**6. Clinical significance of anti-HLA donor-specific antibodies**

cross-match is an absolute contraindication for transplantation whereas B cell cross-match is not routinely performed and therefore not utilized in the decision-making process for trans‐ plantation. With the increasing recognition of the potential importance of a positive CDC B cell cross-match, these results are now often interpreted in the context of solid phase assays.

The basic principle of flow cross-match technique is similar to CDC assay. Since the description of this assay in the early 1980s, this technique has been widely adopted to determine transplant suitability [71]. Similar to CDC assay, flow assay requires the addition of donor cells to recipients' sera, followed by the addition of a secondary fluorescein-labelled antibody allowing for the detection by flow cytometry and quantification of antibodies expressed as channel shifts. Unlike CDC cross-match, flow cytometric cross-match identifies both complementfixing and non-complement-fixing anti-HLA donor-specific antibodies. However, the availa‐ bility of different subtypes of detection antibodies has allowed for the differentiation between complement-fixing versus non-complement-fixing antibodies [72]. Although an universal cutoff value for a positive flow cross-match has not been determined, it is agreed that the use of a low cut-off point will result in increased sensitivity but reduced specificity for predicting graft outcomes (especially in the presence of negative CDC cross-match) as this may identify anti-HLA donor specific antibodies of no clinical significance. Nevertheless, renal transplant recipients with positive flow cross-match but negative CDC cross-match have a significantly greater risk of antibody-mediated rejection (AMR) and early graft loss with a positive predic‐

To avoid problems associated with the viability of the donor cells, which could affect the accuracy of cell-based assays, the introduction of solid-phase assays have largely circumvented these problems and improved the sensitivity of detection of anti-HLA antibodies [74]. The identification of anti-HLA antibodies using ELISA was first described in 1993 where purified HLA antigens were directly immobilized on the surface of microtitre plates but the basic principle of antibody detection was similar to cell-based assays [75]. The Luminex platform is a solid-phase assay that utilizes polystyrene microspheres (beads), each embedded with fluorochromes of differing intensity attached to one (single-antigen beads) or several HLA molecules (screening beads) to determine anti-HLA antibody specificity. Similar to other assays, the addition of recipients' sera containing anti-HLA antibodies are added to the bead mix, these antibodies will bind to the appropriate beads expressing specific antigen(s). A second phycoerytherin-labelled anti-human IgG is then added to this mixture and these antibodies will bind to the primary anti-HLA antibody already attached to the beads. The sample is then passed through lasers, which would independently excite the beads and the phycoerytherin therefore allowing the laser detector to define antibody specificity [76, 77]. Unlike the CDC assays, Luminex assay detect both complement-fixing and non-complementfixing anti-HLA antibodies but does not detect IgM autoantibodies or non-HLA antibodies. With the continued reliance on using cell-based cross-match assays, especially CDC crossmatch assays to determine transplant suitability, a potential disadvantage of virtual crossmatch is that transplants may be excluded based on antibody results with unknown clinical relevance [78]. It is generally accepted that solid phase virtual cross-match to identify anti-

tive value for predicting AMR of 83% [72, 73].

356 Current Issues and Future Direction in Kidney Transplantation

It is well known that the presence of high levels of pre-transplant class I (HLA-A and B) ± II (HLA-DR) donor-specific antibodies (DSA; i.e. anti-HLA antibodies with reactivity against the potential donor leading to positive cross-match often as a result of prior sensitization events including previous HLA-mismatched transplants, blood transfusions or pregnancy) is associated with poorer graft outcomes, including the development of acute AMR, chronic AMR, transplant glomerulopathy and late graft loss (Table 1) [79-81]. However, few studies have suggested that the association between pre-transplant DSA and graft survival was restricted to recipients who had developed early AMR, within the first 30-days post-trans‐ plantation [82]. In addition, the authors queried the cost-effectiveness of pre-transplant screening for preformed DSA by demonstrating that the additional cost associated with quarterly screening for anti-HLA antibodies would be between 3200 to 6700 Euros, which would equate to an additional 83,000 to 130,000 Euros per avoided AMR because of preformed non-lymphocytotoxic DSA in transplant candidates on the transplant wait-list for >5 years [82]. There is also increasing evidence demonstrating that the development of *de novo* DSA (occur‐ ring post-transplantation), especially development of DSA directed against HLA-DQ graft molecules in HLA-class II incompatible graft transplantations, are both associated with acute and subclinical AMR and graft loss in kidney transplant only and/or simultaneous pancreaskidney transplant recipients [80, 83-85]. Although there is no current consensus on the level of clinically significant DSA identified by flow cytometric or Luminex assays, most studies have demonstrated that increasing single, peak or total DSA levels were associated with an incremental risk of rejection and/or graft loss [86, 87]. Recent studies have suggested that the detection of C1q-fixing DSA (i.e. the potential to identify DSA that can activate complements by binding C1q) may be more specific in predicting acute rejection, biopsy C4d-deposition, transplant glomerulopathy and late graft failure following kidney transplantation but this remains controversial and not routinely performed in many transplanting centres [88, 89]. The clinical benefit of routine regular surveillance for *de novo* DSA in improving graft survival following kidney transplantation remains unclear although a recent study of 72 live-donor renal transplant recipients suggested that the appearance of *de novo* DSA was inversely proportional to the amount of maintenance immunosuppressive drugs (especially in the weaning phase of immunosuppression minimization particularly prednisolone) such that DSA monitoring may be highly effective for detecting escape from tolerance and reappearance of the immune response in weaned patients [90]. With the greater understanding of HLA antigens and anti-HLA antibodies, innovative techniques have been established to allow transplantation across positive CDC and/or flow cross-match barriers but this is beyond the scope of this chapter.


donor kidneys in most countries, including Australia and the Eurotransplant group (Germany, The Netherlands, Belgium, Luxembourg, Slovenia, and Austria), is weighted largely on the degree of mismatched antigens at the HLA-A, -B and -DR loci, with less emphasis on other factors such as time on dialysis, prior sensitization and even ischaemic time. When a potential deceased-donor kidney is available in Australia, transplant candidates on the wait-list are ranked according to an allocation score calculated from a combination of factors including the number of HLA-mismatches, age of recipient, degree of sensitization and time on wait-list [91]. Approximately 20% of deceased donor kidneys are allocated on a national level to highly sensitized transplant candidates (around 20% of kidneys allocated) but the remaining 80% of

The Evolution of HLA-Matching in Kidney Transplantation

http://dx.doi.org/10.5772/54747

359

deceased donor kidneys are allocated through individual state allocation algorithms.

but this approach has not been widely adopted by other countries [61].

levels of class II DSA.

Despite efforts to achieve equity of access to transplantation in many countries, the inclusion of HLA matching in the allocation of deceased donor kidneys is believed to disadvantage transplant candidates with uncommon HLA phenotypes [92]. Consequently, indigenous populations and ethnic minorities often have a much longer transplant wait-list time and are less likely to receive well-matched kidneys [97-100]. The elimination of the allocation priority for HLA-B mismatches has been shown to improve the transplant potential of ethnic minorities

In Australia, unacceptable class I HLA-mismatches are defined using the Luminex platform and the presence of class I DSA against HLA-A and -B antigens with >2000 mean fluorescent intensity (MFI) excludes transplant candidates from receiving these donor kidneys, independ‐ ent of the CDC-cross match results. At present, class II DSA is not explicitly considered in the allocation of kidneys from deceased donors in Australia but many centres have already adopted the policy of avoiding transplantation of kidneys into transplant candidates with high

**8. Acceptable HLA-mismatch and highly sensitized transplant candidates**

Highly sensitised transplant candidates (defined as those having a panel reactive antibody [PRA] level of >80%) on the deceased donor transplant wait-list are less likely to receive donor kidneys (greater likelihood of obtaining a positive complement-dependent cytotoxicity [CDC] cross-match result with any given donor) and have a much longer wait-list time compared to unsensitized transplant candidates, resulting in a greater risk of mortality whilst remaining on the transplant wait-list [93]. In Australia, highly sensitized transplant candidates represent approximately 5% of the wait-listed candidates and are more likely to wait on average twice as long as unsensitized transplant candidates despite an increase in the number of deceased

Although HLA matching has traditionally been performed at the broad antigen level, a model considering cross-reacting groups (CREGs) may increase the probability of identifying more compatible kidneys for ethnic minorities and highly sensitized transplant candidates. HLA antigens comprise of multiple serologic epitopes made of polymorphic amino acid residues, and it is these structures and their conformation and position that determine antibody

donors over time (202 donors in 2006 compared to 309 donors in 2010) [6].

HLA – human leukocyte antigen, DD – deceased donor, LD – live-donor, CDC-XM – complement dependent cytotoxicity cross-match, DSA – donor-specific antibodies, SAB – single antigen bead, AMR – antibody mediated rejection, DCGS – death-censored graft survival, MFI – mean fluorescent intensity, PPV – positive predictive value.

**Table 1.** Association between donor-specific antibodies and graft outcomes.
