Retrospective flow cytometry crossmatch study 249 patients transplanted (June 1992 and June 2000) with negative CDC-AHG crossmatch Karpinski et al. JASN 2001
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
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
HLA antibody detection by Luminex assay
A1A2A3A11A23A24A25A26A29A30 HLA antibody detection by Luminex assay
What is the clinical relevance of donor specific HLA antibodies detected pre-transplant by solid phase assay?
Amico et al. Transplantation 2009 Significant increase in biopsy proven AMR in patients with pre-transplant DSA
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.
What about PRA? (probability of a positive crossmatch)
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
Resources, professional resources, choose cPRA calculator from options CPRA Calculator
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
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
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%
Negative virtual crossmatch predicts negative flow crossmatch Crossmatches performed since implementation of flow crossmatch (June 2010 – September 2011) FP rate = 2.5%
Virtual Crossmatch Halifax Lab experience
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.
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
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?
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
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
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
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
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.
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
Living Donor Paired Exchange Donor 1 Recipient 1 Donor 2 Recipient 2 N way exchange Donor 3 Recipient 3 Donor N Recipient N
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