Banff Histopathological Consensus Criteria for Preimplantation Kidney Biopsies

Introduction

Worldwide, the demand for donor kidneys far exceeds the number of available organs; however, the discard rate of recovered kidneys remains >40%. Preimplantation biopsy—also referred to as procurement, harvest or donor biopsy—is often used by transplant centers in addition to clinical data and parameters of machine pump perfusion to make determinations regarding organ acceptance. The Organ Procurement and Transplantation Network (OPTN) policies currently recommend preimplantation biopsy for all kidneys with a Kidney Donor Profile Index (KDPI) >85% or at the request of the accepting surgeon, according to the deceased donor allocation policy implemented in December 2014 within the United States (OPTN Policy 2.12.A) 1. The KDPI is derived from 10 donor factors (age, height, weight, ethnicity, history of diabetes and/or hypertension, cause of death, serum creatinine [SCr], hepatitis C virus [HCV] status and donor after circulatory death status). Under the former allocation system, preimplantation biopsy was previously recommended for all expanded criteria donor (ECD) kidneys, defined as organs from older donors (aged >60 years) and persons aged 50–59 years with hypertension, SCr >1.5 mg/dL or death from cerebrovascular accident 2, 3. These policies recommended that organ procurement organizations (OPOs) provide the receiving transplant program with biopsy information from a wedge biopsy 10 mm long by 5 mm wide and 5 mm deep to be taken from the kidney cortex to capture at least 25 glomeruli. After the implementation of these recommendations, requests for donor biopsy interpretation by hospital pathologists increased significantly. A pathology data form based on the United Network for Organ Sharing (UNOS) guidelines emphasizes the number of glomeruli present in a wedge biopsy and the percentage of global glomerulosclerosis. This form is currently customized by local OPOs. This practice deviates substantially from routine evaluation of postimplantation allograft biopsies for cause (indication biopsies), which are routinely needle biopsies to assess not only the glomeruli but the tubular, interstitial and vascular compartments, following recommendations established by the Banff group. The Banff group previously defined and subsequently refined the pathologic criteria for postimplantation biopsies performed for cause and more recently applied the same criteria for implantation biopsies in centers performing protocol biopsies 4, 5.

A Banff working group on preimplantation biopsy was established in 2010 and initially conducted a survey asking renal pathologists at large for their input through the Renal Pathology Society website. The results of the survey revealed a list of areas to be addressed. These items became the aims of the Banff working group. Preliminary data of the preimplantation renal biopsy working group and a proposal for Banff consensus criteria for histopathologic interpretation were presented at the 11th Banff conference, held in Paris, France, in 2011. The agreed-upon criteria were subsequently applied to a larger cohort of biopsies, and the working group was expanded to include international participants. The results were presented at the 12th Banff conference, held in Brazil in 2013, at which evaluation of the Banff histopathologic consensus criteria with graft outcome was decided. The study concluded with the results of the comparison of histopathologic data with graft outcome at the 13th Banff conference, held in Vancouver, Canada, in 2015. The current investigation is the first Banff study aiming to develop consensus to define the gold standard for interpretation of preimplantation biopsies. The working group's specific aims were defined as follows: (i) Address interobserver variability utilizing frozen sections, (ii) compare wedge and needle core biopsies, (iii) compare frozen and defrosted formalin-fixed tissue sections, (iv) develop histopathologic criteria for the interpretation of preimplantation biopsies, and (v) compare histopathologic and clinical parameters with graft outcome.

Methods

Briefly, the questionnaire included questions on (i) the participants’ experience with the UNOS-recommended preimplantation wedge biopsies, (ii) the perceived accuracy of frozen section interpretation, (iii) possible interobserver variability in the assessment of preimplantation biopsies, and (iv) recommendations for a pathologic gold standard for evaluating ECD kidneys. The survey results strongly recommended assessment of all aspects listed regarding preimplantation biopsy interpretation.

A total of 124 biopsies were used. Of these, 40 biopsies were scanned using the Aperio system (Leica Biosystems, Wetzlar, Germany) at Washington University in St. Louis and at the University of Alberta. Slides were scanned on an Aperio ScanScope XT at ×20 magnification, corresponding to 0.50 μm/pixel.

The study was approved by the Washington University Kidney Translational Research Core and the institutional review board at Washington University in St. Louis (IRB 05-2009). Four groups were created as follows: frozen core biopsies (n = 5), defrosted paraffin core biopsies (n = 5), frozen wedge biopsies (n = 15), and defrosted paraffin wedge biopsies (n = 15).

All cases submitted for scoring by intraclass correlation (ICC) analysis were implanted. Kidneys were from 20 unique donors; both kidneys of these donors were implanted. Donor demographic data of 19 donors implanted at the Washington University kidney transplant center in Saint Louis, Missouri, were retrieved (Table 1). We were unable to retrieve the demographics of one donor for whom both kidneys were implanted. Briefly, median age was 49.2 years (range 46.4–56.0 years), and nine donors were male. Causes of death were anoxia in 16%, cerebrovascular in 58%, “natural causes” in 6% and trauma in 16%. Regarding race, 89% were white, 5% were black and 5% were Hispanic. Calculated terminal creatinine is shown in Table 1.

A representative image of the online images is shown in Figure 1. The virtual slide histopathologic data form included designation as wedge or core biopsy and the following parameters: number of glomeruli, number of globally sclerosed glomeruli, percentage of globally sclerosed, number of arteries, interstitial inflammation, interstitial fibrosis, tubular atrophy, arterial intimal thickening, arteriolar hyalinosis, glomerular thrombi, acute tubular injury, and other lesions. A simplified scoring scheme was chosen as follows: none (<5% of area involved), mild (6–25% of area involved), moderate (26–50% of area involved), severe (>50% of area involved). Arteriolar hyalinosis was scored as mild when at least one arteriole was involved, as moderate when more than one arteriole was involved, and as severe when multiple arterioles were affected. The simplified scoring sheet, as described, is shown in Figure 2.

Slides were evaluated by 32 expert renal pathologists in a blinded fashion with no knowledge of donor demographic data. All biopsies were scored by the same group of pathologists, who were assumed to be a random subset of all pathologists. Perfect correlation is indicated as 1, with excellent >0.75, good as 0.5–0.75, fair as 0.25–0.5 and poor as <0.25.

The following three comparisons were performed: (i) frozen versus paraffin wedge biopsies, (ii) frozen versus paraffin core biopsies, (iii) frozen core versus wedge. Paraffin sections were prepared from the defrosted formalin-fixed and paraffin-embedded wedge or core biopsies.

Data from an additional 74 biopsies were collected for graft outcome submitted by the following transplant centers: Washington University in St. Louis; University of Pittsburgh Medical Center; Johns Hopkins University; Institute for Clinical and Experimental Medicine (IKEM; Prague, Czech Republic); Mayo Clinic (Arizona); Transplantation Institute, Medical University of Warsaw (Poland); INCUCAI (Argentinian national institute for procurement and implants) and CUCAIBA (Buenos Aires institute of procurement and implants; Buenos Aires, Argentina); University Hospital, University of São Paulo (Brazil); and Santa Casa de Misericordia de Porto Alegre Hospital and Universidad Federal de Ciências da Saúde de Porto Alegre (Rio Grande do Sul, Brazil).

A code was assigned to each center, and a worksheet was distributed to participating pathologists. The following demographic and clinicopathologic data of the donors and recipients were obtained: donor age; donor SCr; donor cause of death; cold ischemia time (in minutes); donor type (ECD vs. standard criteria donor [SCD]); recipient age; race; date of transplant; age at transplantation; cause of renal failure before transplantation; delayed graft function (DGF; defined as DGF requiring dialysis); slow graft function (persistent elevation in SCr, does not require dialysis); presence or absence of proteinuria at any time; acute cellular rejection (yes or no, grade and number of events); and SCr at 1, 3, 6, and 12 mo after transplant. Posttransplant biopsy findings were also reported if available. The various variables including sex, race, graft function, graft failure and proteinuria were assigned numeric codes to perform statistical analysis. All patients were anonymized.

Results

Discussion

Numerous studies have addressed various issues related to preimplantation kidney biopsies and recommended a set of histopathologic parameters to be evaluated based on their predictive value for graft outcome 7-13. Other investigators included clinical and additional histopathologic parameters in evaluation of donor kidneys 14-17. Histopathologic scoring systems were suggested to assist decision making by transplant teams regarding organ acceptability for transplantation; however, none of the previously proposed scoring systems were as rigorously tested for reproducibility as in the current study. Furthermore, there is no widely accepted histopathologic scoring system for donor kidney biopsies (recently reviewed by Wang et al) 18. Reproducibility in our study is modest. In this regard, future studies should evaluate new histopathology metrics and tissue-preparation techniques.

The significant disagreements among previous studies exist in part because of different methodologies applied to analysis, procurement and processing of donor biopsies 19-21. The significance of arterial intimal fibrosis was acknowledged both by Mazzucco et al and Bosmans et al 20, 21. A likely reason why different studies find different histologic lesions to be critical is that chronicity in any anatomic compartment is prognostic, but sampling considerations lead to different lesions being overrepresented in different biopsy sets. Thrombi were rare in our cohort; however, a recent study showed that thrombi can be missed due to sampling error even when kidney involvement is extensive 22.

Such discrepant results have caused ambiguity. It is unclear whether donor kidney biopsies are able to minimize inappropriate discard of marginal organs. Many investigators are seeking additional prognostic information beyond histopathology. Reese et al, for example, explored the role of urine biomarkers and showed an association of high urinary neutrophil gelatinase-associated lipocalin and liver-type fatty acid binding protein concentrations with lower recipient estimated glomerular filtration rate at 6 mo and good correlation with donor acute kidney injury but found no predictive value for DGF or early allograft dysfunction after transplantation 23. It appears that the fundamental question of how best to predict deceased donor kidney function after transplantation remains elusive. Our study shows that histopathologic parameters in the donor biopsy may be useful but do not all correlate with graft outcome; however, the study is limited by selection bias. Only allografts that are clinically acceptable for transplantation have outcome data. Nonetheless, our results are in agreement with previously published studies 20, 21, 24, 25. Perhaps the difficulty lies in discriminating between changes related to normal aging without significant kidney dysfunction and changes, possibly related to aging, that are predictive of graft outcome. This line of questioning is an ongoing investigation 26.

The working group developed a consensus scoring sheet for donor biopsies, as shown in Figure 2. Following extensive debate, a simplified scoring schema was determined, distinct from Banff scoring for indication biopsies. During this study, we found nonuniformity across multiple institutions with regard to scoring parameters in actual procurement practices. We found, for example, that most centers use wedge biopsies because of perceived higher risk for bleeding associated with needle core biopsies; however, others found needle biopsies acceptable 27. Our study shows some superiority of frozen wedge versus needle core biopsies. This may be related to the lack of standardization of needle biopsies. Different needle gauges lead to different degrees of sampling that are not apparent in standardized wedge biopsies. Banff participants expressed the opinion that there are technical difficulties in cutting 18-gauge needle core biopsies, resulting in inadequate material for frozen section interpretation (data not shown). Needle biopsies are perceived to be more likely to damage larger caliber medullary vessels. The possibility of using punch biopsies instead was suggested, but most in the working group thought that an ample wedge biopsy with good cortical sampling is more likely to yield sufficient number of glomeruli and is likely superior to needle or punch biopsies. We also found that wedge biopsies were often subcapsular and not deep enough to capture vessels (data not shown).

The value of having an experienced renal pathologist versus a general surgical pathologist with no training in reading donor biopsies was also addressed 28. The authors found that trained renal pathologists improved histopathology scores compared with nonrenally trained pathologists 28, 29. Notably, the study by Azancott et al used formalin-fixed (not defrosted), paraffin-embedded biopsies. One may predict that discordance may be higher if frozen sections are used, but none of the participating centers in our study reported using formalin-fixed, paraffin-embedded tissue for initial evaluation of preimplantation biopsies. Our study found frozen and defrosted formalin-fixed, paraffin-embedded wedge biopsies to be comparable. Our study did not compare interpretation by renal pathologists versus general pathologists, but in our opinion, there is a need for training of pathologists assigned to donor biopsy interpretation. There is also need for closer collaboration and communication of pathologists with the transplant teams, particularly on the issue of appropriate sampling of the donor kidney. To this end, the next step of the Banff working group is to assist in preparing educational material for sample processing and interpretation of donor biopsies. Our study used digitally scanned slides accessed remotely from sites on three continents. A digital system for donor biopsy interpretation has already been implemented in some centers in the United States and Europe 30.

Histopathologic assessment of preimplantation biopsies is one component of donor organ assessment and is not an exclusive determinant to discard or transplant donor kidneys. The positive or negative predictive value of studies such as this one is inherently limited because of organ usage bias (in our study, all implanted kidneys were good kidneys), recipient characteristics and posttransplant variables affecting graft outcome. Our outcome data can be considered preliminary. Nevertheless, knowledge of the structural integrity of the kidney is intuitively an important parameter to consider in organ allocation. Importantly, other lesions in addition to those shown to be reproducible in this study need to be mentioned when observed in biopsy assessment. Examples include severe arteriolar hyalinosis, cholesterol emboli, coagulative acute tubular necrosis, extensive thrombosis, myoglobin cast nephropathy, focal segmental glomerulosclerosis, overt proliferative glomerulonephritis, Kimmelstiel–Wilson nodules of diabetic nephropathy, and bile cast nephropathy associated with end-stage liver disease due to HCV infection. Working group preliminary data (not shown) and personal experience of the participants attest to the existence of lesions in donor biopsies, as described earlier. In addition, published studies are confirmatory 31. The donor biopsy can potentially be incorporated into the now widely used KDPI to better judge the impact of medical factors such as black ethnicity, deceased donor status, hypertension, diabetes mellitus and HCV infection. Comprehensive clinical evaluation such as that required in calculation of KDPI is an important part of the donor evaluation; however, the C-statistic (ability to predict graft failure) for KDPI is only 0.6, and further studies seeking to rigorously evaluate the incremental value of biopsy readings over clinical assessment alone need to be performed. Examples of specific scenarios that warrant investigation include hypertension or diabetes mellitus of unknown duration and donors of black race with normal renal function. In these cases, high KDPI can be misleading. Currently, these donor parameters automatically result in the assignment of a substantial negative clinical score without regard to either duration or severity of clinical symptoms and without an attempt to determine whether the donor kidney is actually affected by any of these factors (Table 6).

The caveats and current problems in this study include the small cohort size and freeze artifact. Freeze artifact appears to be a particularly significant factor in a pathologist's ability to assess arteriolar hyalinosis and interstitial fibrosis. The issue of rapid protocols for formalin fixation and paraffin embedding, now technically much improved and increasingly adapted in clinical practice, was not evaluated in this study but needs to be investigated.

In conclusion, the working group found wedge biopsies to be superior to needle core biopsies. This issue was debated repeatedly in the published literature, and some authors suggested the superiority of needle biopsies. Our study did not confirm the latter suggestion. The histopathologic variables that have good reproducibility—number of glomeruli, number of globally sclerosed glomeruli, percentage of globally sclerosed glomeruli—are all superior using wedge kidney biopsies. Reproducibility is fair for interstitial fibrosis, tubular atrophy, interstitial inflammation, arteriolar thrombi and arterial intimal fibrosis. The current study represents the consensus of the Banff working group on scoring donor kidney biopsies; however, our consensus is provisional and subject to future review.

Disclosure

The authors of this manuscript have no conflicts of interest to disclose as described by the American Journal of Transplantation.