25. HLA Class I antibody analysis
Patient A3,31 B7,60 DR1,14 (52) DQB5,6
Donor A1, B DR7,17 (53,52) DQB2

26. HLA Class I antibody analysis
Patient A3,31 B7,60 DR1,14 (52) DQB5,6
Donor A1, B DR7,17 (53,52) DQB2
Unacceptable antigens: A1, A36, B8

27. HLA Class II antibody analysis
Patient A3,31 B7,60 DR1,14 (52) DQB5,6
Donor A1, B DR7,17 (53,52) DQB2

28. HLA Class II antibody analysis
Patient A3,31 B7,60 DR1,14 (52) DQB5,6
Donor A1, B DR7,17 (53,52) DQB2
Unacceptable antigens: DR7, DR53, DQ2

29. What is the clinical relevance of donor specific HLA antibodies detected pre-transplant by solid phase assay?

30. Amico et al. Transplantation 2009
Significant increase in biopsy proven AMR in patients with pre-transplant DSA

31. 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.

32. What about PRA? (probability of a positive crossmatch)

33. 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

34. CPRA Calculator http://optn.transplant.hrsa.gov/
Resources, professional resources, choose cPRA calculator from options

36. 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

37. 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.

38. 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

39. Negative virtual crossmatch predicts negative flow crossmatch
Crossmatches performed since implementation
of flow crossmatch (June 2010 – September 2011).
157
FP rate = 2.5% 4

40. Virtual Crossmatch Halifax Lab experience

41. 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.

42. 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

43. 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

44. 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

45. 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

46. Highly Sensitized Patient
Case

47. 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?

48. 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)

49. Virtual crossmatch in transplantation from live donors

50. 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

51. 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

52. 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

53. Case 2

54. 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

55. 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

56. 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)

57. 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

58. 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.

59. 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

60. Living Donor Paired Exchange
Donor N
Recipient 1
Recipient 2
Recipient 3
Recipient N
N way exchange

61. 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

62. Thank you