Greg Bennett

Review article: Luminex technology for HLA antibody detection in organ transplantation.

Since its inception in the early 1960s, the serologically based complement‐dependent cytotoxicity (CDC) assay has been the cornerstone technique for the detection of human leucocyte antigen (HLA) antibodies, not only in pre‐transplant renal patients, but also in other forms of organ transplantation. Recently, solid phase assays have been developed and introduced for this purpose, and in particular the Flow‐based bead assays such as the Luminex system. This latter assay has proved to be far more sensitive than the CDC assay and has revealed pre‐sensitization in potential transplant recipients not detected by other methods of HLA antibody detection. However, the clinical implications of this increased sensitivity have not been convincingly demonstrated until recently. This technology for HLA antibody detection permits the evaluation of the clinical importance of antibodies directed at, for example, HLA‐DPB1 and HLA‐DQA1, which has not been possible to date. There are Luminex issues, however, requiring resolution such as the ability to distinguish between complement fixing and non‐complement fixing antibodies and determination of their relative clinical significance. Luminex technology will permit a re‐evaluation of the role of HLA antibodies in both early and late antibody‐mediated rejection.

Anti-HLA donor-specific antibodies detected in positive B-cell crossmatches by Luminex predict late graft loss.

The significance of B‐cell crossmatching in kidney transplantation is controversial. Recipients (n = 471) transplanted in a single centre from 1987 to 2005 with complete T‐ and B‐cell crossmatch records were studied. Sera from 83 patients transplanted across a positive B‐cell crossmatch, with concomitant negative T‐cell crossmatch (T–B+) on either current and/or peak sera were studied using Luminex® to determine presence of donor‐specific antibodies (DSA). Clinical outcomes of T–B+ patients were compared with 386 T–B− patients. T–B+ predicted vascular (p = 0.01), but not cellular (p = 0.82) or glomerular (p = 0.14) rejection. IgG HLA DSA were found in 33% (n = 27) of the T–B+ patients and were associated with higher risk of any (p = 0.047), vascular (p = 0.01) or glomerular (p < 0.001) rejection at 6 months. Of 27 patients with DSA, 18/21 (86%) were the complement‐fixing IgG1 and/or IgG3 subclass antibodies. DSA imposed a statistically significant higher risk of graft loss 5 years posttransplant (1.8 [1.0–3.3], p = 0.045). This study showed that only one‐third of positive B‐cell crossmatch (BXM) was caused by DSA and was associated with late graft loss. Thus, using BXM to preclude kidney transplantation may potentially disadvantage >60% of patients in whom BXM is not indicative of the presence of DSA.

Clinical significance of anti-HLA antibodies detected by Luminex®: Enhancing the interpretation of CDC-BXM and important post-transplantation monitoring tools

B-cell crossmatch (BXM) was originally introduced to increase the sensitivity to detect anti-HLA antibodies of conventional CDC crossmatch in renal transplantation. Newer techniques such as Luminex((R)) have greater sensitivity in detecting anti-HLA antibodies but have not been directly evaluated versus BXM. We discuss our experience with Luminex testing and the significance of donor-specific antibodies (DSA) defined by Luminex in three populations, as compared with the CDC crossmatch. In the general transplant population, Luminex-defined DSA were found in only one third of positive CDC-BXM and were associated with graft rejection. Luminex testing enhanced the interpretation of CDC-BXM and identified patients with clinically relevant BXM. In the highly sensitized transplant population, Luminex-defined DSA were found in two thirds of positive BXM and were a better predictor of graft rejection. Therefore, Luminex assays rather than CDC-BXM should be used to facilitate kidney allocation in highly sensitized patients. In the post-transplantation population, Luminex antibody monitoring for DSA was shown to be important, as it defined low-level de novo DSA that were associated with development of transplant glomerulopathy and a significant predictor of graft loss in those patients. Thus Luminex testing facilitated the interpretation of CDC-BXM and provided a useful predictive tool for the detection of clinically significant DSA in post-transplantation antibody monitoring.

Donor human leukocyte antigen specific antibodies predict development and define prognosis in transplant glomerulopathy

The pathogenesis of transplant glomerulopathy (TG) remains unclear, with evidence of human leukocyte antigen (HLA) antibodies as important contributors to the disease. We studied the risk factors and the associations of HLA antibodies in the development of TG. Sixty-one cases with morphologic features of TG were identified and compared with contemporaneous matched patients (without TG) from a 17-year period, all undergoing renal biopsy in a single center. Univariate risk factors for TG were previous glomerulitis [odds ratio (OR) 3.3, 95% confidence interval (95% CI) [1.2-9.4], p = 0.025), delayed graft function (OR 2.3 [1.0-5.1], p = 0.042), HLA class I presensitization defined by Luminex solid-phase immunoassays (OR 5.0 [2.3-11.0]. p < 0.001), and de novo posttransplant development of donor HLA specific antibody (DSA) (OR 4.7 [1.7-13.2], p = 0.002). Only DSA remained significantly associated with TG after adjustment (OR 3.8 [1.1-12.9], p = 0.032). DSA was detected in >50% of TG patients, suggesting HLA antibodies play a critical role in TG pathogenesis. TG patients with DSA had increased risk of graft loss (median graft survival 4.4-5.2 years), whereas patients with morphologic features of TG without DSA had similar graft survival compared with the non-TG group (median graft survival 15 years). Thus, DSA is a useful predictor for graft failure in TG patients.

C4d-negative antibody-mediated rejection with high anti-angiotensin II type I receptor antibodies in absence of donor-specific antibodies

Acute antibody‐mediated rejection can occur in absence of circulating donor‐specific antibodies. Agonistic antibodies targeting the anti‐angiotensin II type 1 receptor (anti‐AT1R) are emerging as important non‐human leucocyte antigen (HLA) antibodies. Elevated levels of anti‐angiotensin II receptor antibodies were first observed in kidney transplant recipients with malignant hypertension and allograft rejection. They have now been studied in three separate kidney transplant populations and associate to frequency of rejection, severity of rejection and graft failure.

Desensitization for renal transplantation: depletion of donor-specific anti-HLA antibodies, preservation of memory antibodies, and clinical risks

Desensitization protocols reduce donor‐specific anti‐HLA antibodies (DSA) and enable renal transplantation in patients with a positive complement‐dependent cytotoxic cross‐match (CDC‐CXM). The effect of this treatment on protective antibody and immunoglobulin levels is unknown. Thirteen patients with end‐stage renal disease, DSA and positive CDC‐CXM underwent desensitization. Sera collected pre‐ and post‐transplantation were analysed for anti‐tetanus and anti‐pneumococcal antibodies, total immunoglobulin (Ig) levels and IgG subclasses and were compared to healthy controls and contemporaneous renal transplant recipients treated with standard immunosuppression alone. Ten patients developed negative CDC‐CXM and enzyme‐linked immunosorbent assay (ELISA) and underwent successful transplantation. Eight recipients achieved good graft function without antibody‐mediated or late rejection, BK virus or cytomegalovirus infection. One patient had primary non‐function due to recurrent oxalosis, and one patient with immediate graft function died from septicaemia. Seven recipients required post‐operative transfusion and three developed septicaemia. DSA remained negative by ELISA at 12 months, but were detectable by Luminex®. Anti‐tetanus and anti‐pneumococcal antibodies, total Ig and IgG subclasses were below the normal range but comparable to levels in renal transplant recipients who had not undergone desensitization. Desensitization protocols effectively reduce DSA and allow successful transplantation. Post‐operative bleeding and short‐term infectious risk is increased. Protective antibody and serum immunoglobulin levels are relatively preserved.

Angiotensin II type 1 receptor antibody precipitating acute vascular rejection in kidney transplantation

Atypical non HLA antibodies are increasingly recognised as causes of immunological injury in allotransplantation. In this report we describe a non HLA sensitized male renal allograft recipient who developed acute vascular rejection on a “for cause” biopsy (Banff v2, g2, ptc 3) at day 4 post first renal allograft in the presence of elevated angiotensin II type 1 receptor antibodies (AT1R‐Ab level 14.1). The acute rejection was treated with pulse corticosteroid therapy, anti‐thymocyte globulin (ATG × 6), plasma exchange (1.5 plasma volume replacement x6) and oral candesartan. Serum creatinine improved and follow up biopsy confirmed resolution of rejection following treatment. AT1R‐Ab should be considered when rejection is diagnosed in the absence of HLA antibodies.

Human leucocyte antigen DQ alpha heterodimers and human leucocyte antigen DR alleles in tubulointerstitial nephritis and uveitis syndrome.

Tubulointerstitial nephritis and uveitis (TINU) syndrome is a rare clinical entity of acute interstitial nephritis (AIN) associated with uveitis. First described in 1975 by Dobrin et al., over 200 cases have been reported. Although the pathogenesis of TINU syndrome is unknown, the current view is that it is the result of an autoimmune process. Both cell-mediated immune responses and autoantibody production have been implicated. The association between TINU and human leucocyte antigen (HLA) phenotypes (DQ and DR) alleles has been the subject of several small studies. The authors review the literature and present two cases of definite TINU in an Australian population in which the same HLA DQA1*01, DQB1*05 and DRB1*01 haplotype was identified.

Transplant glomerulopathy and rapid allograft loss in the presence of HLA-Cw7 antibodies

Dear Sirs, Transplantation is the preferred modality of renal replacement therapy and as re-transplantation frequency increases, sensitized recipients require additional efforts to provide immunologically compatible grafts. Luminex techniques can be utilized to monitor donor-specific antibodies (DSA) that may influence premature allograft loss. We report the unusual case of anti-HLA-Cw7 antibodies leading to rapid loss of a renal transplant. The patient was a 21-year-old man (HLA-A3; B51,65; C*08:02,12:03; DRB1*01:01,13:02) with end-stage kidney disease because of congenital renal dysplasia. The first renal transplant from his mother (HLA-A3,24; B14[65],39; C*07:02,08:02; DRB1*13:02;15:01) failed after 5.5 years because of medication noncompliance. He made multiple DSA to his first transplanted kidney allograft (A24, C*07:02, DRB1*15:01). The first transplant was subsequently removed. As a consequence of his previous organ transplant and blood transfusion at the time of transplant nephrectomy, the patient was sensitized [complement-dependent cytotoxicity (CDC) panel reactive antibody peak 25%, confirmed by Luminex ]. The pretransplant antibody specificities included HLA Class I anti C*07:01, C*07:02, A*23:02, A*24:02, A*24:03, B*54:01 HLA Class II anti-DR15 [mean fluorescence intensity (MFI) 2062]. The patient’s father was a donor for the subsequent transplant (HLA-A2,3; B49,51; C*07:01,12:03; DRB1* 01:01,04:01). Because of ABO incompatibility (initial anti-A1 titre N/16 into O recipient) ABO desensitization was undertaken using an established protocol with a single dose of rituximab (375 mg/kg day )30), plasmapheresis (five complete 1.3 l plasma volume exchange) and immunoglobulin (0.5 g/kg day )1). HLA-C locus DSAs HLA-C*07:01 were noted in the pre-transplant serum (MFI 5206), but in the presence of a weak CDC B-cell crossmatch and negative T-cell crossmatch (prerituximab) desensitization for ABO incompatibility, with plasma exchange, was undertaken. At transplant, the recipient was CDC cross-match negative with an anti-A1 titre of 1:1 (tested by serial dilution against Dolichos Biflorus Lectin) with no evidence of DSA by enzyme-linked immunosorbent assay [1] or Luminex single antigen (Table 1) [2]. Standard immunosuppression was utilized (basiliximab induction, prednisolone, tacrolimus and mycophenolate mofetil) in addition to three postoperative plasmapheresis treatments, with immediate graft function. Tacrolimus and mycophenolic acid levels were therapeutic throughout the post-transplant period; antiA1 titres remained at 1:1. Graft function deteriorated acutely after 428 days; a biopsy demonstrated widespread transplant glomerulopathy and arteriolopathy that failed to respond to intravenous pulse methylprednisolone (1000 mg for three consecutive days), anti-thymocyte globulin (2.5 mg/kg for 7 days, Fresenius, Bad Homburg, Germany), plasmapheresis (6 · 1.3 plasma volume exchange for 2 weeks) and intravenous immunoglobulin (0.1 g/kg with each plasma exchange). Retrospective analysis of serum samples demonstrated a rise in HLA-Cw7 antibodies predating the deterioration in allograft function (Table 1). This report is the first to identify HLA-Cw7 associated with rapid development of transplant glomerulopathy. HLA-C*07:01 and )C*07:02, the most common alleles of this locus, demonstrate significant homogeneity particularly at the a1 helix antigen recognition site [3]. Antibodies to both (DSA and non-DSA respectively) developed simultaneously post-transplantation and this phenomenon may be because of epitope sharing. In a recent publication by Duquesnoy and Marrari, 56 HLA class I eplets to define antibodies in relation to the HLA-C locus were described [4]. From this report, a review of the patients HLA antibody repertoire identified 13 eplets (9D, 12 AVR, 63REN, 69KRQ, 69RA, 73AS, 77VSN, 79VRN, 113YD, 147L, 151ARA, 193PL, 267QE) specific for C*07:01/07:02, which are not explicable by the presence of shared eplets from other HLA class I specificities. This case is consistent with Duquesnoy and Marrari’s finding that HLA-C antibody producers were exposed to a median of 11 mismatched eplets resulting in increased detection of antibody by solid phase Luminex testing [4]. Only one previous report has implicated anti-HLA-C antibodies as the cause of acute renal allograft rejection

The mean fluorescence intensities of anti-HLA antibodies detected using micro-bead flow cytometry predict the risk of platelet transfusion refractoriness.

There are no accepted methods to predict the development of platelet transfusion refractoriness (PTR) due to human leucocyte antigen (HLA)‐alloimmunization. Hence, matched platelets are usually given only to patients demonstrating PTR, necessarily resulting in some ineffective random donor platelets (RDPLT) transfusions. To assess its utility in predicting PTR, we retrospectively tested samples from 387 patients receiving chemotherapy for acute leukaemia or autologous transplantation using a micro‐bead flow cytometry assay. The average of the mean fluorescence intensities (avgMFI) of the class I beads in the screening assay was correlated with outcomes of RDPLT transfusions during a 2 week period. Antibodies were detected in 57 patients; 66 developed PTR, of whom 28 were alloimmunized. avgMFI usefully predicted the development of PTR (area under the receiver operating curve 0·87, 95% confidence interval: 0·77–0·96). A logistic regression model estimated the probability of PTR to be >90% when avgMFI >5440. These results indicate that micro‐bead flow cytometry assays could inform a risk‐adapted strategy for managing thrombocytopaenic HLA allo‐immunized patients.