**5. Serological and molecular HLA typing and the detection of donorspecific anti-HLA antibodies (Figure 4)**

The evolution in our understanding of the HLA system is closely linked to advancements in technology. Traditional serological-based low resolution HLA typing methods can be com‐ pleted relatively quickly but are dependent on the availability of specific cell types, viability and appropriate anti-sera that are capable of recognising HLA antigens. The emergence of molecular HLA typing techniques over the past two decades has allowed for a more specific, flexible and robust means of high resolution HLA typing. In 1982, *Wake et al* described restriction fragment length polymorphism (RFLPs), which eventually highlighted the short‐ comings of serology-based methods ensuing the establishment of molecular-based HLAtyping for routine clinical practice [64]. Data generated via the genome project and the initiation of polymerase chain reaction (PCR) techniques through the 1980s further refined DNA-based techniques for HLA-typing, which has led to the development of a number of PCR-based techniques still in use to the present day.

Alongside advances in the typing of HLA alleles, the techniques used to detect anti-HLA antibodies has also evolved from CDC assays to more sensitive techniques including flowcytometry and solid-phase assays (e.g. enzyme-linked immunosorbent assay [ELISA] or Luminex),whichhasallowedamoreaccurateassessmentoftransplantcandidate's immunolog‐ ical risk pre-transplantation (e.g. calculated panel reactive antibodies to determine level of sensitizationandapplicationofvirtualcross-matchtodeterminetransplantsuitability)(Figure4).

> in renal transplantation. The inability to correlate all graft losses with anti-HLA antibodies has led to the development of more sensitive cross-match assays, including flow cytometric crossmatch assays. It is noteworthy that 20% of patients transplanted across a positive cross-match did not lose their grafts [68]. Because T cells express class I antigens and B cells express both class I and II antigens, the interpretation of T cell together with B cell cross-match will help to establish whether class I and/or II anti-HLA antibodies are present. A positive B cell CDC crossmatch invariably accompanies a positive T cell CDC cross-match but this may reflect either anti-HLA antibodies to class I antigens and/or multiple antibodies to class I and/or II antigens. However, a positive B cell CDC cross-match may occur in the absence of a positive T cell CDC cross-match and suggest the presence of class II antigens or low levels class I antigens. The presence of a positive T cell CDC cross-match is an absolute contraindication for transplanta‐ tion whereas a positive B cell cross-match is a relative contraindication because of the uncer‐ tainty regarding the clinical significance and the possibility of false-positive results [69, 70]. In the allocation of deceased donor kidneys in Australia, the presence of a positive T cell CDC

**Figure 4.** Detection of anti-HLA antibodies – differences between cell-based and solid-phase assays.

HLA – human leukocyte antigen, CDC-XM – complement-dependent cytotoxicity cross-match, ELISA – enzyme-linked

**Cell-based assays Solid-phase assays** 

• Sensitive with high degree of specificity to donor antigens, luminex more sensitive than ELISA • Capable of quantifying anti-HLA antibodies level

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• CDC-XM reduced the incidence of hyperacute

• Inability to identify the antigen causing positive

rejection

immunosorbent assay.

Since the recognition of the clinical importance of CDC assay in kidney transplantation in the 1960s, CDC cross-match has become the cornerstone of determining transplant suitability in both live and deceased donor renal transplantation [65]. The underlying principle of CDC cross-match is to detect clinically relevant donor-specific anti-HLA antibodies that could result in hyperacute rejection following transplantation. Donor T and B cells are incubated in the presence of recipients' sera and complements. If donor-specific anti-HLA antibodies are present, these will bind to donor cells and initiate the complement cascade resulting in lysis of donor lymphocytes. The percentage of lysis will be quantified and forms the basis of determining transplant candidate's suitability for transplantation. Many laboratories perform CDC assays in the presence of anti-human globulin (enhances the sensitivity of assay by enhancing the number of Fc receptors available to bind with complements) and/or dithio‐ threitol (breaks down the disulfide bonds in IgM antibodies of no clinical significance) to improve the accuracy and reduce the false negative rates associated with these assays [66, 67]. Initial data using the CDC assay revealed that 80% of CDC cross-match–positive transplants and 4% of CDC cross-match–negative transplants were associated with early graft loss (within 48 hours post-transplant), thereby establishing the clinical significance of anti-HLA antibodies

Cw, DP and DQ DSA have been shown to be associated with poorer graft outcomes [62, 63], matching at the HLA-Cw, DP and DQ loci are not routinely performed and therefore is not

explicitly included in the allocation of deceased donor kidneys in any countries.

**specific anti-HLA antibodies (Figure 4)**

354 Current Issues and Future Direction in Kidney Transplantation

PCR-based techniques still in use to the present day.

**5. Serological and molecular HLA typing and the detection of donor-**

The evolution in our understanding of the HLA system is closely linked to advancements in technology. Traditional serological-based low resolution HLA typing methods can be com‐ pleted relatively quickly but are dependent on the availability of specific cell types, viability and appropriate anti-sera that are capable of recognising HLA antigens. The emergence of molecular HLA typing techniques over the past two decades has allowed for a more specific, flexible and robust means of high resolution HLA typing. In 1982, *Wake et al* described restriction fragment length polymorphism (RFLPs), which eventually highlighted the short‐ comings of serology-based methods ensuing the establishment of molecular-based HLAtyping for routine clinical practice [64]. Data generated via the genome project and the initiation of polymerase chain reaction (PCR) techniques through the 1980s further refined DNA-based techniques for HLA-typing, which has led to the development of a number of

Alongside advances in the typing of HLA alleles, the techniques used to detect anti-HLA antibodies has also evolved from CDC assays to more sensitive techniques including flowcytometry and solid-phase assays (e.g. enzyme-linked immunosorbent assay [ELISA] or Luminex),whichhasallowedamoreaccurateassessmentoftransplantcandidate's immunolog‐ ical risk pre-transplantation (e.g. calculated panel reactive antibodies to determine level of sensitizationandapplicationofvirtualcross-matchtodeterminetransplantsuitability)(Figure4). Since the recognition of the clinical importance of CDC assay in kidney transplantation in the 1960s, CDC cross-match has become the cornerstone of determining transplant suitability in both live and deceased donor renal transplantation [65]. The underlying principle of CDC cross-match is to detect clinically relevant donor-specific anti-HLA antibodies that could result in hyperacute rejection following transplantation. Donor T and B cells are incubated in the presence of recipients' sera and complements. If donor-specific anti-HLA antibodies are present, these will bind to donor cells and initiate the complement cascade resulting in lysis of donor lymphocytes. The percentage of lysis will be quantified and forms the basis of determining transplant candidate's suitability for transplantation. Many laboratories perform CDC assays in the presence of anti-human globulin (enhances the sensitivity of assay by enhancing the number of Fc receptors available to bind with complements) and/or dithio‐ threitol (breaks down the disulfide bonds in IgM antibodies of no clinical significance) to improve the accuracy and reduce the false negative rates associated with these assays [66, 67]. Initial data using the CDC assay revealed that 80% of CDC cross-match–positive transplants and 4% of CDC cross-match–negative transplants were associated with early graft loss (within 48 hours post-transplant), thereby establishing the clinical significance of anti-HLA antibodies

HLA – human leukocyte antigen, CDC-XM – complement-dependent cytotoxicity cross-match, ELISA – enzyme-linked immunosorbent assay.

**Figure 4.** Detection of anti-HLA antibodies – differences between cell-based and solid-phase assays.

in renal transplantation. The inability to correlate all graft losses with anti-HLA antibodies has led to the development of more sensitive cross-match assays, including flow cytometric crossmatch assays. It is noteworthy that 20% of patients transplanted across a positive cross-match did not lose their grafts [68]. Because T cells express class I antigens and B cells express both class I and II antigens, the interpretation of T cell together with B cell cross-match will help to establish whether class I and/or II anti-HLA antibodies are present. A positive B cell CDC crossmatch invariably accompanies a positive T cell CDC cross-match but this may reflect either anti-HLA antibodies to class I antigens and/or multiple antibodies to class I and/or II antigens. However, a positive B cell CDC cross-match may occur in the absence of a positive T cell CDC cross-match and suggest the presence of class II antigens or low levels class I antigens. The presence of a positive T cell CDC cross-match is an absolute contraindication for transplanta‐ tion whereas a positive B cell cross-match is a relative contraindication because of the uncer‐ tainty regarding the clinical significance and the possibility of false-positive results [69, 70]. In the allocation of deceased donor kidneys in Australia, the presence of a positive T cell CDC 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.

HLA donor specific antibodies complements the results of cell-based assays to help inform

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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

decision-making process with regards to transplant suitability.

scope of this chapter.

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

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

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 antiHLA donor specific antibodies complements the results of cell-based assays to help inform decision-making process with regards to transplant suitability.
