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Ann Thorac Surg 2000;69:1520-1524
© 2000 The Society of Thoracic Surgeons

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Original articles: General thoracic

Influence of panel-reactive antibodies on posttransplant outcomes in lung transplant recipients

^a Departments of Department of Surgery, Duke University Medical Center, Durham, North Carolina, USA
^b Department of Medicine, Duke University Medical Center, Durham, North Carolina, USA
^c Department of Pathology, Duke University Medical Center, Durham, North Carolina, USA
^d Department of Immunology, Duke University Medical Center, Durham, North Carolina, USA

Address reprint requests to Dr Davis, Department of General and Thoracic Surgery, Duke University Medical Center, Box 3864, Durham, NC 27710
e-mail: davis053{at}mc.duke.edu

Presented at the Forty-sixth Annual Meeting of the Southern Thoracic Surgical Association, San Juan, Puerto Rico, Nov 4–6, 1999.

	Abstract

Top Abstract Introduction Material and methods Results Comment References

 
Background. Panel-reactive antibody (PRA) is used to estimate the degree of humoral sensitization in the recipient before transplantation. Although pretransplant sensitization is associated with increased complications in other solid organ transplant recipients, less is known about the outcome of sensitized lung transplant recipients. Therefore, we sought to determine the impact of elevated pretransplant PRA on clinical outcomes after lung transplantation.

Methods. The records of the first 200 lung transplant operations performed at Duke University Medical Center were reviewed. The outcomes of sensitized patients, PRA greater than 10% before transplantation (n = 18), were compared with the outcomes of nonsensitized patients.

Results. Sensitized patients experienced a significantly greater number of median ventilator days posttransplant (9 ± 8) as compared with nonsensitized recipients (1 ± 11; p = 0.0008). There were no significant differences between the number of episodes of acute rejection; however, there was a significantly increased incidence of bronchiolitis obliterans syndrome occurring in untreated sensitized recipients (56%) versus nonsensitized (23%; p = 0.044). In addition, there was a trend towards decreased survival in the sensitized recipients, with a 2-year survival of 58% in sensitized recipients as compared with 73% in the nonsensitized patients (p = 0.31).

Conclusions. Sensitized lung transplant recipients experience more acute and chronic complications after transplantation. These patients probably warrant alternative management strategies.

	Introduction

Top Abstract Introduction Material and methods Results Comment References

 
Lung transplantation has emerged as a viable therapeutic option in patients with end-stage pulmonary and pulmonary vascular disease. Although overall survival after transplantation has shown significant improvements in recent years as compared with previous years, current 1-year survival remains 70% and 5-year survival remains a disappointing 40% [1]. Primary graft failure and bronchiolitis obliterans have been identified as major causes of early and late posttransplant mortality, respectively. However, the pathogenesis of both these processes is poorly understood, and consequently treatment is often ineffective.

Panel-reactive antibodies (PRA) are used to estimate the degree of humoral sensitization in the recipient before transplantation. Sensitization, defined as a positive panel reactive antibody screen, represents circulating antibodies to a random panel of donor lymphocytes. The development of pretransplant antibodies may be caused by previous blood transfusions, prior pregnancies, connective tissue diseases, or previous transplantation. Although pretransplant sensitization is associated with increased acute and chronic complications in most solid organ transplant recipients [2–5], less is known about the outcome of sensitized lung transplant recipients [6].

Because the presence of antibodies to human leukocyte antigens (HLA) pretransplantation has been associated with increased rates of acute graft failure and chronic rejection in heart and kidney allograft recipients, we hypothesized that sensitized lung transplant recipients with antibodies to HLA as measured by the panel of reactive antibody (PRA) screen would have increased primary allograft dysfunction and/or increased chronic allograft rejection manifesting as obliterative bronchiolitis. In order to determine the impact of elevated pretransplant PRA on outcomes after lung transplantation, we reviewed the outcomes of lung transplants performed in patients with an elevated PRA and compared ventilator days, length of hospital stay, acute rejection episodes, development of chronic rejection, and overall survival in these sensitized patients with nonsensitized recipients. In addition, all posttransplant biopsy specimens were reviewed in the sensitized patients.

	Material and methods

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We retrospectively reviewed the records of the first 200 lung transplant operations (performed at Duke University Medical Center between November 1992 and December 1998) and identified all patients with a PRA screen of greater than 10% before transplantation. The technique for determining PRA during this study was the complement-dependent cell cytotoxicity (CDC) test. Briefly, a panel of T-cell-enriched lymphocytes from donors of known HLA types is screened with recipient sera using the microcytotoxicity technique enhanced with anti-human globulin [7], and the percent of panel lymphocytes lysed by the recipient serum in the presence of complement is reported. Although pretreatment of the recipient’s sera with dithiothreitol can be used to increase the specificity of the PRA test (by inactivating IgM autoantibodies), it was not utilized. In this study, the peak PRA level was used that generally corresponded with the most recent PRA. PRA levels greater than 10 were considered elevated (sensitized) in this analysis.

Ventilatory days
Ventilator days posttransplant in sensitized lung transplant recipients were compared with those in nonsensitized recipients. Patients on a ventilator pretransplant were not considered in this analysis.

Length of hospital stay
The number of days recipients remained hospitalized after transplantation was compared between the sensitized and the nonsensitized groups. All patients were considered in this analysis.

Diagnosis of acute and chronic rejection (bronchiolitis obliterans syndrome [BOS])
All patients underwent surveillance bronchoscopes at 1, 3, and 6 months after transplantation. In addition, bronchoscopic or open biopsies were performed if patients developed clinical indications such as a fall in forced expiratory volume in 1 second, hypoxia, or new abnormality on chest roentgenogram. Acute rejection was defined by histologic criteria found on biopsy and based on the uniform grading system revised in 1996 [8]. All episodes were documented and the number of grade 1 or greater rejection in the first 100 days was used for the analysis. Episodes of acute rejection were treated with intravenous methylprednisolone at 500 mg/d for 3 days followed by a prednisone taper.

The diagnosis of BOS was made based on pulmonary function data and histological evidence from lung biopsy results according to International Society of Heart and Lung Transplantation criteria [9]. The diagnosis of BOS requires a 20% or greater decline in pulmonary function from a patient’s baseline and the exclusion of acute rejection or infection. Recipients were considered at risk for BOS only if they survived 6 or more months after transplantation. For the analysis, patients with a BOS score of 1 or greater were considered to be positive, while a BOS score of 0 was considered negative.

Immunosuppression
All patients received similar immunosuppression after transplantation. From 1992 to 1997, all patients received induction with intravenous methylprednisolone and were maintained on long-term immunosuppression with cyclosporine (5 to 10 mg/kg/d), azathioprine (1 to 2 mg/kg/d), and prednisone (initially at 20 mg/d). From 1997 to 1998, patients received either azathioprine or mycophenolate (1 g twice daily) as part of an ongoing randomized study, in addition to cyclosporine or prednisone. Tacrolimus was not used as a primary agent in any patient, but patients were used in certain cases of refractory acute rejection, chronic rejection, or suspected cyclosporine intolerance. Induction with rabbit antithymocyte globulin (RATG) was employed in 22 patients from 1994 to 1996 as part of previous randomized study. RATG or antithymocyte immunoglobulin (ATGAM) therapy was also used in certain cases of refractory acute rejection or chronic rejection.

Pathology
All transbronchial, surgical, or autopsy specimens in sensitized patients were reviewed by an experienced pulmonary pathologist (D.N.H.).

Statistical analysis
Kaplan-Meier actuarial survival curves were performed using the life table method on SAS software (version 6.12; SAS Institute, Cary, NC). Differences between the groups were compared using the Wilcoxon rank sum test, ² analysis, or Fisher’s exact test, as appropriate, performed on SAS software. Values were reported as mean ± SEM.

	Results

Top Abstract Introduction Material and methods Results Comment References

 
Clinical outcomes are shown in Table 1. Of the 200 lung transplants performed between November 1992 and December 1998, 18 (9%) were in patients with at least one pretransplant PRA screen of greater than 10%. The mean PRA, donor, and recipient ages, gender, race, and underlying disease of sensitized and nonsensitized patients are shown in Table 2.

Survival
Survival by Kaplan-Meier analysis of the sensitized cases was not significantly different from nonsensitized cases: 65% (high PRA) versus 79% (low PRA) at 1 year, and 58% (high PRA) versus 73% (low PRA) at 2 years (Fig 1); p = 0.31 by log rank test. Kaplan-Meier analysis of all 200 lung transplant patients showed 1-, 2-, and 3-year survivals to be 78%, 71%, and 61%, respectively. There was also no difference in survival for high (>10) versus intermediate (1 to 10) PRA.

View larger version (11K): [in this window] [in a new window]   Fig 1. Kaplan-Meier comparison of survival between high and low PRA.

Length of stay
The number of days recipients remained hospitalized after lung transplantation did not differ significantly between the high PRA patients (37 ± 6 days) and the low PRA group (29 ± 3 days) (p = 0.41).

Acute rejection
The number of acute rejection per 100 days posttransplant did not differ significantly between the two groups. The sensitized recipients experienced an average of 0.5 ± 0.2 episodes of rejection during the first 100 days after transplantation, while the nonsensitized group experienced 0.8 ± 0.07 episodes (p = 0.18).

BOS
The incidence of BOS in patients who survived 6 months or greater posttransplantation was compared among sensitized patients not treated for the elevated PRA (n = 14), sensitized patients treated postoperatively with plasmapheresis and intravenous immunoglobulin (IVIG) to reduce the PRA (n = 4), and eligible nonsensitized patients (n = 139). BOS occurred in 56% of untreated sensitized patients versus 23% of nonsensitized patients (p = 0.044). In contrast, among treated sensitized patients, all 3 who survived greater than 6 months are free from BOS (grade 0). The fourth patient died of primary allograft failure and sepsis 2 months posttransplant. Because of the small number, treated sensitized patients were not included in the statistical analysis.

Pathology
Histological samples were reviewed in all sensitized patients. We have seen evidence of humoral-mediated injury in posttransplant biopsies from patients with high PRAs. In these patients, biopsies taken during episodes of allograft dysfunction showed evidence of vascular injury in a pattern distinct from acute cellular rejection. In 1 patient, a biopsy obtained 1 day posttransplant showed marked alveolar capillaritis accompanied by endothelial swelling and margination of inflammatory cells in arterioles (Fig 2). Immunofluorescent staining of a portion of this biopsy preserved in the fresh state revealed deposition of C3 in a pattern outlining capillary walls (Fig 3). Staining for IgG and IgM was also present, but in a more diffuse pattern. In a second patient, a biopsy taken 1 week posttransplant showed extensive swelling of endothelial cells in small arteries and arterioles, again accompanied by margination of mixed inflammatory cells including neutrophils. In a third patient, a biopsy obtained several months posttransplant showed extensive endothelial reactivity and intimal myxoid change in the absence of significant inflammation. In all biopsies, perivascular mononuclear inflammatory cell infiltrates were minimal. Our rationale for plasmapheresis in these patients was based on the resolution of these pathologic changes after treatment, adding additional credence to these findings being antibody mediated.

View larger version (86K): [in this window] [in a new window]   Fig 2. Immunohistochemical staining for complement (C3) showing deposition in a pattern outlining capillary walls from a patient with a high PRA. This staining is evidence of humoral-mediated lung injury.

View larger version (148K): [in this window] [in a new window]   Fig 3. Histopathologic examination of biopsy from same patient as Figure 2 showing marked alveolar capillaritis accompanied by endothelial swelling and margination of inflammatory cells (hematoxylin and eosin, x520).

	Comment

Top Abstract Introduction Material and methods Results Comment References

In comparison, Gammie and colleagues [6] found elevated PRA did not predict decreased survival or the development of acute rejection or bronchiolitis obliterans in lung transplant recipients. This paper retrospectively evaluated PRA levels in 247 lung transplant recipients and found 21 (8.5%) had preoperative PRA values greater than 10%. The paper concluded that PRA testing before lung transplantation should be used selectively only for those potential recipients with specific risk factors for humoral sensitization.

We further evaluated the impact of elevated pretransplant PRA on clinical outcomes after lung transplantation. As expected, the majority of recipients with elevated PRA values had risk factors for humoral sensitization (parous female, previous blood transfusions, previous transplants, connective tissue diseases). Sensitized patients had a significantly greater number of median ventilator days posttransplant (9 ± 8) as compared with nonsensitized recipients (1 ± 11; p = 0.0008). Although not significant, there was a trend towards decreased survival in the sensitized recipients, with 2-year survival of 58% in sensitized recipients as compared with 73% in the nonsensitized patients (p = 0.31). One explanation for this is that antibody-induced injury is seen early in the course posttransplant. Humoral injury could prolong ventilation in these patients and contribute towards a trend of worse survival. Elevated PRA places recipients at higher risks for hyperacute rejection in other solid organs, but the role of antibodies in lung allograft rejection remains controversial. The pathology we reported suggests antibody-mediated injury may be important in lung transplant recipients, particularly those with elevated pretransplant PRAs.

Interestingly, despite our immunohistochemical findings of humoral injury in some recipients with high PRA, to our knowledge, antibody-mediated injury to the lung has been reported only anecdotally [12]. In fact, Saint Martin and colleagues [13] performed immunofluorescence with C3, immunoglobin M, and immunoglobin G and found no evidence of humoral rejection in 106 biopsies, but in contrast to our study, only 1 patient had a high pretransplant PRA; therefore, their results are not inconsistent with ours.

In agreement with Gammie and colleagues [6], we also found elevated PRA levels pretransplant did not correlate with increased number of acute rejection episodes. In contrast to Gammie and colleagues, our study showed BOS appeared in a significantly greater percent of high PRA recipients (56%) compared with low PRA patients (23%; p = 0.044) undergoing lung transplant. Several reasons exist for the difference in the two studies and, importantly, both studies only evaluated a small number of patients with high PRAs. The treatment course of patients with high PRAs may have differed between the studies. Our study concluded before all recipients had completed the 6-month period posttransplant and were therefore not analyzed for a BOS score, while in the study by Gammie and colleagues, follow-up time was sufficient for all patients eligible to receive a BOS score (they used as criteria a survival of greater than 60 days). In support of our finding that elevated pretransplant PRA levels predict BOS occurrence is the paper by Andres and colleagues [14], suggesting the development of anti-HLA antibodies after lung transplantation play an important role in the development of BOS.

In conclusion, elevated pretransplant PRA values in recipients undergoing lung transplantation may identify a high-risk population (for early humoral-mediated injury and chronic allograft dysfunction). Alternative management strategies designed to lower anti-HLA antibodies peri- and postoperatively may be indicated in this population. The scheme we now utilize at Duke University Medical Center is shown in Figure 4.

View larger version (15K): [in this window] [in a new window]   Fig 4. Proposed treatment scheme for lung transplant recipients with elevated pretransplant PRA.

	References

Top Abstract Introduction Material and methods Results Comment References

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2. Charco R., Vargas V., Balsells J., et al. Influence of anti-HLA antibodies and positive T-lymphocytotoxic crossmatch on survival and graft rejection in human liver transplantation. J Hepatol 1996;24:452-459.[Medline]
3. Smith J.D., Danskine A.J., Laylor R.M., Rose M.L., Yacoub M.H. The effect of panel reactive antibodies and the donor specific crossmatch on graft survival after heart and heart-lung transplantation. Transplant Immunol 1993;1:60-65.[Medline]
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6. Gammie J.S., Pham S.M., Colson Y.L., et al. Influence of panel-reactive antibody on survival and rejection after lung transplantation. J Heart Lung Transplant 1997;16:408-415.[Medline]
7. Southeastern Organ Procurement Foundation Tissue Typing Manual. Vol. section B-20. Richmond, VA: SEOPF, 1993:12–3.
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11. Kerman R., Susskind B., Buelow R., et al. Correlation of ELISA-detected IgG and IgA anti-HLA antibodies in pretransplant sera with renal allograft rejection. Transplantation 1996;62:201-205.[Medline]
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13. Martin G.A.S., Reddy V.B., Garrity D.R., et al. Humoral (antibody-mediated) rejection in lung transplantation. J Heart Lung Transplant 1996;15:1217-1222.[Medline]
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