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Nephrology 20, Suppl. 2 (2015) 2–8

Review

Banff 2013 update: Pearls and pitfalls in transplant renal pathology SHIGEO HARA Department of Diagnostic Pathology, Kobe University Graduate School of Medicine, Kobe, Japan

KEY WORDS: C4d, isolated v-lesion, polyomavirus BK nephropathy, transplant glomerulitis, transplant glomerulopathy. Correspondence: Shigeo Hara, Department of Diagnostic Pathology, Kobe University Graduate School of Medicine. 7-5-2, Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan. Email: shara@med. kobe-u.ac.jp Accepted for publication 14 March 2015. doi:10.1111/nep.12474 Conflict of Interest: The authors have no conflict of interest to declare.

ABSTRACT: The pathological classification of rejection in renal allografts (Banff classification) has undergone substantial evolution for more than 20 years, and has been the diagnostic gold standard in clinical practice. The 2013 updated Banff classification encompasses a revised scheme of antibody-mediated rejection (ABMR) that consists of donor-specific antibody (DSA) positivity, characteristic histological manifestations for both acute and chronic ABMR, and DSA-induced endothelial cell injury which is represented by either C4d positivity, microvascular inflammation or expression of activated endothelial gene transcripts. Other modified criteria include a C4d positivity threshold, and histological definition of transplant glomerulitis and transplant glomerulopathy. Morphologically, glomerulonephritis, either recurrent or de novo, can be challenging to differentiate from ABMR-mediated transplant glomerulitis. Endothelial arteritis by itself does not warrant the diagnosis of acute T-cell mediated rejection; ABMR should also be considered based on the DSA test results. With regard to polyomavirus BK-associated nephropathy, immunohistochemical examination using antisimian virus (SV) 40 antibody can be a promising method to assess the quantitative viral load of polyomavirus BK and graft survival. In summary, the 2013 updated Banff classification strictly defines ABMR with histopathological and serological criteria irrespective of C4d positivity. Inclusion of gene expression data relevant to ABMR highlights that the Banff criteria have entered the era of ‘Seeing the Unseen’ schemes, reflecting recent advances in understanding the molecular events in allograft injury.

The 12th Banff Conference on Allograft Pathology was held August 19–23, 2013, in Comandatuba, Brazil. The meeting report was published in November 2013, highlighting the inclusion of C4d-negative antibody-mediated rejection (ABMR).1 In the present review, the updated version of the Banff classification (Banff 2013) is summarized, especially focusing on the practical guide it offers on pathological diagnosis and pitfalls. Table 1 shows an adapted summary of the updated ABMR classification.1 Diagnosis of both acute/active and chronic, active ABMR requires all three pathological and laboratory manifestations: morphologic evidence of acute or chronic tissue injury, evidence of current/recent antibody interaction with vascular endothelium, and serologic evidence of donorspecific antibody (DSA). Endarteritis, which has been considered as a histological clue for suspecting T-cell mediated rejection (TCMR), may be regarded as the pathological mani2

festation of suspicion for both ABMR and TCMR, based on the DSA test. The application of microarray data into one of the diagnostic criteria for suggesting endothelial cell injury by DSA was a landmark leap, reflecting recent advancements in the immunobiology of renal transplant pathology.

C4d POSITIVITY One of the most relevant outcomes of Banff 2013 was that C4d positivity is no longer a requirement for the diagnosis of ABMR. Since being adopted into the Banff 2005 classification, C4d positivity has been a cardinal feature for the diagnosis of both acute and chronic ABMR.2 In light of the accumulation of recent evidence in C4d-negative ABMR,3,4 C4d staining was considered as one of the criteria to suggest evidence of endothelial activation triggered by DSA interaction.1 © 2015 Asian Pacific Society of Nephrology

Banff 2013 update

Table 1. Revised classification of antibody-mediated rejection1 Acute/active ABMR; all three features must be present for diagnosis 1. Histologic evidence of acute tissue injury, including one or more of the following: Microvascular inflammation (g > 0 and/or ptc > 0) Intimal or transmural arteritis (v > 0) Acute thrombotic microangiopathy, in the absence of any other cause Acute tubular injury, in the absence of any other apparent cause 2. Evidence of current/recent antibody interaction with vascular endothelium, including at least one of the following: Linear C4d staining in ptc (C4d2 or C4d3 by IF, or C4d > 0 by IHC) At least moderate microvascular inflammation ([g + ptc] ≥ 2) Increased expression of gene transcripts indicative of endothelial injury 3. Serologic evidence of DSAs (HLA or other antigens) Chronic, active ABMR; all three features must be present for diagnosis 1. Morphologic evidence of chronic tissue injury, including one or more of the following: Transplant glomerulopathy (cg > 0), if no evidence of chronic TMA Severe ptc basement membrane multilayering (requires EM) Arterial intimal fibrosis of new onset, excluding other causes 2. Evidence of current/recent antibody interaction with vascular endothelium, including at least one of the following: Linear C4d staining in ptc (C4d2 or C4d3 by IF, or C4d > 0 by IHC) At least moderate microvascular inflammation ([g + ptc] ≥ 2) Increased expression of gene transcripts indicative of endothelial injury 3. Serologic evidence of DSAs (HLA or other antigens) C4d staining without evidence of rejection; all three features must be present for diagnosis 1. Linear C4d staining in ptc (C4d2 or C4d3 by IF, or C4d > 0 by IHC) 2. g = 0, ptc = 0, cg = 0 (by light microscopy and by EM if available), v = 0; no TMA, no ptc basement membrane multilayering, no acute tubular injury 3. No acute cell-mediated rejection (Banff 97 type 1A or greater) or borderline changes Abbreviations: ABMR, antibody-mediated rejection; DSAs, donor specific antibodies; EM, electron microscopy; HLA, human leukocyte antigen; IF, immunofluorescence; IHC, immunohistochemistry; ptc, peritubular capillaries; TMA, thrombotic microangiopathy.

The threshold for C4d positivity has been modified. In a 4-tiered grading system that ranged from 0 to 3+, ‘C4d positive’ was originally defined as 3+ in both frozen and paraffin sections.5 The clinical significance of 2+ in frozen sections was unknown, while 2+ in paraffin sections did not warrant positivity and further studies using frozen sections were recommended. In Banff 2013, the criteria for C4d positivity were revised to 2+ or 3+ in frozen sections and >0 in paraffin sections (Fig. 1). However, 1+ in frozen sections was not unanimously approved as a criterion for C4d positivity. Further investigation will be required to establish biological and clinical significance of a minimum level of positive staining in frozen sections. Evaluation of C4d staining can be affected by the density of peritubular capillaries in the renal cortex. As a kidney allograft undergoes progressive interstitial fibrosis and tubular atrophy (IF/TA), the peritubular capillaries are obliterated by the accumulating sclerotic matrix, rendering C4d scoring ostensibly lower. A renal allograft in an advanced stage of IF/TA should be cautiously evaluated when scoring its C4d positivity grade. Double immunostaining using anti-CD34 antibody can improve the accuracy in estimating the C4dpositive fraction of peritubular capillaries.6

TRANSPLANT GLOMERULITIS Transplant glomerulitis is histologically characterized by glomerular microvascular inflammation and enlargement of © 2015 Asian Pacific Society of Nephrology

score

area

C4d 0

0%

C4d 1

1-10%

Neg

Unknown

C4d 2

10-50%

Unknown

Positive ?

C4d 3

>50%

IF

IHC

IF

Neg

IH C Neg

Neg

Positive Positive

Positive

Positive

Banff 2007

Banff 2013

Abbreviations: IF, immunofluorescence; IHC, immunohistochemistry Fig. 1 Revised criteria for C4d positivity. Comparison of previous (Banff 2007) and updated (Banff 2013) threshold level of C4d positivity is presented. Abbreviations: IF, immunofluorescence; IHC, immunohistochemistry

endothelial cells (Fig. 2a). These infiltrative changes are triggered by the interaction of DSA and endothelial cells, followed by complement activation and recruitment of inflammatory cells.7 However, the alloimmune response is not the sole culprit for glomerular inflammation in a kidney allograft. For instance, acute T-cell mediated rejection is a potential cause for glomerulitis which is predominated by T cells.8,9 In addition, intraglomerular inflammation without 3

S Hara

(a)

(b)

(c)

C4d deposition and any evidence of rejection (‘isolated transplant glomerulitis’) can be observed in protocol biopsies of non-DSA renal allografts.10 Despite these diagnostic pitfalls, transplant glomerulitis has been considered as one of the potent histological clues for the suspicion of ABMR. In Banff 2013, the grading of transplant glomerulitis (g) is classified according to the percentage of glomeruli involved, which was originally defined in the Banff 97 classification as: 1–25%, 26–50% and >50% for g1, g2 and g3, respectively.11 In addition to these conventional criteria, endothelial swelling and capillary occlusion were adopted as defining transplant glomerulitis in Banff 2013.1 However, it can be challenging to distinguish enlarged endothelial cells from hematopoietic cells adherent to the glomerular capillary wall. In support of this difficulty, Sis et al. demonstrated at the Banff 2013 meeting that the reproducibility of transplant glomerulitis was generally quite low (κ = 0.2–0.3).1 In addition, immunostaining with anti-CD68 antibody did not improve interobserver agreement regarding transplant glomerulitis. More reliable methods for scoring transplant glomerulitis are required to establish a strict pathological evaluation of ABMR. Another issue to be solved is the refinement of guidelines for cut-off level between what is within a normal limit and what constitutes slight transplant glomerulitis.1 The current Banff scheme sets minimum threshold levels for various inflammatory reactions. In tubulitis, at least 1 mononuclear cell infiltration within a single renal tubule is adequate to define t1.11 Up to 3–4 inflammatory cells within >10% of cortical peritubular capillaries are required for scoring mild peritubular capillaritis (ptc1).5 However, the minimum 4

Fig. 2 (a) Transplant glomerulitis. The glomerular capillaries were occluded with enlarged endothelial cells and inflammatory cells including monocytes and neutrophils. (b) Mild transplant glomerulopathy (cg1b). Double contours were confined almost exclusively to 30 min, citrate (pH 5–7) or EDTA (pH 7–9) buffers, monoclonal antibody to SV40 large T antigen (PAb416 being the most recommended) at a dilution of

Banff 2013 update: Pearls and pitfalls in transplant renal pathology.

The pathological classification of rejection in renal allografts (Banff classification) has undergone substantial evolution for more than 20 years, an...
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