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Presentation on theme: "1 8 1 8 1 1 1 1 1 1 1 1 8 1 1 8 8 8 1 1 1 1 1 1 8 8 8 1 1 1 1 1 1 1 1 1 1 1 8 1 8 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 8 1 8 8 8 1 1 1 1 1 1 8 8 8 8."— Presentation transcript:

1 PRA = 36% (21/58)Anti-A11 and B44

2 PRA = 36% (21/58)Anti-A11 and B44

3 PRA = 95% (55/58)Specificity?

4 T cell B cell Flow Cytometry Crossmatch

5 T cell B cell FITC-  -IgG Flow Cytometry Crossmatch

6 T cell B cell Flow Cytometry Crossmatch FITC-  -IgG

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

8 T cell B cell Anti-CD3Anti-CD19 Flow Cytometry Crossmatch Detect fluorescent labels by flow cytometry FITC-  -IgG

9 Flow Crossmatch Implemented in Halifax, June 2010

10 Negative Weak positive Strong positive T cell X-matchB cell X-match Flow Cytometry Crossmatch Gating strategy FITC-  -IgG

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

12

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 Tells the instrument how much antibody is bound to the bead 2 lasers

15 HLA antibody detection by Luminex assay

16 A1A2A3A11A23A24A25A26A29A30 HLA antibody detection by Luminex assay

17 A1 A2 A3 A11 A23 A24 A25 A26 A29 A30 2 A2 1 A1 3 A3 4 A11 5 A23 6 A24 7 A25 8 A26 9 A29 10 A30 HLA antibody detection by Luminex assay

18 A1 A2 A3 A11 A23 A24 A25 A26 A29 A30 2 A2 1 A1 3 A3 4 A11 5 A23 6 A24 7 A25 8 A26 9 A29 10 A30 HLA antibody detection by Luminex assay

19 A1 A2 A3 A11 A23 A24 A25 A26 A29 A30 2 A2 1 A1 3 A3 4 A11 5 A23 6 A24 7 A25 8 A26 9 A29 10 A30 HLA antibody detection by Luminex assay

20 A1 A2 A3 A11 A23 A24 A25 A26 A29 A30 2 A2 1 A1 3 A3 4 A11 5 A23 6 A24 7 A25 8 A26 9 A29 10 A30 HLA antibody detection by Luminex assay

21 A1 A2 A3 A11 A23 A24 A25 A26 A29 A30 2 A2 1 A1 3 A3 4 A11 5 A23 6 A24 7 A25 8 A26 9 A29 10 A30 PE-  -IgG HLA antibody detection by Luminex assay

22 A1 A2 A3 A11 A23 A24 A25 A26 A29 A30 2 A2 1 A1 3 A3 4 A11 5 A23 6 A24 7 A25 8 A26 9 A29 10 A30 HLA antibody detection by Luminex assay

23 Patient Case

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

25 Patient A3,31 B7,60 DR1,14 (52) DQB5,6 Donor A1, B8 DR7,17 (53,52) DQB2 HLA Class I antibody analysis

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

27 Patient A3,31 B7,60 DR1,14 (52) DQB5,6 Donor A1, B8 DR7,17 (53,52) DQB2 HLA Class II antibody analysis

28 Patient A3,31 B7,60 DR1,14 (52) DQB5,6 Donor A1, B8 DR7,17 (53,52) DQB2 Unacceptable antigens: DR7, DR53, DQ2 HLA Class II antibody analysis

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 Resources, professional resources, choose cPRA calculator from options CPRA Calculator

35

36 Correlation between virtual and Flow crossmatch Some allele specific non-DSA Some weak DSA FP 3.1% FN 14% Non-HLA abs False pos FCXM Tambur et al. AJT 2009

37 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. Tambur et al. AJT 2009

38 A Virtual Crossmatch Protocol Significantly Increases Access of Highly Sensitized Patients to Deceased Donor Kidney Transplantation. Bingaman et al. Transplantation 2008 FP = 3% Cost effective Decreased TAT Increases access to transplantation of highly sensitized patients 12%

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

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 Virtual Crossmatch Patient 1A1,3B8,50DR4,17A11,A24,A25,B18,B44,DR12 Patient 2A2,3B44,62DR7,8A1,A26,A33,B52,DR15 Patient 3A3,11B8,18DR4,15A2,A31,A66,B7,B52 Patient 4A1,24B7,45DR9,12A3,A30,B60,B61,DR15,DR16 Patient 5A23,24B27,35DR10,16A1,B8,B44,DR7 Patient 6A2,23B51,55DR9,17A3,B60,B61,B62,B63,DR12,DR13 Patient 7A1,30B7,60DR11,13A2,DR1,DR7,DR8 Patient 8A3,31B27,61DR4,7A1,A23,A24,B18,B45,DR11,DR12 Patient 9A2,24B7,45DR4,8B27,B51,DR15,DR16 Patient 10A2,2B37,44DR9,12B8,B60,B61,DR10 VXMHLA typingHLA antibodies identified

43 Virtual Crossmatch Patient 1A1,3B8,50DR4,17A11,A24,A25,B18,B44,DR12 Patient 2A2,3B44,62DR7,8A1,A26,A33,B52,DR15 Patient 3A3,11B8,18DR4,15A2,A31,A66,B7,B52 Patient 4A1,24B7,45DR9,12A3,A30,B60,B61,DR15,DR16 Patient 5A23,24B27,35DR10,16A1,B8,B44,DR7 Patient 6A2,23B51,55DR9,17A3,B60,B61,B62,B63,DR12,DR13 Patient 7A1,30B7,60DR11,13A2,DR1,DR7,DR8 Patient 8A3,31B27,61DR4,7A1,A23,A24,B18,B45,DR11,DR12 Patient 9A2,24B7,45DR4,8B27,B51,DR15,DR16 Patient 10A2,2B37,44DR9,12B8,B60,B61,DR10 DonorA1,2B7,8DR4,17 VXMHLA typingHLA antibodies identified

44 Virtual Crossmatch Patient 1A1,3B8,50DR4,17NegA11,A24,A25,B18,B44,DR12 Patient 2A2,3B44,62DR7,8PosA1,A26,A33,B52,DR15 Patient 3A3,11B8,18DR4,15PosA2,A31,A66,B7,B52 Patient 4A1,24B7,45DR9,12NegA3,A30,B60,B61,DR15,DR16 Patient 5A23,24B27,35DR10,16PosA1,B8,B44,DR7 Patient 6A2,23B51,55DR9,17NegA3,B60,B61,B62,B63,DR12,DR13 Patient 7A1,30B7,60DR11,13PosA2,DR1,DR7,DR8 Patient 8A3,31B27,61DR4,7PosA1,A23,A24,B18,B45,DR11,DR12 Patient 9A2,24B7,45DR4,8NegB27,B51,DR15,DR16 Patient 10A2,2B37,44DR9,12NegB8,B60,B61,DR10 DonorA1,2B7,8DR4,17 VXMHLA typingHLA antibodies identified

45 Virtual Crossmatch Patient 1A1,3B8,50DR4,17NegA11,A24,A25,B18,B44,DR12 Patient 4A1,24B7,45DR9,12NegA3,A30,B60,B61,DR15,DR16 Patient 6A2,23B51,55DR9,17NegA3,B60,B61,B62,B63,DR12,DR13 Patient 9A2,24B7,45DR4,8NegB27,B51,DR15,DR16 Patient 10A2,2B37,44DR9,12NegB8,B60,B61,DR10 DonorA1,2B7,8DR4,17 VXMHLA typingHLA antibodies identified

46 Highly Sensitized Patient Case

47 PRA = 95% (55/58)Specificity?

48 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) Highly sensitized patient, Case 1

49 Virtual crossmatch in transplantation from live donors

50 Case 1 Potential recipientMother Potential donorSon Recipient HLA typing A3,3 B7,7 Cw7,7 DR4,15 DQ6,7 Donor HLA typing A1,3 B7,8 Cw7,7DR4,17DQ2,7

51 Class I HLA antibody analysis Recipient HLA typing A3,3 B7,7 Cw7,7 DR4,15 DQ6,7 Donor HLA typing A1,3 B7,8 Cw7,7DR4,17DQ2,7 Donor specific antibodies: A1, B8

52 Class II HLA antibody analysis Recipient HLA typing A3,3 B7,7 Cw7,7 DR4,15 DQ6,7 Donor HLA typing A1,3 B7,8 Cw7,7DR4,17DQ2,7 Donor specific antibodies: DR17, DQ2?

53 Case 2

54 RecipientSisterBrotherMotherFather A B (62)4915(62)0835 C (10)0703(10)0704 Bw DRB DRB3/4/ DQB103(7)03(8)0603(8)03(7)03(8) 0603(7) DQA

55 Case 2 RecipientSisterBrotherMotherFather A B (62)4915(62)0835 C (10)0703(10)0704 Bw DRB DRB3/4/ DQB103(7)03(8)0603(8)03(7)03(8) 0603(7) DQA MM4/103/10 4/10

56 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) Class I HLA antibody analysis

57 Family Study RecipientSisterBrotherMotherFather A B (62)4915(62) C (10)0703(10)0704 Bw DRB DRB3/4/ DQB103(7)03(8)0603(8)03(7)03(8) 0603(7) DQA MM4/103/10 4/10 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 Donor 1 Group A HLA-A1,3 Recipient 1 Group B No HLA abs Donor 2 Group O HLA-A2,3 Recipient 2 Group A Anti-HLA-A2 XX 2 way exchange

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

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


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