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Keywords:

  • Human leukocyte antigen antibodies;
  • kidney transplantation;
  • sensitized patients;
  • waiting list

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. References

Identification of factors responsible for an increase in the breadth or strength of HLA-specific antibody (HSA) is critical to the continued successful management and transplantation of sensitized patients. A retrospective review of our HLA registry identified 107 patients with known HSA and sufficient information in their electronic patient record to determine the presence or absence of a proinflammatory event. The patients were stratified according to transplant status [sensitized and on the transplant waitlist (n = 65); immunosuppressed recipients of a positive crossmatch (+XM) transplant (n = 42)]. Eighty-three percent of waitlist candidates and 55% of sensitized kidney transplant recipients with a documented proinflammatory event had an associated increase in HSA. Interestingly, among patients with a culture-proven infection, 97% of the waitlist patients and 54.8% of +XM recipients had an associated rise in HSA. Overall, proinflammatory events were associated with a greater increase among waitlist patients than +XM recipients, 5.3-fold [IRR 5.25, (95% CI 4.03–6.85), p < 0.001] versus 2.5-fold [IRR 2.54, (95% CI 1.64–3.95), p < 0.001] increase in HSA. Therefore, sensitized patients known to have an infection or undergoing surgery should be monitored for expansion of HSA.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. References

An estimated 30% of patients on the renal transplant waiting list were sensitized to HLA antigens, and since 1995 the number of highly sensitized (PRA>80%) renal transplant registrations has more than doubled (based on OPTN data, accessed June 17, 2008). Sensitized patients on the renal transplant waiting list face many obstacles both before and after transplantation. On average, these patients wait longer to receive a kidney transplant (1). Patients wait-listed for a deceased donor transplant who experience an increase in the breadth of HLA-specific antibodies (HSA) have a further reduced opportunity for transplantation. Importantly, without frequent monitoring, such an increase in HSA may go undetected and result in an unexpected positive crossmatch (+XM). Sensitized patients are known to have poorer outcomes after transplantation (2–10). The increased incidence of acute rejection and graft loss is often antibody mediated and is related to the patient's prior sensitization (11). This may have particular relevance for patients who have undergone desensitization to overcome a +XM inasmuch as an antibody surges during the critical period of engraftment can be catastrophic. If events that are associated with an increase in HSA can be identified in sensitized patients awaiting transplantation and those desensitized for a +XM, then they can be appropriately monitored and treated. In turn, this will permit a more accurate assessment of risk for a potential transplant and aid in the accurate identification of unacceptable antigens.

Recently, we have observed among our sensitized patients changes in breadth and strength of HSA temporally associated with systemic proinflammatory events, such as infection, and hypothesized that such events may evoke a significant HSA increase. Given that more than 80% of renal transplant recipients will develop an infection during their first post-transplant year (12), further characterization of the relationship between proinflammatory events and rises in HSA is critical particularly in the era of transplantation across a known HLA incompatibility. To test this hypothesis, we reviewed sensitized waitlist patients and patients transplanted following desensitization for a possible association between known proinflammatory events and increases in HSA. Our results, while preliminary, suggest that specific proinflammatory events play a primary role in the elevation of HSA levels.

Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. References

Patient selection

The initial study population included 266 adult (≥18 years) sensitized patients who were either awaiting kidney transplant or had received a kidney transplant across a known HLA incompatibility with desensitization treatment using plasmapheresis and low-dose IVIg between January 2000 and December 2007. We excluded (a) patients without sufficient data in the electronic patient record (n = 31); (b) patients without a documented proinflammatory event (n = 71) and (c) patients in whom HSA was not measured within 1 month of the documented proinflammatory event (n = 57). We identified 107 patients with a documented proinflammatory event occurring within 1 month of blood sample collection and HSA measurement.

We then stratified patients according to transplant status [waitlist candidates (n = 65); +XM transplant recipients (n = 42)]. Proinflammatory events were categorized as either infection or other potential proinflammatory events (PPE). In each instance, bacterial infections were culture proven. Minor surgeries, major medical events (e.g. acute myocardial infarction) and traumatic injuries were considered PPEs.

HSA measurement—ELISA/Luminex™

Antibodies were assessed by solid-phase immunoassays. Tests were performed using commercial kits in either the ELISA (Quik ID class I and Quik ID class II, GTI, Brookfield, WI) or Luminex multianalyte bead platform (LifeMatch ID class I and Life Match ID class II, Tepnel Lifecodes, Stamford, CT) according to manufacturers’ instructions as previously described (13–15). Targets were HLA class I and class II phenotypes from panels of 41–50 and 20–26 subjects, respectively. Sequential sera from any patient were tested on the same platform against the same panel so that these sera had comparable evaluations. Panel reactive antibodies (PRA) values were given as the percentage of phenotypes yielding a positive reaction. Test metrics included optical density ratios (ODR) for sera tested by ELISA, median fluorescence intensity (MFI) tested by Luminex and the inverse of the last 2-fold dilution yielding a positive result in cytotoxicity crossmatch assays.

Statistical methods

HSA scores were found to be overdispersed count data, as the variance was found to be larger than the mean. Therefore, Poisson regression was not used to estimate the change in HSA. Instead, negative binomial regression was used to estimate the change in HSA as well as the coefficient of overdispersion, and a random intercept was used to account for correlation between repeated measurements in the same patient. Statistical significance was set at the α= 0.05 level. All analyses were performed using Stata 9.1 (StataCorp, College Station, TX).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. References

Sensitized waitlist candidates

Sixty-five patients were identified as having a documented proinflammatory event within 1 month of HSA measurement. Eighty-three percent of these patients (n = 54) had an associated rise in HSA from baseline. Thirty-five patients were determined to have an infection. Of those patients, 97.1% (n = 34) had an associated rise in HSA with a mean change of 37.7 ODR (range, 3.7–108.9) (Figure 1A). More than 50% of the documented infections were related to dialysis access. In addition, a total of 30 waitlist candidates had a documented PPE, 67% of whom were found to have an associated rise in HSA (n = 20) with a mean change of 34.6 ODR (range, 3.7–90.7) (Figure 1B). Examples of PPEs included myocardial infarction and surgical procedures such as AV-graft placement, thyroidectomy, native nephrectomy and cholecystectomy. Among sensitized waitlist candidates, a proinflammatory event was associated with a 5.25-fold increase in HSA from baseline (95% CI 4.03–6.85, p < 0.001).

image

Figure 1. Association between infection (panel A) and other potential proinflammatory events (PPE) (panel B) and increase in strength of HLA-specific antibodies (HSA) among sensitized waitlist candidates. A total 97% of waitlist candidates with a culture-proven infection had an associated rise in HSA with a mean change of 37.7 ODR (n = 34). In addition, 67% of waitlist candidates with a documented PPE were found to have an associated rise in HSA with a mean change of 34.6 ODR (n = 20). Examples of PPEs included myocardial infarction and surgical procedures, such as AV-graft placement, thyroidectomy, native nephrectomy and cholecystectomy.

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To provide an appropriate comparison population, 25 waitlist patients were identified who had multiple HSA measurements over a 6-month time period and sufficient electronic records to exclude with reasonable certainty the presence of a proinflammatory event. Their average baseline HSA level was 9.59 ODR (range, 0–38.8) with a mean change from baseline over that same time period of 1.49 ODR (range, 0–10.03). This is in contrast to the mean rise of 37.7 ODR that occurred among patients with a documented infection and 34.6 among patients with a documented PPE.

+XM transplant recipients

Forty-two patients were identified as having a culture-proven infection within 1 month of HSA measurement. Fifty-five percent of these patients (n = 23) had an increase in the breadth of HSA and a change in HSA specificity from baseline. In fact, the mean change in percentage of PRA was 45.8% (range, 11–90%). With regard to HSA specificity, the mean percentage increase in a donor-specific antibody (DSA) was 29.7 MFI (range, 1.3–95.4), representing an 8-fold increase in the cytotoxic crossmatch titer (baseline geometric mean 3.03, time of infection geometric mean 24.3) (Figure 2). The increase in the breadth of HSA was due primarily to expansion of reactivity among other antigens of a cross-reactive group (CREG) (data not shown). Among +XM kidney transplant recipients, a proinflammatory event was associated with 2.54-fold increase in HSA from baseline (95% CI 1.64–3.95, p < 0.001). Comparing +XM kidney transplant recipients with waitlist candidates, the baseline HSA was 1.93-fold higher (95% CI 1.17–3.19, p = 0.011) and the rise in HSA associated with a proinflammatory event was 2-fold lower (95% CI 1.27–3.33, p = 0.004).

image

Figure 2. Increase in strength of donor-specific antibodies (DSA) among incompatible kidney transplant recipients with a culture-proven infection. Strength of DSA was measured by percentage change in median fluorescence intensity (MFI) as measured by Luminex or a fold increase in cytotoxic crossmatch titer as measured by the change in a geometric mean. The mean change in the percentage of MFI from baseline was 29.7 MFI, and the fold increase from baseline in cytotoxic crossmatch titer was 8 (n = 23).

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. References

Sensitization has a significant impact on both access and outcome in transplantation, as it has been well documented that these patients wait longer to receive a transplant (1) and are more likely to experience an acute rejection episode in the post-transplant period (4,5). The presence of HSA prior to transplantation is correlated with higher rates of AMR and graft loss after transplantation (7–10). Regardless of the timing of HSA detection, it is clear that elevated HSA has substantial clinical and therapeutic implications, highlighting the importance of understanding the potential relationship between proinflammatory events, such as infection, that may result in an elevation in HSA (15–17).

The epidemiology of infectious diseases in the immunosuppressed transplant population has been well characterized (18). It is estimated that up to 80% of renal transplant recipients will develop an infection during the course of their first post-transplant year, emphasizing the contribution of infection to the morbidity and mortality of renal transplant patients (19,20). Further, several studies have demonstrated plasma cell infiltrates and C4d deposition on acute rejection biopsies of patients with viral infections (CMV and EBV) (21,22), supporting the observed association between infection and elevation in HSA among highly sensitized patients in our study. This relationship between infection and a rise in HSA persists even among immunosuppressed transplant recipients, suggesting the presence of a robust memory B-cell response to the release of proinflammatory cytokines independent of cognate antigen recognition.

Our data demonstrate a strong association between the development of infection and increases in both breadth and strength of HSA. This increase in HSA may prove to have a significant impact on the timing of transplantation for sensitized waitlist candidates and on the development of acute rejection among sensitized kidney transplant recipients. The data indicate that more frequent monitoring of HSA among sensitized patients may be prudent. Currently, however, there are federal regulations in place that limit reimbursement for antibody testing, which could hamper efforts to optimize the management of these patients. The vigilance required when monitoring and treating highly sensitized end-stage renal disease (ESRD) patients who have infectious complications cannot be overstated.

The impact of proinflammatory events on sensitized patients appears to extend beyond infection and may include surgical procedures, as noted in our study. Specifically, elevations in HSA have been documented after transplant nephrectomy (TN) (23). This elevation in HSA seen after TN can be explained by the removal of an organ capable of absorbing an antibody rather than the proinflammatory environment created by surgery. However, our findings suggest that surgical procedures may at least in part contribute to rises in HSA, as surgical procedures in addition to TN resulted in elevations in HSA, including AV-graft placement, thyroidectomy, native nephrectomy and cholecystectomy.

We must acknowledge that these data are preliminary and are subject to the many confounders and biases inherent in any retrospective review. While our manuscript is limited by its retrospective nature, based on prior studies it appears as though rises in HSA have the potential for significant clinical impact, including an increased risk for acute rejection in the transplant period. Clearly, future prospective studies need to be performed in order to more accurately correlate rises in HSA with clinical outcome. Given these results and the knowledge of increased infectious complications among ESRD patients and immunosuppressed transplant recipients, it is imperative that both pre- and post-transplant HSA screens be performed in response to documented patient infections or surgical procedures. Physicians, surgeons, nurses and coordinators should be educated about the association of proinflammatory events, particularly infection, with increases in HSA and with the potential deleterious consequences if left untreated.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. References