Objectives: Prognostic implications of early protocol biopsies have been studied; however, the value of late protocol biopsy in predicting graft outcome has not been well defined. Here, we compared the effects of early and late protocol biopsy histologic findings in stable kidney allografts and aimed to understand the significance of “borderline” rejection on allograft function.

Materials and Methods: We studied 261 biopsies from 159 renal transplant recipients who were on a steroid-free, calcineurin inhibitor and mycophenolate mofetil regimen and who received transplants between 2004 and 2012 with mean follow-up of 5 years. Early (between 3 and 9 mo) and subsequent late (between 12 and 24 mo) protocol biopsies were performed. Biopsies were classified as normal, interstitial fibrosis and/or tubular atrophy, subclinical acute rejection with interstitial fibrosis and/or tubular atrophy, and borderline rejection with interstitial fibrosis and/or tubular atrophy. A linear mixed-effects model was used to determine the effects of early and late protocol biopsies on estimated glomerular filtration rate changes, with baseline time for estimated glomerular filtration rate fixed at 12 months.

Results: The adjusted model showed that estimated glomerular filtration rate at 3 months, donor age, delayed graft function, and early protocol biopsies were associated with baseline estimated glomerular filtration rate at 12 months. Estimated glomerular filtration rate changes over time were associated with findings of interstitial fibrosis and/or tubular atrophy at early biopsy and subclinical acute rejection and borderline rejection at late biopsy. At last follow-up, final estimated glomerular filtration rate was significantly associated with interstitial fibrosis and/or tubular atrophy at early biopsy and with subclinical acute rejection at late biopsy.

Conclusions: Although early protocol biopsy predicted baseline estimated glomerular filtration rate, late biopsy was important for predicting changes in function over time. In addition, a diagnosis of “borderline” rejection on protocol biopsies predicted long-term graft function.

Key words : Borderline rejection, Interstitial fibrosis and/or tubular atrophy, Subclinical acute rejection, Surveillance biopsies

Introduction

Protocol biopsies at fixed intervals after renal transplant have long been used to diagnose and treat subclinical rejection in an attempt to improve long-term graft survival. These have also served as an important research tool to understand the pathophysiology of allograft injury.1 Protocol biopsy is a relatively safe procedure and is performed in an outpatient setting with discharge after 4 hours of bed rest.2 Some of the common pathologies identified on protocol biopsies in the first 2 years after transplant are subclinical acute rejection, borderline rejection, and interstitial fibrosis and/or tubular atrophy (IF/TA). The role of early protocol biopsy in kidney transplant recipients is well studied; however, the predictive ability of late protocol biopsies has not been well elucidated.

It has been suggested that subclinical acute rejection found on 1-year posttransplant protocol biopsy is predictive of long-term graft survival,3,4 but there are other studies with contradictory results.5 Stegall and associates demonstrated that mild fibrosis present at 1 year progressed to more severe forms at 5 years in only 23% of allografts,6 thereby contradicting assertions by Nankivell and colleagues that chronic allograft nephropathy was a continuum of events whereby tubulointerstitial and glomerular damage, once established, are irreversible, resulting in declining renal function and graft failure.7 Interstitial fibrosis and/or tubular atrophy on its own do not predict long-term renal allograft survival,but the combination of inflammation and fibrosis has been shown to be a surrogate marker for progressive graft functional decline.8,9 Therefore, it becomes critical to examine the predictive ability of histologic abnormalities in early and in late protocol biopsies on long-term renal allograft function.

There is a paucity of literature comparing the effects of early versus late protocol biopsy findings on graft function in renal transplant recipients. In this study, we examined the effects of histologic phenotypes in early and late protocol biopsies on long-term renal allograft function.

Materials and Methods

Patient and clinical data
This is a single-center retrospective cohort study of adult kidney-only transplant recipients, from both living and deceased donors, maintained on a steroid-free regimen of tacrolimus and mycophenolate mofetil (MMF) after alemtuzumab induction therapy, and transplanted between 2004 and 2012. Exclusions were (1) high immunologic risk (panel reactive antibody ≥ 20), (2) absence of at least 1 protocol biopsy with adequate material according to Banff criteria,10 and (3) recipients receiving other types of immunosuppression. Our study included 159 stable renal transplant recipients. Allograft function was monitored at 3, 12, 24, 36, 48, and 60 months and was defined as the optimal Modification of Diet in Renal Disease equation11 calculated as estimated glomerular filtration rate (eGFR) based on creatinine and patient characteristics within 6 ± 3 days from biopsy day.

Immunosuppression and treatment of rejection
All recipients received alemtuzumab induction consisting of a loading dose of 30 mg at the time of implant followed by 15 mg on postoperative day 1, methylprednisolone at 250 and 125 mg intravenously before the first and second doses of induction, tacrolimus (trough of 5-8 ng/dL), and MMF (maximum total daily dose of 2 g daily).12 Recipients with subclinical acute cellular rejection (Banff ≥ 1A) were treated with steroid pulse with or without depleting antibody therapy. Patients with antibody-mediated rejection, defined by histologic evidence of antibody activity as evidenced by peritubular capillaritis and/or glomerulitis with or without C4d positivity and donor-specific antibody, were treated, in addition to steroids, with plasmapheresis followed by intravenous immunoglobulin. Mixed acute cellular rejection and antibody-mediated rejection were treated with a combination of the above treatments. Borderline rejection found on protocol biopsies was treated by optimizing maintenance immunotherapy to therapeutic trough levels of calcineurin inhibitors and increasing MMF to maximum tolerated dose. No pulse steroids were given for borderline rejections found on protocol biopsy.

Analyses of renal allograft biopsies
Early protocol biopsy was performed between 3 and 9 months and then sequential late protocol biopsy was performed between 12 and 24 months, with a 3-month minimum gap between biopsies. A total of 261 specimens were submitted for analysis. Specimens were placed in Zamboni fixative and stained for microscopic examination with hematoxylin and eosin stain, periodic acid-Schiff stain, and Gomori trichrome stain. The stains enhanced the identification of glomerulitis and tubulitis and any destruction of tubular base membranes and also enhanced the recognition of chronic features such as arteriolar hyaline, increased mesangial matrix, double contours in glomerular capillaries, and thickened tubular basement membranes. The pathologist evaluated sections under light microscopy for glomerulitis, tubulitis, and peritubular capillaritis to evaluate for acute rejection. In addition, sections underwent C4d immunohistochemical staining to evaluate for antibody-mediated rejection. The trichrome-stained fragment was analyzed by the pathologist for ascertainment of the modified Banff chronicity score.13,14 The modified Banff chronicity score was calculated after the sum glomerulosclerosis, interstitial fibrosis, tubular atrophy, and chronic vascular changes, as specified in the Banff classification.10 We grouped our biopsies as normal, interstitial fibrosis and/or tubular atrophy (IF/TA) alone, and inflammation with varying degree of IF/TA, grouped as either subclinical acute rejection (SAR+IF/TA) or borderline rejection (BDR+IF/TA) depending on the type of inflammation score. Both acute cellular rejection and antibody-mediated rejection were grouped together into the subclinical acute rejection group (SAR+IF/TA).

Covariates analyzed were recipient cofactors of age, sex, ethnicity (White vs African American), retransplant status, cytomegalovirus status (CMV immunoglobulin G), hepatitis C status, pretransplant history of cancer, coronary artery disease, diabetes mellitus, dyslipidemia defined by statin therapy, hypertension, and cause of end-stage renal disease (ESRD). Posttransplant covariates were CMV status, new-onset diabetes after transplant, bone density scan diagnosis of osteopenia, dyslipidemia, protocol biopsy IF/TA mean score, and eGFR at 3 months. Donor covariates were age, sex, living versus deceased donor, extended criteria donor versus standard criteria donor, and donor CMV status. Transplant characteristics included HLA mismatch number, DR match, delayed graft function (defined by need for ≥ 1 dialysis after transplant), indication biopsy-proven rejection, and cold ischemia time.

Statistical analyses
Normal quantitative variables were described using mean and standard deviation (SD). Categorical data were described using frequency and proportion. Not normal data were log-transformed to induce normality. One-way analysis of variance was used to compare the quantitative baseline data according to early and late biopsy categories, whereas Fisher exact test was used to compare the categorical data according to different types of biopsies. Linear mixed-effects modeling was used to determine the effects of early and late protocol biopsy findings on eGFR at 12 months and over the follow-up period. The rate of decline in eGFR was summarized using average change and standard error. Univariate and multivariate linear mixed-effects models were developed to examine the effects of protocol biopsy on changes in eGFR. Three adjusted models were developed: (1) adjusted model for dichotomized early and late biopsy (abnormal vs normal), (2) adjusted model for 4 histology categories of early and late biopsy (normal, SAR+IF/TA, BDR+IF/TA, IF/TA), and (3) separate adjusted model for early and late biopsy (normal, SAR+IF/TA, BDR+IF/TA, IF/TA). Random intercepts and fixed slopes were considered in linear mixed model analysis. In adjusted model, we included all clinically relevant variables that were found to be significant in the univariate analysis at 10% level of significance and selected a final model retaining as many variables as possible using the Akaike Information Criterion. The effects of protocol biopsies on final eGFR were also determined using linear regression analysis. Results of regression analysis were presented using estimates, its 95% confidence intervals (CI), and P values. P values less than 5% were regarded as significant. All statistical analyses were carried out using SAS 9.3 (SAS Institute Inc, Cary, NC).

Results

Baseline data
Of the 159 kidney transplant recipients who qualified for the study, 261 protocol biopsies were performed. Of the 159 recipients, 102 recipients (64.2%) underwent both early and subsequent late protocol biopsies. In both early and late protocol biopsy groups, the mean (SD) duration of recipient follow-up after biopsy was 5.02 years (range, 0.24-8.67 years). Early protocol biopsy histologic findings were significantly associated with recipient age (P = .0331), recipient CMV status (P = .0238), older donor age (P = .0388), living-donor kidney (P = .0340), eGFR at 3 months (P = .0102), and prolonged cold ischemia time (P = .044; Table 1). Late protocol biopsy histologic findings were borderline associated toward recipient CMV status (P = .0744) and donor age (P = .0814; Table 2). In both early and late protocol biopsies, mean IF/TA (SD) scores remained significantly higher in the IF/TA group alone for both early 1.86 (1.22) and late 2.09 (1.35) protocol biopsies compared with normal, SAR+IF/TA, and BDR+IF/TA histology (P < .0001; Table 3).

Biopsy and biopsy-related data
In early protocol biopsies performed before 12 months, normal pathology was found in 105 recipients (66%) and abnormal pathology was seen in the remaining 54 recipients (34%), of which SAR+IF/TA was identified in 7 recipients (4.4%), BDR+IF/TA in 17 recipients (10.69%), and IF/TA in 30 recipients (18.87%). Late protocol biopsies performed after 12 months in the 102 recipients revealed normal pathology in 59 recipients (58%) and abnormal pathology in the remaining 43 recipients (42%), which were distributed as SAR+IF/TA in 4 recipients (4%), BDR+IF/TA in 8 recipients (9%), and IF/TA in 30 recipients (29%). All data are summarized in Figure 1.

Association between histologic findings and allograft function
The eGFR declined in all recipients from 12 months on a yearly basis to the approximate 5-year follow-up visit with a mean slope of -0.61 ± 0.47 mL/min/1.73 m² per year in which slope was determined by applying the least-squares regression for each patient. In application of the linear mixed-effects model, when varying intercept and slope for each patient, the decline was -1.26 ± 0.40 mL/min/1.73 m² per year. The changes in eGFR are shown in Figure 2 according to early and late protocol biopsies. In early protocol biopsies, SAR+IF/TA (34.06 ± 13.24 mL/min/1.73 m²) and IF/TA (37.35 ± 19.08 mL/min/1.73 m²) had lower eGFR than the normal (50.35 ± 18.61 mL/min/1.73 m²) and BDR+IF/TA groups (59.70 ± 55.89 mL/min/1.73 m²) over the mean follow-up period (Figure 2, top). For allografts with any abnormal pathology on late protocol biopsy, eGFR results in SAR+IF/TA (25.65 ± 14.28 mL/min/1.73 m²), BDR+IF/TA (42.17 ±19.24 mL/min/1.73 m²), and IF/TA (45.26 ± 16.13 mL/min/1.73 m²) were lower than in the normal group (51.77 ± 16.96 mL/min/1.73 m²) over the mean follow-up period (Figure 2, bottom).

Unadjusted analysis showed that ethnicity (non-White), recipient CMV status, ESRD from diabetic or immunoglobulin A nephropathy, donors of older age, female donors, living-type donors, prolonged cold ischemia time, IF/TA seen on early protocol biopsy, early rejection by indication biopsy, SAR+IF/TA seen in early and late protocol biopsy, eGFR at 3 months, and abnormal histology in early protocol biopsy were all associated with baseline eGFR at 12 months. Changes in the slope of eGFR over time were associated with recipients with > 1 transplant, history of dyslipidemia, ESRD from immunoglobulin A nephropathy, HLA mismatch, delayed graft function, SAR+IF/TA seen in early protocol biopsy, BDR+IF/TA and IF/TA in late protocol biopsy, and abnormal histology in late protocol biopsy. All data are summarized in Table 4 and Table 5.

In the adjusted model based on 4 histology categories of early and late biopsies, eGFR at 3 months (P < .0001), donor age (P = .0127), and abnormal findings on early protocol biopsy of both BDR+IF/TA (P = .029) and IF/TA (P = .047) versus normal were associated with baseline eGFR at 12 months. With association of abnormal histology in protocol biopsies in relation to decline of eGFR in early protocol biopsy, those identified with IF/TA (P = .0038) had significantly higher rates of decline in eGFR over the mean follow-up than those with normal biopsy. In addition, late protocol biopsy findings of SAR+IF/TA (P = .0605) and BDR+IF/TA (P = .0495) compared with normal were significantly associated with the rate of decline in eGFR over time (Table 6). Thus, early protocol biopsy regression model showed that abnormal early protocol biopsy was independently associated with baseline eGFR but not with rate of change in the eGFR, whereas the late protocol biopsy regression model showed that findings of any abnormal late protocol biopsy were associated with rate of decline in eGFR. Furthermore, the final eGFR model with early and late biopsies demonstrated that IF/TA in early protocol biopsy (P = .0009) and SAR+IF/TA in late protocol biopsy (P = .0035) predicted eGFR at final follow-up (Table 7).

Discussion

Our study demonstrated that SAR+IF/TA seen in early protocol biopsy did not influence baseline eGFR or rate of change of eGFR decline but that SAR+IF/TA seen in subsequent late protocol biopsies predicted the slope of eGFR decline and was significantly associated with final eGFR at 7 years posttransplant. The term “subclinical acute rejection” coined by Rush and associates using the Banff schema showed that subclinical acute rejection occurred in 30% to 45% of well-functioning grafts by 3 months after transplant and resulted in increased IF/TA by 12 months.7,15,16 In addition, early protocol subclinical acute rejection findings were shown to have lower eGFR and were linked to increased risk of graft loss at 5 years.3,8 In contrast, our study did not show an association between early protocol biopsy SAR+IF/TA and long-term graft function, likely because evidence favors treating SAR+IF/TA when diagnosed early after transplant. Early protocol biopsy and treatment of subclinical acute rejection before occurrence of renal dysfunction have been shown to improve renal allograft function and lead to improvement of short-term allograft outcomes.^4,17,18 Hence, it is not surprising that aggressive treatment of SAR+IF/TA seen in early protocol biopsy did not affect graft function in our study.

Our findings are in concordance with others who have shown that subclinical acute rejection after 1 year on protocol biopsies was associated with deteriorating graft function and survival during intermediate-term follow-up.^3,12,19 There is also a paucity of literature on late protocol biopsies performed after 12 months in kidney transplant recipients, and our study fills the scarcity of knowledge in this area with our findings that late protocol SAR+IF/TA is significantly associated with rate of eGFR decline over long-term follow-up. Other studies have shown that interstitial fibrosis alone in early protocol biopsy may not predict graft functional decline in allografts with good baseline function; however, ongoing inflammation with interstitial fibrosis leads to graft functional decline,⁹ which was also shown in our study where there was increasing percentage of recipients with fibrosis in the inflammation groups in late protocol biopsy and thus the presence of progressive fibrosis in inflamed kidneys predicted graft function. It could be speculated that, although early protocol biopsies are a useful tool, late protocol biopsies more likely have prognostic value in predicting long-term graft function. In many of the existing studies, the group “subclinical inflammation” consisted of protocol biopsies and included both borderline changes and lesions meeting the criteria for Banff acute rejection scores (Banff ≥ 1A). Ours is the first study to group SAR+IF/TA and BDR+IF/TA separately and evaluate the significance of each finding to graft outcome.

The influence of borderline changes or its progression to acute rejection on long-term graft function remains unclear.²⁰ More importantly; the pathogenic role of borderline inflammation found on protocol biopsy has not been established. In our study, early protocol biopsy BDR+IF/TA histology was significantly associated with baseline eGFR at 12 months, and subsequent late protocol biopsy BDR+IF/TA histology was associated with the slope of eGFR decline. Several studies on indication biopsy findings have associated lesions defined in the Banff classification with severity of rejection, response to therapy, and allograft survival²¹; however, histologic analyses of stable renal allografts by protocol biopsies based on Banff criteria showed substantial inter- and intraobserver variations for scoring and grading of borderline rejection,²² which can limit precise management decisions. A recent study investigated the significance of treating early borderline changes identified by early protocol biopsy in relation to progression of chronic IF/TA, showing that BDR diagnosed by protocol biopsy negatively affected follow-up allograft histology, leading the authors to recommend treating BDR in protocol biopsy to prevent chronic graft injury.²³ This is similar to findings in our study, which indicated that the persistence of borderline infiltrates can negatively impact graft function over time. Since then, our practice protocols have been modified to include steroid pulse therapy to treat BDR+IF/TA.

More recently, Halloran and associates analyzed the molecular phenotype of borderline biopsies compared with biopsies with T-cell-mediated rejection and nonrejection using microarray analyses.²⁴ Atrophy and scarring were unusually high in the biopsies classified as borderline but showed molecular features of T-cell-mediated rejection by microarray. These findings indicate that scarring reduced the amount of tissue that can be used for the assessment of interstitial Banff score, thus misclassifying some T-cell-mediated rejection as borderline category. Borderline rejection seen on protocol biopsies may reflect early T-cell-mediated rejection and progress to more severe lesions, whereas others may reflect lesions that do not require treatment.^25-27

Thus, it is now conceivable that borderline changes in renal allograft likely represent early alloimmune activity. It has been shown that nonspecific inflammatory infiltrates outside the Banff localization of cortical tissue and infiltrative pattern in areas of scarred tissue may not be harmless.8 The study by Mengel and associates looked at the significance of nonspecific and minimal cellular infiltrates in renal allograft biopsy and found that the incidence of borderline rejection was 13.9%, where both nonspecific and specific infiltrative histologic categories were identified as predictors of allograft function at 1 and 2 years after transplant.²⁹ Thus, nonspecific inflammatory infiltrates below Banff threshold may play a role in development of IF/TA and graft function decline in the long-term, as shown in our study.

In our study, we examined the impact of IF/TA found on early and subsequent late protocol biopsies in relation to long-term allograft function. Interstitial fibrosis and/or tubular atrophy found on early protocol biopsy was associated with worse baseline eGFR and influenced the rate of eGFR decline over time. Our findings are similar to others who described early protocol biopsies findings of IF/TA on long-term graft survival to be modulated by subclinical rejection. Heilman and associates performed serial early protocol biopsies and concluded that persistent inflammation diagnosed by protocol biopsy as subclinical inflammation increased the risk of IF/TA at 1 year.²⁹ In the study by Moreso and associates, protocol biopsies, performed during the first 6 months in stable grafts, were evaluated according to Banff criteria. Cox regression analysis showed that subclinical rejection with fibrosis was an independent predictor of graft survival.³⁰ However, these studies did not address the detrimental effects of early protocol IF/TA on graft function. The IF/TA scores for the various histologic groups in our early protocol biopsy showed varying degree of IF/TA in the groups, with subclinical inflammation most notably in the BDR+IF/TA group. Early IF/TA has the potential to be superimposed on subclinical rejection or chronic arteriopathy and/or glomerulopathy with long-term follow-up.⁸ We hypothesize that, in the present study, the association of early IF/TA on graft function could be the consequence of early subclinical rejection, thus adversely affecting long-term graft function. Thus, IF/TA could be both preceded and worsened by subclinical or acute rejection leading to deterioration in function and worse graft outcome in the long-term.^31-32

In our study, there was no association of subsequent late protocol biopsy finding of IF/TA on rate of change of eGFR or graft function at final follow-up. Our findings are similar to the DeKAF study, which tried to differentiate clinical and pathologic entities that caused late graft dysfunction. There was no difference in graft survival between patients with and without chronic allograft nephropathy or IF/TA alone, and thus IF/TA or chronic allograft nephropathy in late biopsies was of no prognostic value. Hence, late protocol IF/TA does not appear to carry much prognostic implication in late allograft dysfunction without additional active lesions.^33,34

Several limitations should be considered while interpreting the study’s results. The major limitations of our study are sample size and the retrospective nature of data collection. The small sample size produced low frequencies in some of the important variables, yielding sometimes wide confidence intervals and not providing sufficient data for comparison of some of the categorical variables. Caution should be used in the extrapolation of our findings to kidney transplant recipients on other immunosuppressive regimens. The main strengths of our study are longitudinal assessment of eGFR, comprehensive data collection, and length of follow-up. Despite being a retrospective study, we evaluated all important confounders in the study. This is the first study, to our knowledge, that evaluated the significance of late protocol biopsy compared with early protocol biopsy on graft function based on long-term follow up. This is also the first study that studied the implications of BDR+IF/TA on long-term graft function.

In conclusion, late protocol SAR+IF/TA was predictive of long-term graft function. In addition, BDR+IF/TA findings in both early and subsequent late protocol biopsies that were managed by optimizing maintenance therapy alone were associated with lower graft function in this cohort of kidney transplant recipients on a steroid-free regimen. We also demonstrated that early, but not late protocol IF/TA, was prognostic for long-term graft function. Long-term kidney allograft function is influenced by immunologic and nonimmunologic factors, such as acute rejection, chronic rejection, IF/TA, infections, and de novo and recurrent disease. Significant proportions of these conditions are insidious and cause a slow decline in renal function. These can be detected by protocol biopsies at prespecified time points after kidney transplant. In this study, we have shown that both early and late protocol biopsies carry prognostic significance to graft outcomes based on various histologic phenotypes. The disease entities that affect graft function are indolent in nature and often represent an ongoing injury pattern. Hence, early protocol biopsy alone is insufficient to detect the continuum of factors that influence long-term graft outcome. The evaluation of these biopsies thus aids in grading various subclinical injuries and helps in the modulation of immunosuppressive regimens. There is a need to validate the findings of our study by designing further studies that are prospective in design. Future direction for this study includes translating the information obtained to determine the optimal timing of these protocol biopsies, development of more sensitive markers of subclinical rejection and inflammation, and further understanding the management strategies for various pathologic findings.

References:

1. Henderson LK, Nankivell BJ, Chapman JR. Surveillance protocol kidney transplant biopsies: their evolving role in clinical practice. Am J Transplant. 2011;11(8):1570-1575.
   CrossRef - PubMed
   
2. Schwarz A, Gwinner W, Hiss M, Radermacher J, Mengel M, Haller H. Safety and adequacy of renal transplant protocol biopsies. Am J Transplant. 2005;5(8):1992-1996.
   CrossRef - PubMed
   
3. Thierry A, Thervet E, Vuiblet V, et al. Long-term impact of subclinical inflammation diagnosed by protocol biopsy one year after renal transplantation. Am J Transplant. 2011;11(10):2153-2161.
   CrossRef - PubMed
   
4. Choi BS, Shin MJ, Shin SJ, et al. Clinical significance of an early protocol biopsy in living-donor renal transplantation: ten-year experience at a single center. Am J Transplant. 2005;5(6):1354-1360.
   CrossRef - PubMed
   
5. Roberts IS, Reddy S, Russell C, et al. Subclinical rejection and borderline changes in early protocol biopsy specimens after renal transplantation. Transplantation. 2004;77(8):1194-1198.
   CrossRef - PubMed
   
6. Stegall MD, Park WD, Larson TS, et al. The histology of solitary renal allografts at 1 and 5 years after transplantation. Am J Transplant. 2011;11(4):698-707.
   CrossRef - PubMed
   
7. Nankivell BJ, Borrows RJ, Fung CL, O'Connell PJ, Allen RD, Chapman JR. The natural history of chronic allograft nephropathy. N Engl J Med. 2003;349(24):2326-2333.
   CrossRef - PubMed
   
8. Cosio FG, Grande JP, Wadei H, Larson TS, Griffin MD, Stegall MD. Predicting subsequent decline in kidney allograft function from early surveillance biopsies. Am J Transplant. 2005;5(10):2464-2472.
   CrossRef - PubMed
   
9. Park WD, Griffin MD, Cornell LD, Cosio FG, Stegall MD. Fibrosis with inflammation at one year predicts transplant functional decline. J Am Soc Nephrol. 2010;21(11):1987-1997.
   CrossRef - PubMed
   
10. Racusen LC, Solez K, Colvin RB, et al. The Banff 97 working classification of renal allograft pathology. Kidney Int. 1999;55(2):713-723.
    CrossRef - PubMed
    
11. Levey AS, Bosch JP, Lewis JB, Greene T, Rogers N, Roth D. A more accurate method to estimate glomerular filtration rate from serum creatinine: a new prediction equation. Modification of Diet in Renal Disease Study Group. Ann Intern Med. 1999;130(6):461-470.
    CrossRef - PubMed
    
12. Zachariah M, Nader ND, Brar J, et al. Alemtuzumab and minimization immunotherapy in kidney transplantation: long-term results of comparison with rabbit anti-thymocyte globulin and standard triple maintenance therapy. Transplant Proc. 2014;46(1):94-100.
    CrossRef - PubMed
    
13. Kambham N, Nagarajan S, Shah S, Li L, Salvatierra O, Sarwal MM. A novel, semiquantitative, clinically correlated calcineurin inhibitor toxicity score for renal allograft biopsies. Clin J Am Soc Nephrol. 2007;2(1):135-142.
    CrossRef - PubMed
    
14. Solez K, Colvin RB, Racusen LC, et al. Banff 07 classification of renal allograft pathology: updates and future directions. Am J Transplant. 2008;8(4):753-760.
    CrossRef - PubMed
    
15. Rush DN, Henry SF, Jeffery JR, Schroeder TJ, Gough J. Histological findings in early routine biopsies of stable renal allograft recipients. Transplantation. 1994;57(2):208-211.
    CrossRef - PubMed
    
16. Rush D, Nickerson P, Gough J, et al. Beneficial effects of treatment of early subclinical rejection: a randomized study. J Am Soc Nephrol. 1998;9(11):2129-2134.
    PubMed
    
17. Kurtkoti J, Sakhuja V, Sud K, et al. The utility of 1- and 3-month protocol biopsies on renal allograft function: a randomized controlled study. Am J Transplant. 2008;8(2):317-323.
    CrossRef - PubMed
    
18. Kee TY, Chapman JR, O'Connell PJ, et al. Treatment of subclinical rejection diagnosed by protocol biopsy of kidney transplants. Transplantation. 2006;82(1):36-42.
    CrossRef - PubMed
    
19. Ciancio G, Burke GW, Gaynor JJ, et al. A randomized trial of thymoglobulin vs. alemtuzumab (with lower dose maintenance immunosuppression) vs. daclizumab in renal transplantation at 24 months of follow-up. Clin Transplant. 2008;22(2):200-210.
    CrossRef - PubMed
    
20. Meehan SM, Siegel CT, Aronson AJ, et al. The relationship of untreated borderline infiltrates by the Banff criteria to acute rejection in renal allograft biopsies. J Am Soc Nephrol. 1999;10(8):1806-1814.
    PubMed
    
21. Marcussen N, Olsen TS, Benediktsson H, Racusen L, Solez K. Reproducibility of the Banff classification of renal allograft pathology. Inter- and intraobserver variation. Transplantation. 1995;60(10):1083-1089.
    CrossRef - PubMed
    
22. Veronese FV, Manfro RC, Roman FR, et al. Reproducibility of the Banff classification in subclinical kidney transplant rejection. Clin Transplant. 2005;19(4):518-521.
    CrossRef - PubMed
    
23. Min SI, Park YS, Ahn S, et al. Chronic allograft injury by subclinical borderline change: evidence from serial protocol biopsies in kidney transplantation. J Korean Surg Soc. 2012;83(6):343-351.
    CrossRef - PubMed
    
24. de Freitas DG, Sellares J, Mengel M, et al. The nature of biopsies with “borderline rejection” and prospects for eliminating this category. Am J Transplant. 2012;12(1):191-201.
    CrossRef - PubMed
    
25. Dahan K, Audard V, Roudot-Thoraval F, et al. Renal allograft biopsies with borderline changes: predictive factors of clinical outcome. Am J Transplant. 2006;6(7):1725-1730.
    CrossRef - PubMed
    
26. Mengel M, Chang J, Kayser D, et al. The molecular phenotype of 6-week protocol biopsies from human renal allografts: reflections of prior injury but not future course. Am J Transplant. 2011;11(4):708-718.
    CrossRef - PubMed
    
27. Sellares J, de Freitas DG, Mengel M, et al. Inflammation lesions in kidney transplant biopsies: association with survival is due to the underlying diseases. Am J Transplant. 2011;11(3):489-499.
    CrossRef - PubMed
    
28. Mengel M, Gwinner W, Schwarz A, et al. Infiltrates in protocol biopsies from renal allografts. Am J Transplant. 2007;7(2):356-365.
    CrossRef - PubMed
    
29. Heilman RL, Devarapalli Y, Chakkera HA, et al. Impact of subclinical inflammation on the development of interstitial fibrosis and tubular atrophy in kidney transplant recipients. Am J Transplant. 2010;10(3):563-570.
    CrossRef - PubMed
    
30. Moreso F, Ibernon M, Goma M, et al. Subclinical rejection associated with chronic allograft nephropathy in protocol biopsies as a risk factor for late graft loss. Am J Transplant. 2006;6(4):747-752.
    CrossRef - PubMed
    
31. Shishido S, Asanuma H, Nakai H, et al. The impact of repeated subclinical acute rejection on the progression of chronic allograft nephropathy. J Am Soc Nephrol. 2003;14(4):1046-1052.
    CrossRef - PubMed
    
32. Nankivell BJ, Chapman JR. The significance of subclinical rejection and the value of protocol biopsies. Am J Transplant. 2006;6(9):2006-2012.
    CrossRef - PubMed
    
33. Gourishankar S, Leduc R, Connett J, et al. Pathological and clinical characterization of the 'troubled transplant': data from the DeKAF study. Am J Transplant. 2010;10(2):324-330.
    CrossRef - PubMed
    
34. Matas AJ, Leduc R, Rush D, et al. Histopathologic clusters differentiate subgroups within the nonspecific diagnoses of CAN or CR: preliminary data from the DeKAF study. Am J Transplant. 2010;10(2):315-323.
    CrossRef - PubMed