**3. Association between anti-HLA donor-specific antibodies and transplant outcomes (Table 1)**

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


unclear with suggestions that acute AMR may account for up to 7% of all acute rejections (and up to 50% of acute rejection episodes experienced by pre-sensitized patients with positive cross-match); whereas the prevalence of chronic AMR manifesting as transplant glomerulop‐ athy may be as high as 20% at 5 years post-transplant [19, 20]. The growing incidence may be attributed to a number of plausible reasons including: greater acceptance of highly-sensitized candidates for transplantation, the use of non-calcineurin-inhibitor-based immunosuppres‐ sive regimen such as mammalian target of rapamycin inhibitors, better detection techniques for DSA, availability of markers of antibody injury such as C4d staining and a greater under‐ standing of AMR, which may have been misinterpreted as chronic allograft nephropathy or

Detection of Antibody-Mediated Rejection in Kidney Transplantation and the Management of Highly Sensitised…

http://dx.doi.org/10.5772/54735

111

In most countries, a large proportion of renal transplant candidates on the transplant waitlist are sensitized with high PRA levels and have multiple anti-HLA antibodies, which often result in protracted wait-list time [22]. In Australia, 23% of transplant candidates have a peak class I PRA of >20% and these sensitized transplant candidates often have twice as long a waiting time as unsensitized candidates [23]. Pre-transplant DSA is a major immunologi‐ cal hurdle for successful kidney transplantation. The clinical importance of pre-transplant DSA has been clearly established over the past decade and the presence of high levels of pre-transplant class I (HLA-A and B) ± II (HLA-DR) DSA, typically occurring as a result of prior sensitizing events including previous blood transfusions, HLA-mismatched trans‐ plants and/or pregnancy, is associated with inferior graft outcomes, including an increased risk of developing acute and chronic antibody-mediated rejection (AMR), transplant glomer‐ ulopathy and late graft loss (Table 1) [24-27]. However, few studies have suggested that the association between pre-transplant DSA and graft survival was restricted to recipients who had developed early AMR or those with high levels of DSA as determined by peak HLA-DSA strength expressed as mean fluorescent intensity (MFI) using Luminex technology and that pre-transplant screening for preformed DSA may not be cost-effective [28, 29]. *Lefau‐ cheur C et al* demonstrated in a large single centre study that renal transplant recipients with a peak pre-transplant DSA >465 MFI determined by Luminex have a significantly higher risk of developing AMR and that recipients with peak DSA >3000 have almost a four-fold increase in the risk of graft loss compared to recipients with peak DSA MFI of <3000 high‐ lighting the importance of using DSA strength to more accurately assess the immunological risk of transplant recipients [29]. There is also increasing evidence demonstrating that the development of *de novo* DSA may occur in over 50% of renal transplant recipients at 2-years post-transplant suggesting that regular monitoring of de novo DSA post-transplant may help identify those at risk of developing poorer graft outcome [30]. Several studies have shown that the development of *de novo* DSA (occurring post-transplantation), especially DSA directed against HLA-DQ graft molecules in HLA-class II incompatible graft transplan‐ tations, are both associated with acute and subclinical AMR and graft loss in kidney trans‐ plant only and/or simultaneous pancreas-kidney transplant recipients and post-transplant monitoring of DSA could potentially help clinicians to individualize the amount of immuno‐

undefined rejection in the past [21].

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, BPAR – biopsy-proven acute rejection, PPV – positive predictive value.

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

unclear with suggestions that acute AMR may account for up to 7% of all acute rejections (and up to 50% of acute rejection episodes experienced by pre-sensitized patients with positive cross-match); whereas the prevalence of chronic AMR manifesting as transplant glomerulop‐ athy may be as high as 20% at 5 years post-transplant [19, 20]. The growing incidence may be attributed to a number of plausible reasons including: greater acceptance of highly-sensitized candidates for transplantation, the use of non-calcineurin-inhibitor-based immunosuppres‐ sive regimen such as mammalian target of rapamycin inhibitors, better detection techniques for DSA, availability of markers of antibody injury such as C4d staining and a greater under‐ standing of AMR, which may have been misinterpreted as chronic allograft nephropathy or undefined rejection in the past [21].

**Study Cohort Rejection Graft survival**

(p=0.01)

glomerular rejection

categorized by MFI: MFI <465 – prevalence 1% MFI 466 to 3000 – prevalence 19% MFI 3001 to 6000 – prevalence 36% MFI >6000 – prevalence 51% Peak DSA MFI predicted AMR better

than current DSA MFI

Incidence AMR 31%

greater risk of AMR

vs >9500 36%

negative 35%

0%

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

Total MFI and AMR: <9500 7%

Class II DSA but not class I DSA

Overall incidence of clinical/subclinical rejection including AMR and/or acute Tcell mediated rejection at day 200 posttransplant: DSA-positive 71% vs DSA-

BPAR: DSA-positive 56% vs DSA-negative

Class II > class I DSA higher incidence of AMR: 100% vs 22% Class II DSA MFI of 4487 predicted AMR with sensitivity of 100% and specificity of 87%.

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, BPAR – biopsy-proven acute rejection, PPV – positive

vs no DSA 3% (p < 0.001)

Vascular: 19% T-B- XM vs 32% T-B+ XM

Graft loss: T-B+ 44% vs T-B-

Non-sensitized - 89% and 84% Sensitized with no DSA - 92%

DSA-positive - 71% and 61 Relative risk for graft loss if AMR 4.1 (95% CI 2.2 to 7.7) vs

Overall graft survival at 8 years:

Graft survival lower in patients with DSA and AMR compared to DSA and no AMR and in non-DSA patients

3-year graft survival was 100% for total MFI <9500 vs 76% for

5-year DCGS: No DSA 89% vs DSA without AMR 87% vs DSA

No difference in graft survival

DSA-positive 68% DSA-negative 77%

total MFI >9500.

with AMR 68%

5 and 8-year DCGS:

and 92%

no AMR

27%

DSA+ significantly predict vascular or

PPV for AMR with peak DSA 35% vs current DSA 32% Prevalence of AMR

Incidence of AMR: preformed DSA 35%

*Eng H et al* [24]

*Lefaucheur C et al* [25]

*Lefaucheur C et al* [26]

*Mujtaba M et al* [34]

*Amico P et al* [94]

*Song EY et al* [95]

predictive value.

N=471 DD renal transplant

110 Current Issues and Future Direction in Kidney Transplantation

83 T-B+ XM vs 386 T-B- XM IgG DSA in 33% of T-B+ XM

N=402 DD renal transplant

Peak sera: positive DSA 21%

N=237 LD/DD renal transplant

All negative T and B-cell

27% class I or II anti-HLA antibody with 52% anti-HLA antibody being DSA

N=44 desensitized LD transplant recipients Negative CDC T-cell XM Sensitization = CDC B+ & T+ ± B+ flow XM

N=334 LD and DD renal transplant recipients 332 negative T and B cell CDC-XM 67 DSA vs 267 no DSA

N=28 LD and DD renal transplant recipients Positive flow XM but negative CDC-T cell XM, 57% positive DSA

(Luminex)

Current sera: positive

recipients

patients

recipients

(Luminex)

DSA 19%

recipients

CDC-XM

In most countries, a large proportion of renal transplant candidates on the transplant waitlist are sensitized with high PRA levels and have multiple anti-HLA antibodies, which often result in protracted wait-list time [22]. In Australia, 23% of transplant candidates have a peak class I PRA of >20% and these sensitized transplant candidates often have twice as long a waiting time as unsensitized candidates [23]. Pre-transplant DSA is a major immunologi‐ cal hurdle for successful kidney transplantation. The clinical importance of pre-transplant DSA has been clearly established over the past decade and the presence of high levels of pre-transplant class I (HLA-A and B) ± II (HLA-DR) DSA, typically occurring as a result of prior sensitizing events including previous blood transfusions, HLA-mismatched trans‐ plants and/or pregnancy, is associated with inferior graft outcomes, including an increased risk of developing acute and chronic antibody-mediated rejection (AMR), transplant glomer‐ ulopathy and late graft loss (Table 1) [24-27]. However, few studies have suggested that the association between pre-transplant DSA and graft survival was restricted to recipients who had developed early AMR or those with high levels of DSA as determined by peak HLA-DSA strength expressed as mean fluorescent intensity (MFI) using Luminex technology and that pre-transplant screening for preformed DSA may not be cost-effective [28, 29]. *Lefau‐ cheur C et al* demonstrated in a large single centre study that renal transplant recipients with a peak pre-transplant DSA >465 MFI determined by Luminex have a significantly higher risk of developing AMR and that recipients with peak DSA >3000 have almost a four-fold increase in the risk of graft loss compared to recipients with peak DSA MFI of <3000 high‐ lighting the importance of using DSA strength to more accurately assess the immunological risk of transplant recipients [29]. There is also increasing evidence demonstrating that the development of *de novo* DSA may occur in over 50% of renal transplant recipients at 2-years post-transplant suggesting that regular monitoring of de novo DSA post-transplant may help identify those at risk of developing poorer graft outcome [30]. Several studies have shown that the development of *de novo* DSA (occurring post-transplantation), especially DSA directed against HLA-DQ graft molecules in HLA-class II incompatible graft transplan‐ tations, are both associated with acute and subclinical AMR and graft loss in kidney trans‐ plant only and/or simultaneous pancreas-kidney transplant recipients and post-transplant monitoring of DSA could potentially help clinicians to individualize the amount of immuno‐ suppression to better assess immune reactivity [25, 30-33]. Although there is no current con‐ sensus 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 [29, 34]. Recent stud‐ ies 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 accurate in predicting acute rejection, biopsy C4d-deposition, transplant glomerulopathy and late graft failure following kidney transplantation and the authors suggested that the absence of C1q-positive de novo DSA has a high negative predictive value for transplant glomerulopathy (100%) and graft failure (88%) [35]. However, a recent retrospective study showed that the identification of strong complement-activating DSA (of IgG subclasses 1 and 3) pre-transplant was unlikely to improve AMR risk stratification compared to patients with a combination of both strong and weak/no complement-activating DSA (of IgG subclasses 2 and 4) [36]. The clinical im‐ portance of C1q-specific DSA in predicting graft outcome remains controversial and not routinely performed in many transplanting centres [35, 37]. 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 by removing circulating DSA and/or B or plasma cells and the success and outcomes of these initiatives will be discussed later in this chapter.

**Antibodies HLA-**

Anti-angiotensin type 1–receptor antibody

Anti-endothelial cell antibody [39,99]

Glutathione-S-transferase

Antibodies to MIG (also called CXCL9), ITAC (also called CXCL11), IFN-γ, and glial-derived neurotrophic

factor [103]

Anti-HLA-Ia antibody [105]

Protein kinase Czeta antibody [104]

HLA – human leukocyte antigen

T1 antibody [40]

[96,97]

**antigen (Yes/No)**

MICA antibody [98] Yes Major histocompati-

Vimentin antibody [100] No Intermediate filament

Agrin antibody [101] No Highly purified GBM

Anti-GBM antibody [102] No Alpha-3 chain (the

No Angiotensin type I

ELISA)

(Luminex)

precursors (flow cytometry)

heparan sulphate proteoglycans (ELISA)

transferase T1 enzyme

Goodpasture antigen) and alpha-5 chain of type IV collagen (ELISA)

protein (flow cytometry)

No Endothelial cell

No Glutathione-S-

(ELISA)

No Chemokine or cytokine (ELISA)

(microarray)

No Protein kinase

receptor (cell-based

bility-complex class I related chain A antigens

**Target antigen Location Transplant outcomes**

Detection of Antibody-Mediated Rejection in Kidney Transplantation and the Management of Highly Sensitised…

Endothelial cells (also fibroblasts, epithelial cells)

Circulating proteins

Kidney and lymphocytes

Yes HLA-Ia alleles Endothelial cells Correlate with poorer graft

Abbreviations: MICA – major histocompatibility complex class I chain-related gene A, ACR – acute cellular rejection, AMR – antibody mediated rejection, GBM – glomerular basement membrane, ELISA – enzyme-linked immunosorbent assay,

**Table 2.** Association between non-HLA-DSA and non-HLA antibodies and renal transplant outcomes.

Endothelial cells Increased risk of ACR, vascular

hypertension

Endothelial cells Increased risk of acute and

Endothelial cells Increased risk of rejection

GBM Increased risk of transplant glomerulopathy

Endothelial cells Increased risk of C4d-negative

GBM Increased risk of vascular

acute and chronic AMR

rejection (Alport patients)

Association with chronic renal

Increased risk of graft loss

survival, possibly mediated via anti-HLA-E IgG antibody

allograft injury

chronic rejections

rejection and AMR ± malignant

http://dx.doi.org/10.5772/54735

113

Increased risk of rejection and graft failure, remains debatable
