**2. Evolution of techniques to detect donor-specific anti-HLA antibodies (Figure 1)**

HLA forms part of the major histocompatibility complex (MHC) in humans and MHC antigens are an integral component of the normal functioning of the human immune system. HLA antigens play a crucial role in the recognition of self-antigens and are therefore crucial in the defence of foreign antigens, including donor antigens in solid organ transplantation. HLA antigens are comprised of both class I and II antigens, with class I antigens being expressed on all nucleated cells, whereas class II antigens are being expressed on antigen presenting cells, B cells and endothelial cells [1]. The evolution in our understanding of the HLA system is closely linked to advancements in technology. Traditional serological-based (i.e. antibodybased) low-resolution techniques have been the standard method for HLA typing, enabling efficient and effective anti-HLA antibody detection. However, these techniques are dependent on the availability of specific cell types, cell 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 and robust method of high resolution HLA typing. In 1982, *Wake et al* described restriction fragment length polymorphism (RFLP), which eventually highlighted the shortcomings of serology-based methods ensuing the establish‐ ment of molecular-based HLA-typing for routine detection of anti-HLA antibodies pretransplantation [2]. 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 with the advances in the typing of HLA alleles, the techniques used to detect anti-HLA antibodies has evolved from CDC assays to the more sensitive techniques including flowcytometry and solid-phase assays (e.g. enzyme-linked immunosorbent assay [ELISA] or Luminex), allowing for accurate assessment of pre-transplant immunological risk (e.g. calculated panel reactive antibodies to determine level of sensitization and application of virtual cross-match to determine transplant suitability) [3] (Figure 4).

Since the recognition of the clinical importance of CDC assay in kidney transplantation in the 1960s, CDC cross-match has become the foundation of determining transplant suitability in kidney transplantation [4]. CDC cross-match can detect donor-specific anti-HLA antibodies that may have the potential to induce an anti-HLA antibody-associated hyperacute rejection following transplantation. Donor T and B cells are isolated from peripheral blood mononuclear cells using density gradient separation and incubated in the presence of recipients' sera and complements. If donor-specific anti-HLA antibodies are present, these will bind to specific antigen(s) expressed on donor cells, and with the addition of rabbit serum as a source of exogenous complement, will result in the initiation of the classical complement cascade causing direct damage to the donor cell membrane and therefore making these cells permeable to an important dye. The percentage of cell lysis is quantified and forms the basis of deter‐ mining transplant candidate's suitability for transplantation with a lysis score of 20% generally considered a contraindication for transplantation. Many laboratories perform CDC assays in

the presence of anti-human globulin, which augments the sensitivity of this assay by increasing the number of Fc receptors available to bind complements, and/or dithiothreitol (which breaks

HLA – human leukocyte antigen, CDC-XM – complement-dependent cytotoxicity cross-match, FCXM – flow cytometric

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**Figure 1.** Detection of anti-HLA antibodies – differences between cell-based and solid-phase assays.

cross-match, ELISA – enzyme-linked immunosorbent assay.

Detection of Antibody-Mediated Rejection in Kidney Transplantation and the Management of Highly Sensitised… http://dx.doi.org/10.5772/54735 107

**2. Evolution of techniques to detect donor-specific anti-HLA antibodies**

HLA forms part of the major histocompatibility complex (MHC) in humans and MHC antigens are an integral component of the normal functioning of the human immune system. HLA antigens play a crucial role in the recognition of self-antigens and are therefore crucial in the defence of foreign antigens, including donor antigens in solid organ transplantation. HLA antigens are comprised of both class I and II antigens, with class I antigens being expressed on all nucleated cells, whereas class II antigens are being expressed on antigen presenting cells, B cells and endothelial cells [1]. The evolution in our understanding of the HLA system is closely linked to advancements in technology. Traditional serological-based (i.e. antibodybased) low-resolution techniques have been the standard method for HLA typing, enabling efficient and effective anti-HLA antibody detection. However, these techniques are dependent on the availability of specific cell types, cell 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 and robust method of high resolution HLA typing. In 1982, *Wake et al* described restriction fragment length polymorphism (RFLP), which eventually highlighted the shortcomings of serology-based methods ensuing the establish‐ ment of molecular-based HLA-typing for routine detection of anti-HLA antibodies pretransplantation [2]. 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

Alongside with the advances in the typing of HLA alleles, the techniques used to detect anti-HLA antibodies has evolved from CDC assays to the more sensitive techniques including flowcytometry and solid-phase assays (e.g. enzyme-linked immunosorbent assay [ELISA] or Luminex), allowing for accurate assessment of pre-transplant immunological risk (e.g. calculated panel reactive antibodies to determine level of sensitization and application of

Since the recognition of the clinical importance of CDC assay in kidney transplantation in the 1960s, CDC cross-match has become the foundation of determining transplant suitability in kidney transplantation [4]. CDC cross-match can detect donor-specific anti-HLA antibodies that may have the potential to induce an anti-HLA antibody-associated hyperacute rejection following transplantation. Donor T and B cells are isolated from peripheral blood mononuclear cells using density gradient separation and incubated in the presence of recipients' sera and complements. If donor-specific anti-HLA antibodies are present, these will bind to specific antigen(s) expressed on donor cells, and with the addition of rabbit serum as a source of exogenous complement, will result in the initiation of the classical complement cascade causing direct damage to the donor cell membrane and therefore making these cells permeable to an important dye. The percentage of cell lysis is quantified and forms the basis of deter‐ mining transplant candidate's suitability for transplantation with a lysis score of 20% generally considered a contraindication for transplantation. Many laboratories perform CDC assays in

virtual cross-match to determine transplant suitability) [3] (Figure 4).

**(Figure 1)**

106 Current Issues and Future Direction in Kidney Transplantation

use to the present day.

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

**Figure 1.** Detection of anti-HLA antibodies – differences between cell-based and solid-phase assays.

the presence of anti-human globulin, which augments the sensitivity of this assay by increasing the number of Fc receptors available to bind complements, and/or dithiothreitol (which breaks

down the disulfide bonds in IgM antibodies believed to be of no clinical significance) to reduce the false positivity of these assays [5, 6]. Initial studies evaluating the clinical validity of CDC assays demonstrated that 80% of CDC cross-match–positive kidney transplants and 4% of cross-match–negative kidney transplants were associated with early graft loss, thereby verifying the clinical significance of anti-HLA antibodies in renal transplantation. It is note‐ worthy that 20% of patients transplanted across a positive cross-match did not lose their grafts [3]. Given that 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 assist in establishing whether class I and/or II anti-HLA antibodies are present. A positive B cell CDC cross-match 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 transplantation whereas a positive B cell cross-match is a relative contraindication because of the uncertainty regarding the clinical significance and the chance of false-positive results [7, 8]. The presence of a positive T cell crossmatch is an absolute contraindication for transplantation within the deceased donor kidney allocation algorithm in Australia and New Zealand. \On the contrary, 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 immunological risk of potential renal transplant candidates are established by regular monitoring and storage of their sera to establish peak and current immune reactivity against a panel of donor cells, termed peak and current panel reactive antibodies. When a potential donor becomes available, donor cells are incubated in the presence of both peak and current sera. The presence of a positive CDC cross-match with peak sera even in the presence of a negative CDC cross-match with current sera poses a contraindication to transplantation, as this suggests suggest immunological memory to donor antigens from prior sensitizing events.

off value may disadvantage many transplant candidates as it may detect anti-HLA donor specific antibodies of no clinical significance, especially in the presence of negative CDC crossmatch. Nevertheless, several studies have shown that the presence of a positive flow cytometric cross-match with a negative CDC cross-match is associated with a significantly greater risk of AMR and early graft loss with a positive predictive value for predicting AMR of 83% [10, 11]. To avoid problems associated with the availability and viability of donor cells that could affect the accuracy of cell-based assays, solid-phase assays were introduced which have largely circumvented these problems and improved the sensitivity of detection of anti-HLA antibodies [12]. 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 [13]. 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. The Luminex assay has been used in many transplant centres to select the appropriate desensitization regimen according to DSA strength and to establish an acceptable DSA cut-off that may allow kidney transplantation to proceed following desensitization [14, 15]. 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 single or multiple specific antigen(s). A phycoerytherin-labelled secondary 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 expressing the antigens. 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 [16, 17]. Unlike the CDC assays, Luminex assay detect both complementfixing and non-complement-fixing anti-HLA antibodies but does not detect IgM autoantibod‐ ies or non-HLA antibodies. The concept of virtual cross-match using solid phase assays relies on accurate HLA typing accompanied by evaluation of anti-HLA antibodies. The presence of a negative solid phase virtual cross-match reliably excludes the presence of donor-specific anti-HLA antibodies and is capable of predicting a negative flow cytometric cross-match in >90% of cases and CDC cross-match in 75% of cases. With the continued reliance on using cell-based cross-match assays, especially CDC cross-match assays to determine transplant suitability, a potential disadvantage of virtual cross-match is that transplants may be excluded based on antibody results with unknown clinical relevance [18]. It is generally accepted that solid phase virtual cross-match to identify anti-HLA donor specific antibodies complements the results of cell-based assays to help inform decision-making process with regards to transplant suitability.

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**3. Association between anti-HLA donor-specific antibodies and transplant**

Despite technological advances in detecting pre-transplant DSA, the incidence of acute and chronic AMR appears to increase over time. However, the true incidence of AMR remains

**outcomes (Table 1)**

The inability to correlate all graft losses to anti-HLA antibodies detected using CDC assays (i.e. an inability of CDC assays to detect low levels of clinically significant anti-HLA antibodies) has led to the development of the more sensitive cell-based flow cytometric cross-match assays. The fundamental principle that forms the basis of the flow cytometric cross-match assay is similar to that of the CDC assay. Since the description of this assay in the early 1980s, this technique has been widely adopted to determine transplant suitability in many countries [9]. Similar to the CDC assay, flow cytometric cross-match assays require the addition of donor cells to recipients' sera, followed by the addition of a fluorescein-labelled secondary antibody allowing for the detection and quantification of anti-HLA antibodies by flow cytometer expressed as mean channel shifts. Unlike CDC cross-match, flow cross-match identifies both complement-fixing and non-complement-fixing anti-HLA donor-specific antibodies. Howev‐ er, the availability of different subtypes of detection antibodies has allowed clinicians to differentiation between complement-fixing versus non-complement-fixing anti-HLA antibod‐ ies [10]. Although an universal mean channel shifts cut-off value corresponding to positive flow cross-match has not been determined, it is generally accepted that the use of a low cutoff value may disadvantage many transplant candidates as it may detect anti-HLA donor specific antibodies of no clinical significance, especially in the presence of negative CDC crossmatch. Nevertheless, several studies have shown that the presence of a positive flow cytometric cross-match with a negative CDC cross-match is associated with a significantly greater risk of AMR and early graft loss with a positive predictive value for predicting AMR of 83% [10, 11].

down the disulfide bonds in IgM antibodies believed to be of no clinical significance) to reduce the false positivity of these assays [5, 6]. Initial studies evaluating the clinical validity of CDC assays demonstrated that 80% of CDC cross-match–positive kidney transplants and 4% of cross-match–negative kidney transplants were associated with early graft loss, thereby verifying the clinical significance of anti-HLA antibodies in renal transplantation. It is note‐ worthy that 20% of patients transplanted across a positive cross-match did not lose their grafts [3]. Given that 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 assist in establishing whether class I and/or II anti-HLA antibodies are present. A positive B cell CDC cross-match 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 transplantation whereas a positive B cell cross-match is a relative contraindication because of the uncertainty regarding the clinical significance and the chance of false-positive results [7, 8]. The presence of a positive T cell crossmatch is an absolute contraindication for transplantation within the deceased donor kidney allocation algorithm in Australia and New Zealand. \On the contrary, 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 immunological risk of potential renal transplant candidates are established by regular monitoring and storage of their sera to establish peak and current immune reactivity against a panel of donor cells, termed peak and current panel reactive antibodies. When a potential donor becomes available, donor cells are incubated in the presence of both peak and current sera. The presence of a positive CDC cross-match with peak sera even in the presence of a negative CDC cross-match with current sera poses a contraindication to transplantation, as this suggests suggest immunological memory to donor antigens from prior sensitizing events.

108 Current Issues and Future Direction in Kidney Transplantation

The inability to correlate all graft losses to anti-HLA antibodies detected using CDC assays (i.e. an inability of CDC assays to detect low levels of clinically significant anti-HLA antibodies) has led to the development of the more sensitive cell-based flow cytometric cross-match assays. The fundamental principle that forms the basis of the flow cytometric cross-match assay is similar to that of the CDC assay. Since the description of this assay in the early 1980s, this technique has been widely adopted to determine transplant suitability in many countries [9]. Similar to the CDC assay, flow cytometric cross-match assays require the addition of donor cells to recipients' sera, followed by the addition of a fluorescein-labelled secondary antibody allowing for the detection and quantification of anti-HLA antibodies by flow cytometer expressed as mean channel shifts. Unlike CDC cross-match, flow cross-match identifies both complement-fixing and non-complement-fixing anti-HLA donor-specific antibodies. Howev‐ er, the availability of different subtypes of detection antibodies has allowed clinicians to differentiation between complement-fixing versus non-complement-fixing anti-HLA antibod‐ ies [10]. Although an universal mean channel shifts cut-off value corresponding to positive flow cross-match has not been determined, it is generally accepted that the use of a low cutTo avoid problems associated with the availability and viability of donor cells that could affect the accuracy of cell-based assays, solid-phase assays were introduced which have largely circumvented these problems and improved the sensitivity of detection of anti-HLA antibodies [12]. 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 [13]. 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. The Luminex assay has been used in many transplant centres to select the appropriate desensitization regimen according to DSA strength and to establish an acceptable DSA cut-off that may allow kidney transplantation to proceed following desensitization [14, 15]. 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 single or multiple specific antigen(s). A phycoerytherin-labelled secondary 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 expressing the antigens. 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 [16, 17]. Unlike the CDC assays, Luminex assay detect both complementfixing and non-complement-fixing anti-HLA antibodies but does not detect IgM autoantibod‐ ies or non-HLA antibodies. The concept of virtual cross-match using solid phase assays relies on accurate HLA typing accompanied by evaluation of anti-HLA antibodies. The presence of a negative solid phase virtual cross-match reliably excludes the presence of donor-specific anti-HLA antibodies and is capable of predicting a negative flow cytometric cross-match in >90% of cases and CDC cross-match in 75% of cases. With the continued reliance on using cell-based cross-match assays, especially CDC cross-match assays to determine transplant suitability, a potential disadvantage of virtual cross-match is that transplants may be excluded based on antibody results with unknown clinical relevance [18]. It is generally accepted that solid phase virtual cross-match to identify anti-HLA donor specific antibodies complements the results of cell-based assays to help inform decision-making process with regards to transplant suitability.
