Craig J. Taylor

Consensus guidelines on the testing and clinical management issues associated with HLA and non-HLA antibodies in transplantation.

Background The introduction of solid-phase immunoassay (SPI) technology for the detection and characterization of human leukocyte antigen (HLA) antibodies in transplantation while providing greater sensitivity than was obtainable by complement-dependent lymphocytotoxicity (CDC) assays has resulted in a new paradigm with respect to the interpretation of donor-specific antibodies (DSA). Although the SPI assay performed on the Luminex instrument (hereafter referred to as the Luminex assay), in particular, has permitted the detection of antibodies not detectable by CDC, the clinical significance of these antibodies is incompletely understood. Nevertheless, the detection of these antibodies has led to changes in the clinical management of sensitized patients. In addition, SPI testing raises technical issues that require resolution and careful consideration when interpreting antibody results. Methods With this background, The Transplantation Society convened a group of laboratory and clinical experts in the field of transplantation to prepare a consensus report and make recommendations on the use of this new technology based on both published evidence and expert opinion. Three working groups were formed to address (a) the technical issues with respect to the use of this technology, (b) the interpretation of pretransplantation antibody testing in the context of various clinical settings and organ transplant types (kidney, heart, lung, liver, pancreas, intestinal, and islet cells), and (c) the application of antibody testing in the posttransplantation setting. The three groups were established in November 2011 and convened for a “Consensus Conference on Antibodies in Transplantation” in Rome, Italy, in May 2012. The deliberations of the three groups meeting independently and then together are the bases for this report. Results A comprehensive list of recommendations was prepared by each group. A summary of the key recommendations follows. Technical Group: (a) SPI must be used for the detection of pretransplantation HLA antibodies in solid organ transplant recipients and, in particular, the use of the single-antigen bead assay to detect antibodies to HLA loci, such as Cw, DQA, DPA, and DPB, which are not readily detected by other methods. (b) The use of SPI for antibody detection should be supplemented with cell-based assays to examine the correlations between the two types of assays and to establish the likelihood of a positive crossmatch (XM). (c) There must be an awareness of the technical factors that can influence the results and their clinical interpretation when using the Luminex bead technology, such as variation in antigen density and the presence of denatured antigen on the beads. Pretransplantation Group: (a) Risk categories should be established based on the antibody and the XM results obtained. (b) DSA detected by CDC and a positive XM should be avoided due to their strong association with antibody-mediated rejection and graft loss. (c) A renal transplantation can be performed in the absence of a prospective XM if single-antigen bead screening for antibodies to all class I and II HLA loci is negative. This decision, however, needs to be taken in agreement with local clinical programs and the relevant regulatory bodies. (d) The presence of DSA HLA antibodies should be avoided in heart and lung transplantation and considered a risk factor for liver, intestinal, and islet cell transplantation. Posttransplantation Group: (a) High-risk patients (i.e., desensitized or DSA positive/XM negative) should be monitored by measurement of DSA and protocol biopsies in the first 3 months after transplantation. (b) Intermediate-risk patients (history of DSA but currently negative) should be monitored for DSA within the first month. If DSA is present, a biopsy should be performed. (c) Low-risk patients (nonsensitized first transplantation) should be screened for DSA at least once 3 to 12 months after transplantation. If DSA is detected, a biopsy should be performed. In all three categories, the recommendations for subsequent treatment are based on the biopsy results. Conclusions A comprehensive list of recommendations is provided covering the technical and pretransplantation and posttransplantation monitoring of HLA antibodies in solid organ transplantation. The recommendations are intended to provide state-of-the-art guidance in the use and clinical application of recently developed methods for HLA antibody detection when used in conjunction with traditional methods.

Banking on human embryonic stem cells: estimating the number of donor cell lines needed for HLA matching

BACKGROUND Human embryonic stem (hES) cells are a promising source for transplantation to replace diseased or damaged tissue, but their differentiated progeny express human leucocyte antigens (HLAs) that will probably cause graft rejection. The creation of a bank of HLA-typed hES cells, from which a best match could be selected, would help reduce the likelihood of graft rejection. We investigated how many hES cell lines would be needed to make matching possible in most cases. METHODS The number of hES cell lines needed to achieve varying degrees of HLA match was estimated by use of, as a surrogate for hES-cell donor embryos, blood group and HLA types on a series of 10,000 consecutive UK cadaveric organ donors. The degree of blood group compatibility and HLA matching for a recipient population consisting of 6577 patients registered on the UK kidney transplant waiting list was determined, assuming all donor hES cell lines could provide a transplant for an unlimited number of recipients. FINDINGS A bank of 150 consecutive donors provided a full match at HLA-A, HLA-B, and HLA-DR for a minority of recipients (<20%); a beneficial match (defined as one HLA-A or one HLA-B mismatch only) or better for 37.9% (range 27.9-47.5); and an HLA-DR match or better for 84.9% (77.5-90.0). Extending the number of donors beyond 150 conferred only a very gradual incremental benefit with respect to HLA matching. A panel of only ten donors homozygous for common HLA types selected from 10,000 donors provided a complete HLA-A, HLA-B and HLA-DR match for 37.7% of recipients, and a beneficial match for 67.4%. INTERPRETATION Approximately 150 consecutive blood group compatible donors, 100 consecutive blood group O donors, or ten highly selected homozygous donors could provide the maximum practical benefit for HLA matching. The findings from these simulations have practical, political, and ethical implications for the establishment of hES-cell banks.

Alemtuzumab (CAMPATH 1H) Induction Therapy in Cadaveric Kidney Transplantation—Efficacy and Safety at Five Years

Alemtuzumab is a powerful lymphocyte depleting antibody currently being evaluated in solid organ transplantation. This paper describes 5‐year results of a single center study of alemtuzumab as induction in renal transplantation.

A second major histocompatibility complex susceptibility locus for multiple sclerosis

Variation in the major histocompatibility complex (MHC) on chromosome 6p21 is known to influence susceptibility to multiple sclerosis with the strongest effect originating from the HLA‐DRB1 gene in the class II region. The possibility that other genes in the MHC independently influence susceptibility to multiple sclerosis has been suggested but remains unconfirmed.

Generating an iPSC Bank for HLA-Matched Tissue Transplantation Based on Known Donor and Recipient HLA Types

The likelihood for immunological rejection of Human Leukocyte Antigens (HLA)-mismatched induced pluripotent stem cells (iPSCs) limits their therapeutic potential. Here we show how a tissue bank from 150 selected homozygous HLA-typed volunteers could match 93% of the UK population with a minimal requirement for immunosuppression. Our model provides a practical approach for using existing HLA-typed samples to generate an iPSC stem cell bank that circumvents prospective typing of a large number of individuals.

Immunoglobulin class and specificity of antibodies causing positive T cell crossmatches: relationship to renal transplant outcome

A group of 42 renal transplants performed in the presence of a T-cell-positive crossmatch were analyzed to determine the class and specificity of the donor-reactive cytotoxic antibodies. Dithiothreitol (DTT) was used to reduce IgM antibodies and a monoclonal antibody directed at a monomorphic determinant present on all HLA class I antigens (PA2.6) was used to inhibit cytotoxicity of anti-HLA class I antibodies. Sera from 26 of the positive crossmatches were considered to be autoreactive, and the positive crossmatch proved to be due to IgM and not directed at HLA class I in each case. One year graft survival was 100% in the 5 living-related and 60% in the 21 cadaver donor transplants, of which 10 were regrafts. Of the 42 positive crossmatches, 16 were not due to autoantibody. One was positive in the current serum taken at the time of transplantation, and this graft was rejected hyperacutely, while 15 were positive with peak but not current serum samples. Of the positive crossmatches, 12 were inhibited by PA2.6 demonstrating that they were directed at HLA class I antigens. PA2.6 inhibition could not be shown in 3 and in 1 DTT reduction was technically unsatisfactory. While 4 of the 7 positive crossmatches due to IgM antibodies were successful, the 7 transplants performed with positive crossmatches due to IgG antibodies all failed. DTT reduction and inhibition of cytotoxicity by PA2.6 helps to define positive crossmatches with donor T cells that are not associated with graft failure. Transplantation in the presence of a peak positive T cell crossmatch due to an anti-HLA antibody might only be successful if the antibody in the peak serum is of the IgM class.

Toward the development of a global induced pluripotent stem cell library

The ability to preselect the donor genotype of iPSC lines provides important opportunities for immune matching in cell therapy. Here we propose that an international assessment should be made of how immune incompatibility can best be managed and how a network of GMP HLA homozygous haplobanks could be operated.

Characterization of lymphocytotoxic antibodies causing a positive crossmatch in renal transplantation. Relationship to primary and regraft outcome.

In a series of 123 renal transplants performed in the presence of a positive crossmatch (either peak positive-current positive or peak positive-current negative), we have analyzed the immunoglobulin class and specificity of the donor-reactive antibodies. The immunoglobulin class was determined by dithiothreitol reduction and the specificity by cytotoxicity inhibition using monomorphic antibodies specific for HLA class I, DR, and DQ antigens. There was good primary graft and regraft survival in the presence of peak positive and current positive crossmatches due to IgM non-HLA antibodies. There was also acceptable primary graft and regraft survival with peak positive-current negative crossmatches due to T and B cell IgM HLA class I antibodies, but not with IgG HLA class I antibodies. Positive B cell crossmatches due to IgM or IgG HLA antibodies were associated with good primary graft survival but poor regraft survival.

Rapid HLA-DQB typing by eight polymerase chain reaction amplifications with sequence-specific primers (PCR-SSP)

Molecular genotyping of HLA class II genes using group-specific DNA amplification by the PCR followed by probing with (PCR-SSO) probes is too time consuming for the typing of cadaveric organ donors. Recently, amplification of DNA using PCR-SSP has proved a reliable and rapid method for typing HLA-DRB1 genes. PCR-SSP takes 2 hours to perform and is therefore suitable for the genotyping of cadaveric donors. We have designed a set of primers that in eight PCR reactions will positively identify the HLA-DQB1 alleles corresponding to the serologically defined series HLA-DQ2, DQ4, DQ5, DQ6, DQ7, DQ8, and DQ9. Presently, 30 homozygous cell lines and 138 individuals have been typed by the DQB1 PCR-SSP technique and compared with a combination of serology and RFLP with 100% concordance. No false-negative or false-positive amplifications were recorded. All combinations of DQB1 can be readily identified. DQB1 PCR-SSP can take as little as 130 minutes from start to finish, including DNA preparation.

Screening minVDD outliers using feed-forward voltage testing

MinVDD testing using full vector set search routines consumes too much test time. A 3-step process is proposed using: (1) a reduced vector set (RVS) binary search to measure the intrinsic (defect free) minVDD for a die; (2) a feed-forward to the full vector set (FVS) for low voltage testing; and (3) delta VDD and nearest neighbor residual statistical post-processing (SPP) are applied to the data to screen the minVDD outliers that are identified using the RVS binary search. RVS vs. FVS correlation data is shown on 3 products. Data shows minVDD yield fallout of 0.2-0.8% and 20% of the minVDD outliers shows significant VDD shifts in burn-in.