Objectives: The role of panel reactive antibody has gained universal acceptance in solid-organ transplant. This parameter is used to gauge the level of sensitization of prospective solid-organ recipients. More than one-third of patients on wait lists for kidney transplant are sensitized. Most have previously formed donor-specific and non–donor-specific serum antibodies and/or positive crossmatch by complement-dependent cytotoxicity and/or flow cytometry. We present the rate of positivity at our institution for human leukocyte antigen antibodies and describe the condensation of antibodies in human leukocyte antigens for renal pretransplant recipients.

Materials and Methods: Between January 2011 and December 2012, six hundred twenty consecutive renal transplant recipients on the wait list at the Baskent University were evaluated for this retrospective study. Panel reactive antibody screening and definition tests were studied with Luminex assays for the combination of class I (A, B, C) and class II antigens (DR, DQ).

Results: We found a panel reactive antibody screening positivity in 20.4% of our patients on renal transplant waiting list. Panel reactive antibody defining tests were meaningful in 12.2% of the whole list. We observed that only panel reactive antibody class I positivity was seen in 2.2%, only panel reactive antibody class II positivity was seen in 2.7%, and both panel reactive antibody class I and class II positivities were seen in 7.2% of the defining tests.

Conclusions: The estimated risk of sensitization for patients with a living donor is determined from the combined results of the crossmatch with the donor and those of the recipient’s panel reactive and donor-specific antibodies. Compared with complement-dependent cytotoxicity crossmatch, Luminex assays provide greater sensitivity and specificity in detection of donor-specific antibodies.

Key words : Donor specific antibody, Solid-organ transplant, Crossmatch

Introduction

Pretransplant antibodies against human leukocyte antigens (HLA) predispose one to hyperacute rejection and remain a significant obstacle in clinical renal transplant.^1,2 Blood transfusions, previous transplants, and pregnancy-induced alloimmunization; thus, this sensitization leads to an increase in the incidence of acute rejection. It is well-known that waiting time for sensitized patients is longer than it is for who are not sensitized; therefore, graft survival was affected negatively because of the prolonged dialysis treatment.³

After the development of new immunosuppressive treatments, T-cell–mediated acute rejection has been prevented. However, these new immuno­suppressive medicines, designed to prevent B-cell–mediated humoral rejection, could not accomplished successfully. Pretransplant antibodies against HLA molecules have been known risk factors of acute rejection and graft loss, since the early transplant investigations. Plasmapheresis, and intravenous immunoglobulin treatments to prevent the acute rejection, is difficult and expensive practices. Therefore, the importance of the HLA antibodies, non-HLA antibodies, and some of the autoantibodies (eg, vimentin) have been increasing, and thus, investigations have increased.^{4, 5}

Because the number of transplant patients increases year after year, the search for anti-HLA antibodies before transplant is of substantial clinical interest. These antibodies may be quantified by panel reactive antibody (PRA) estimation. Panel reactive antibody is defined as the percentage of HLA antigens singly or in combination out of a panel reacting with a patient’s serum, and may reflect the percentage of donors expected to react with the patient’s serum. To evaluate the PRA levels according to donor specificity is significant to reduce the possibility of rejection after transplant. We want to present at our institution the rate of positivity for HLA antibodies, and describe the condensation of antibodies in HLA locus for renal pretransplant recipients.

Materials and Methods

Between January 2011 and December 2012, six hundred twenty consecutive renal transplant recipients on the waiting list at the Baskent University Istanbul Practice and Research Hospital. The Transplantation Department was evaluated for this retrospective study. Panel reactive antibody screening and definition tests were studied with Gen-Probe-Lifecodes Screen Kits and Luminex-200, (Luminex Corporation, Austin, TX, USA) procedures from the sera obtained from freshly drawn blood samples in sterile Vacutainers from 620 patients with end-stage renal disease who had been worked-up for renal transplant. These kits contain a panel of fluorescence-labeled microbeads coated with combination of class I (A, B, and C) and class II antigens (DR and DQ) to estimate only IgG anti-HLA antibodies. Panel reactive antibody procedure was done according to product insert.

Results

A total of 620 patients diagnosed with end-stage renal disease and waiting for a renal transplant were analyzed in this retrospective study. The study was approved by the Ethical Review Committee of the Institute. All of the protocols conformed to the ethical guidelines of the 1975 Helsinki Declaration. Written informed consent was obtained from all subjects. We found panel reactive antibody screening positivity in 20.4% of our patients on the renal transplant waiting list (Table 1). Panel reactive antibody defining tests were evaluated in patients with positive PRA screening tests. Panel reactive antibody defining tests were meaningful in 12.2% of the whole list. We observed that PRA class I positivity seen in only 2.2%, PRA class II positivity seen in only 2.7%, and both PRA class I and class II positivity seen in 7.2% of the defining tests (Table 2).

We noted that antibodies formed against the HLA-B and HLA-DRB1 zone, which is known as the most polymorphic zone in the pretransplant recipients, whose defining tests were positive (Table 3 and 4). We did not observe any relation between them when we investigate the mean fluorescent index of antibodies.

Discussion

The role of antidonor antibodies remains an impor­tant issue in renal transplant and in solid-organ transplant in general. It is well-recognized that antigen-specific immune mechanism and non­immunologic factors play an important role in allograft rejection. The number of HLA antigen mismatches defines the degree of HLA incompatibility of between donor and recipient tissues, and the number of HLA mismatches correlates with graft outcomes.^1,2 Anti-HLA antibodies are significantly associated with pretransplant sensitization, nonprimary transplant, and posttransplant acute rejection episodes. Donor-specific antibodies are categorized into 2 major groups: antibodies against the major histocompatibility complex HLA, and antibodies against the non-HLA minor histo­compatibility complex. The major histocompatibility complex HLA antibodies involve anti-HLA class 1 (A, B, and C) and class 2 (DP, DQ, and DR) antibodies.^4,5

Crossmatch and antibody detection techniques have improved over time with increasing sensitivity and specificity. Complement-dependent cytotoxicity assay has remained the criterion standard for determining preformed antibodies after they were first described in the 1960s.³ Although this technique was used many years, it was seen that there were patients with a negative complement-dependent cytotoxicity assay experiencing humoral rejection. Because of this negation, it was understood that complement-dependent cytotoxicity fails to detect some clinically significant antibodies. The new technique that was known as solid phase technology was introduced to detect anti-HLA antibodies more accurately.

Human leukocyte antigen class I and class II specific antibodies can be individually identified using microbead assay or analyzed on the Luminex platform. Using flow beds or Luminex assays to define antibody specificity, the sensitivity of a virtual crossmatch ranged from 78% to 98% for flow T-cell crossmatch and from 88% to 98% for flow B-cell crossmatch. The specificity of virtual crossmatch ranged from 93% to 100% of flow T-cell crossmatch and 91% to 100% for flow B-cell crossmatch.^6-8 The other important advantage of the Luminex platform is introducing a single-antigen identification.

El-Awar⁹ reports that a single antigen bead allows complex sera reacting with many HLA molecules to be dissected and accurate detect antibody specificity. Among the techniques that we used to detect anti-HLA antibodies and donor-specific antibodies, we choose the Luminex screen, because it has been shown to be a sensitive and specific method to detect antibodies.

Currently, 35% patients on the transplant waiting list in the United States are sensitized with panel reactive antibody levels > 0%, and 15% patients are highly sensitized with PRA levels > 80%.10 Approximately 33% of transplant recipients have donor-specific antibodies, with approximately 75% having anti-HLA and 25% having anti-MICA antibodies.¹¹ We found that panel reactive screening positivity in 20.4% of our renal transplant waiting list. This rate decreased to 12.2% for PRA defining tests. We identified 65.2% antibodies positivity formed against the HLA-B and 69.8% for HLA-DRB1 zone.

Our study has several inherent limitations. This study was an incidence study to determine the panel reactive antibody positivity and determine the subgroups positivity for class I and class II antigens.In highly sensitized patients with antibodies against many different HLA alleles, the Luminex-supported single antigen bead test, because of its high resolution ability, is currently the only technique that allows the precise characterization of HLA antibody specificities. Those highly sensitized patients should preferably be transplanted from a deceased donor, rather than a living donor, because of the increased risk of antibody-mediated rejection and early graft failure and loss.^12-14 Highly sensitized recipients may be referred to the national waiting priority list and informed about the financial and medical risks that may occur with transplant.

Conclusions

The presence of donor-specific antibodies is associated with acute and chronic rejection and is predictive of graft loss. The estimated risk of sensitization for patients with a living donor is determined from the combined results of the crossmatch (complement-dependent cytotoxicity and flow cytometry) with the donor and those of the recipient’s PRA and donor-specific antibodies. Compared with complement-dependent cytotoxicity crossmatch, Luminex assays provide greater sensitivity and specificity in detection of donor specific antibodies.

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