The role of electron microscopy in renal allograft biopsy evaluation.

REVIEW URRENT C OPINION

The role of electron microscopy in renal allograft biopsy evaluation Hanneke de Kort a,b, Linda Moran c, and Candice Roufosse d

Purpose of review To review and discuss the use of electron microscopy in the examination of renal transplant biopsies, in particular its role in the diagnosis of glomerular disease and antibody-mediated rejection. Recent findings Electron microscopy can detect recurrent and de-novo glomerular disease at early stages, in particular for focal and segmental glomerulosclerosis and thrombotic microangiopathy. Ultrastructural features are an integral part of the Banff definition of chronic, active antibody-mediated rejection, which has been recently modified to include ultrastructural-only glomerular double contours. In addition, the threshold of peritubular capillary basement membrane multilayering diagnostic for chronic, active antibody-mediated rejection has been changed. As an area for further investigation, ultrastructuralonly glomerular and peritubular capillary features could become tools in the early detection of antibodymediated rejection. Summary Electron microscopy is important in the diagnosis of glomerular disease and chronic, active antibodymediated rejection, both of which contribute to late graft loss. Early detection and treatment may help prolong graft survival. More data are needed on the early ultrastructural features of antibody-mediated injury, so that the usefulness of this technique can be compared with emerging technologies such as transcript analysis. Keywords antibody-mediated rejection, electron microscopy, glomerular disease, kidney transplantation, pathology

INTRODUCTION Ultrastructural examination is central to the interpretation of native kidney biopsies, with previous reviews establishing a crucial contribution of electron microscopy to the final diagnosis in 21% of cases, and a significant contribution in a further 21% [1,2]. In transplant renal biopsies, electron microscopy is useful in the investigation of glomerular disease, including transplant glomerulopathy [3]. Glomerular disease increases in frequency with time post-transplant, and has a negative impact on graft outcome [4,5]. As we become better at dealing with early graft dysfunction and prolonging graft survival, we can expect that early detection and treatment of glomerular disease will become important for increasing later graft survival, and electron microscopy may be a useful tool in this endeavour. However, the use of electron microscopy in transplant biopsy interpretation is not widespread. A survey of practice in 2007 found that only 22% of

laboratories always collected a sample from transplant biopsies for electron microscopy, whereas the majority (75%) took a sample on the basis of clinical features, thereby precluding the chance of early detection of subclinical disease [6]. This paper will discuss the role of electron microscopy in the diagnosis of nonalloimmune glomerular disease, and in alloimmune injury. a

Department of Experimental Immunology, bRenal Transplant Unit, Academic Medical Centre, Amsterdam, The Netherlands, cElectron Microscopy Unit, Department of Histopathology, Imperial College Healthcare NHS Trust, Charing Cross Hospital and dDepartment of Cellular Pathology, Imperial College Healthcare NHS Trust, Hammersmith Hospital, London, UK Correspondence to Dr Candice Roufosse, MD, PhD, Department of Cellular Pathology, Hammersmith Hospital, G Block 1st Floor, Du Cane Rd W12 0HS, London, UK. Tel: +44 20 83833280; fax: +44 20 83833228; e-mail: [email protected] Curr Opin Organ Transplant 2015, 20:333–342 DOI:10.1097/MOT.0000000000000183

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Pathology

KEY POINTS The main indication for the use of electron microscopy in the examination of renal transplant biopsies is glomerular disease. Electron microscopy can help detect glomerular disease at an early stage, potentially more amenable to treatment, in particular for FSGS and TMA. Electron microscopy features are part of the Banff definition of chronic, active ABMR, which will be underdiagnosed if pathologists rely on light microscopy alone to assess the cause of late graft dysfunction. Ultrastructural-only glomerular and PTC endothelial and basement membrane changes may have a role to play in the early detection of antibody-mediated injury, before chronic damage is established. Performing electron microscopy analysis is costly and time-consuming, and its use is limited by the availability of equipment and trained staff.

ELECTRON MICROSCOPY IN THE DIAGNOSIS OF NONALLOIMMUNE GLOMERULAR DISEASE Glomerular disease in renal transplant biopsies is not infrequent and has a negative impact on graft survival, particularly in the long-term [7]. Data from the Australian registry and others have shown that recurrent glomerulonephritis is the third most frequent cause of late graft loss, after death with a functioning graft and chronic rejection [4,5,8]. Focal and segmental glomerulosclerosis (FSGS) and membranoproliferative glomerulonephritis have the greatest incidence of graft loss [8,9 ]. For many glomerular diseases, a diagnosis of recurrence in the allograft can be reached using only light microscopy and immunofluorescence, especially if the nature of the glomerular disease has been established in a native biopsy, and this information has been made available to the pathologist. However, as in native biopsies, electron microscopy is required or very useful for some diagnoses, such as fibrillary/immunotactoid glomerulonephritis, monoclonal immunoglobulin deposition disease and C3 glomerulopathies, including dense deposit disease. As an example, electron microscopy was crucial in a case report diagnosis of recurrent fibronectin glomerulopathy, in a patient with endstage renal failure of unknown cause [10]. Electron microscopy is particularly useful in glomerular diseases with a membranoproliferative pattern, in which the differential diagnosis is between transplant glomerulopathy and an immune complex glomerulonephritis or C3 glomerulopathy. &

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In addition, there is evidence that electron microscopy can detect glomerular diseases either at an early stage (FSGS) or at a stage not visible on light microscopy [early and/or subacute/chronic forms of thrombotic microangiopathy (TMA)]. The important question here is whether diagnosis and treatment of these conditions based on the electron microscopy-only findings will modify the course of the glomerular disease. Primary FSGS recurs in about 25–50% of adult cases, either as an early recurrence with massive proteinuria, or later, generally more insidiously, and is associated with increased graft loss [11,12]. Foot process effacement (FPE) is the first pathological finding in recurrent FSGS, occurring before segmental glomerular scarring on light microscopy. There is evidence that FPE is fully recoverable either through appropriate treatment, such as plasma exchange or rituximab, or, as was illustrated in an unusual case report by Gallon et al. [15], following removal of the graft from a patient with early recurrent FSGS, and reimplantation into another recipient [13–15]. In patients at risk of recurrent FSGS, when light microscopy is normal and in the presence of proteinuria or graft dysfunction, electron microscopy should be performed, and extensive FPE interpreted as likely recurrent podocytopathy, even in biopsies taken immediately postreperfusion [16]. TMA is a tricky diagnosis in the post-transplant setting. Sometimes the clinical picture is incomplete, with renal involvement but no systemic signs of TMA [17]. Most cases of TMA are related to antibody-mediated rejection (ABMR) or immunosuppressive medication (calcineurin inhibitors and mammalian target of rapamycin inhibitors); however, the possibility of recurrent atypical haemolytic/uraemic syndrome (aHUS) related to dysregulation of the alternative pathway of complement activation should be considered, especially in cases in which the cause of end-stage renal failure is recorded as unknown or ‘hypertensive nephrosclerosis’ [18]. In fact, ABMR and genetic abnormalities in complement regulation can occur together [19]. Although most cases of TMA have frank thrombotic lesions on light microscopy, subacute/smouldering forms may be barely visible as subtle segmental glomerular capillary wall double contours on light microscopy, and ultrastructural examination may reveal more frank endothelial swelling and subendothelial rarefaction with new glomerular basement membrane layers. We have seen cases in which these early ultrastructural changes helped diagnose clinically unsuspected antiphospholipid syndrome or recurrent aHUS. Finally, extended use of electron microscopy post-transplantation would help obtain unified data Volume 20 Number 3 June 2015


Electron microscopy in renal allograft biopsies de Kort et al.

on the recurrence rates of various glomerulonephritides, and would inform trials for the effective management of these diseases [20]. Knowledge gleaned from observing the natural history (modified by immunosuppression) of recurrent disease and its response to early treatment may also benefit patients with native glomerular disease. As new diseases are discovered and characterized, so ultrastructural features of their recurrence should be described, as was recently illustrated in a case of recurrent complement factor H-related 5 nephropathy [21], and in a series of recurrent C3 glomerulonephritis [22 ]. &

microscopy (one or more glomerular capillary loops have double contours, affecting 60%)

Y (>50%)

Y (>75%)

Y (60%)

Circumferential ¼ Y (cut-off%)/N(score used)



337

338

NS 3


Type of biopsy N ¼ native T ¼ transplant. BM, basement membrane; N/A, not applicable; NS, not specified; PTC, peritubular capillary.

2–3 PTC with 3 layers T Martinez and Gil [52]

Number of BM layers (1–3)

NS NS T Bro ¨ cker et al. [51 ] &

T


0 ¼ 1 layer; 1 ¼ 5 layers

N (highest number of BM layers on overall PTC circumference) 25

ULTRASTRUCTURAL GLOMERULAR FEATURES IN ACUTE/ACTIVE OR EARLY CHRONIC/ACTIVE ANTIBODY-MEDIATED REJECTION Although electron microscopy is important in defining cABMR, we know that this diagnosis is associated with poor therapeutic responses and graft loss [53]. Recent research has focussed on the identification of antibody-mediated damage at an earlier stage. Endothelial cells, including those of the renal microcirculation, are the predominant target of antibody-mediated injury [54,55,56 ]. Ultrastructural parameters indicative of glomerular endothelial cell activation (detailed in Table 2 and illustrated in Fig. 1) have been found either in association with histological, immunohistochemical (C4d), and/or serological features of acute/active ABMR [48,51 ,57 ], or preceding the development of cABMR/transplant glomerulopathy in subsequent biopsies [40,48,58]. The earliest changes include glomerular endothelial cell swelling (GESw), glomerular endothelial vacuolation, glomerular endothelial serration, and expansion of the lamina rara interna (LRIE). At a later stage, widespread loss of glomerular endothelial fenestration, glomerular basement membrane multilayering, glomerular basement membrane double contours, and podocyte FPE occur (Fig. 1). In a cohort including many presensitized patients, the combination of three features (GESwþLRIEþglomerular basement membrane double contours) was found to be specific for acute ABMR [48]. Conversely, the absence of these features can be a reassuring sign of the absence of acute ABMR, especially in biopsies where C4d immunohistochemistry is unhelpful. For example, in highly sensitized patients, terminal complement pathway inhibition with eculizumab led to an absence of ultrastructural features of endothelial activation, in contrast to biopsies from a control group not treated with eculizumab [59]. In recipients of an ABO-incompatible (ABOi) transplantation, the absence of these features despite circulating anti-AB antibodies and C4d-positivity could be interpreted as a marker of endothelial accommodation [51 ]. However, in our own (unpublished) experience and that of others [51 ], a modest degree of GESw, LRIE, and glomerular basement membrane &

Roufosse et al. [50]


Numbers of PTC with 3 and 5 layers

Y (50%) 15–25 Categories A, B and C1, C2, and C3 based on 3 worst affected PTC N/T Liapis et al. [33]


10

Y (>75%)

clinician’s attention towards a patient at risk, with features suspicious for antibody-mediated damage warranting heightened clinical, histological and serological surveillance, and/or avoidance of immunosuppressive minimization, then a lower threshold may be useful.

Moderate ¼ 3 PTC with 5–6 layers; severe ¼ 1 PTC with 7 layers Number of BM layers (1–7) T Ivanyi et al. [49]

Reference

Table 1 (Continued)

Type of renal biopsy

Expression of score

Score

Number PTC counted

Circumferential ¼ Y (cut-off%)/N(score used)

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&

&

&

&

Volume 20 Number 3 June 2015


Electron microscopy in renal allograft biopsies de Kort et al. Table 2. List of ultrastructural features in the renal microcirculation in antibody-mediated damage Ultrastructural features examined

Abbreviations

References

Foot process effacement

FPE

Bro ¨ cker et al. [51 ]

Expansion of lamina rara interna

LRIE

Wavamunno et al. [40], Haas and Mirocha [48], & & Papadimitriou et al. [57 ], Bro ¨ cker et al. [51 ]

Glomerular basement membrane multilayering

GBML

Haas and Mirocha [48], Bro ¨ cker et al. [51 ]

Double contours of glomerular basement membranes

DC

Papadimitriou et al. [57 ], Bro ¨ cker et al. [51 ]

Glomerular endothelial cell swelling

GESw

Wavamunno et al. [40], Haas and Mirocha [48], & & Papadimitriou et al. [57 ], Bro ¨ cker et al. [51 ]

Loss of glomerular endothelial fenestration

LGEF

Wavamunno et al. [40], Papadimitriou et al. [57 ], & Bro ¨ cker et al. [51 ]

Adhesion of inflammatory cells to glomerular endothelium

AGE


Glomerular endothelial serration

GESr

Wavamunno et al. [40]

Glomerular endothelial vacuolation

GEV

Wavamunno et al. [40], Haas and Mirocha [48]

Mesangial cell area

MCA


Mesangial matrix area

MMA


Peritubular capillary basement membrane multilayering

PTCBML

Wavamunno et al. [40], Haas and Mirocha [48], & Bro ¨ cker et al. [51 ], Roufosse et al. [50]

Adhesion of inflammatory cells to peritubular capillary endothelium

APE


Peritubular capillary endothelial cell swelling

PESw

Wavamunno et al. [40], Bro ¨ cker et al. [51 ]

Peritubular capillary endothelial serration

PESr


Loss of peritubular capillary endothelial fenestration

LPEF


GLOMERULAR CAPILLARIES &

&

&

&

&

&

PERITUBULAR CAPILLARIES

&

&

Reference column denotes publications in which these features are correlated to concurrent evidence of active/acute antibody-mediated rejection, or the future development of chronic, active antibody-mediated rejection. AGE, adhesion of inflammatory cells to glomerular endothelium; APE, adhesion of inflammatory cells to peritubular capillary endothelium; DC, double contours; FPE, foot process effacement; GBML, glomerular basement membrane multilayering; GESr, glomerular endothelial serration; GESw, glomerular endothelial cell swelling; GEV, glomerular endothelial vacuolation; LPEF, loss of peritubular capillary endothelial cell fenestration; LGEF, loss of glomerular endothelial fenestration; LRIE, expansion of the lamina rara interna; MMA, mesangial matrix area; MCA, mesangial cell area; PESw, peritubular capillary endothelial swelling; PESr, peritubular capillary endothelial serration; PTCBML, peritubular capillary basement membrane multilayering.

multilayering, either in isolation or together, can be seen even in patients without DSA, for example in hyperfiltration injury [28], and in acute T-cell mediated rejection and acute calcineurin inhibitor toxicity [48]. In addition, these features may not add information to light microscopic features of microcirculation inflammation [48]. So the exact role and thresholds of glomerular endothelial changes remain to be defined. In the recent Banff classification update, both acute/active and chronic/active ABMR require evidence of current/recent antibody interaction with the vascular endothelium, including either C4d positivity, or, for cases of C4d-negative ABMR, microcirculation inflammation score [glomerulitis (g)þ peritubular capillaritis] at least two, or increased expression of transcripts indicative of endothelial injury [30 ]. Ultrastructural endothelial features should be evaluated as potential candidates for this list. &&

Tubuloreticular inclusions (TRIs) are sometimes noted in allograft biopsies on electron microscopy (Fig. 1h). TRIs are intracellular organelles characterized by small clusters of anastomosing tubule-like structures that arise from the granular endoplasmic reticulum. TRI are thought to be associated with IFN-b and IFN-a activity, and correlate clinically with systemic IFN treatment or endogenous overproduction of IFNs. Recent investigations have suggested a possible association between TRI in renal allograft biopsies and sensitization/DSA [60,61].

EARLY ULTRASTRUCTURAL PERITUBULAR CAPILLARY FEATURES IN ANTIBODYMEDIATED REJECTION In the context of acute ABMR, PTC shows endothelial cell lysis, apoptosis, and fragmentation, but no PTCBML, confirming this feature as a marker of




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chronicity [48,62]. However, peritubular capillary endothelial swelling and peritubular capillary endothelial serration are seen early in the development of transplant glomerulopathy [40], and low levels of PTCBML may also precede transplant glomerulopathy [27,43,50]. In a vintage-matched case control study comparing 16 patients who developed transplant glomerulopathy versus controls, we found that when at least 10 PTC had at least three layers of basement membrane in an early biopsy, this was predictive of transplant glomerulopathy in a later biopsy [50]. In a cohort of kidney transplant recipients with de-novo DSA, we found that a mean number of basement membrane layers in PTC of at least 2.5 around 1 year post-transplantation was associated with transplant glomerulopathy development (unpublished data). Although low levels of PTCBML may not be specific for rejection, in the context of patients with a known DSA, they may be useful as a marker of developing chronic antibodymediated damage. PTCBML challenges the notion of ABMR as a binary acute/active versus chronic/ active diagnosis, hinting at a continuous process of endothelial damage, often subclinical, and hard to diagnose.

RECOMMENDATIONS FOR ELECTRON MICROSCOPY SAMPLING AND EXAMINATION IN TRANSPLANT BIOPSIES Based on previous recommendations and our own experience, in Table 3, we outline an approach to procuring and analyzing electron microscopy in transplant biopsies [30 ,63]. Chronic ABMR will be underdiagnosed without the use of electron microscopy, particularly if centres have no &&

protocolized DSA testing. However, there are valid objections to the use of electron microscopy, including restricted access, cost, and lack of definitive proof of a benefit in terms of graft survival [64]. Collecting a sample for electron microscopy and taking it to the stage of a resin block for potential future use is neither costly nor time-consuming. A toluidine blue stained, 1-mm-thick section of the tissue can also be examined by light microscopy, in order to avoid missing a diagnostic lesion (e.g. an artery with intimal arteritis) that could be present in this sample. This is also not especially expensive, and can be performed on an older ultramicrotome without a diamond knife. In the context of trials seeking to prolong long-term graft survival, for example through new drugs or interventions such as protocol biopsies, and where time lapses are such that surrogate markers are needed, electron microscopy to detect the earliest signs of cABMR may prove a useful tool [65].

CONCLUSION Electron microscopy is important in the diagnosis of glomerular disease and cABMR, which are important causes of late graft loss. As a potential tool in the early detection and treatment of these conditions, the cost of electron microscopy examination will in the long run need to be justified by a prolongation of graft survival. Electron microscopy is used in the Banff definition of cABMR, but consensus is needed on PTCBML methodology. The potential role for electron microscopy in detection of early antibody-mediated damage should be considered in the context of other competing technologies, such as transcript analysis.

Table 3. Recommendations for the use of electron microscopy in renal transplant biopsy interpretation Indications for collecting EM 1. In all cases, if possible 2. OR If glomerular disease was the cause of ESRF If there is a clinical suspicion of glomerular disease (proteinuria/haematuria) If the patient is at risk for ABMR (high risk transplantation, de-novo DSA, previous T-cell or antibody-mediated mediated rejection) In late indication biopsies (>1 year) Indications for performing EM If glomerular disease was the cause of ESRF If there is clinical or pathological suspicion of glomerular disease, in particular in the case of membranoproliferative pattern glomerular changes on light microscopy If the patient is at risk for or has histological evidence of acute ABMR, in order to establish a diagnosis of ultrastructure-only chronic, active ABMR In late indication biopsies (>1 year) ABMR, antibody-mediated rejection; DSA, donor-specific antibodies; EM, electron microscopy; ESRF, end-stage renal failure; GN, glomerulonephritis.

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Acknowledgements The authors acknowledge the support and knowledge imparted to them all in the preparation of this manuscript by Dr Jill Moss. Financial support and sponsorship The authors would like to acknowledge the European Renal Association—European Dialysis and Transplant Association (ERA/EDTA) and Dutch Kidney Foundation. We are grateful for support from the NIHR Biomedical Research Centre funding scheme. Conflicts of interest C.R. is currently receiving a grant (#857917463) from the Roche Organ Transplantation Research Foundation. H.d.K. is currently receiving a grant (KSTP12.06) from the Dutch Kidney Foundation. The remaining author has no conflicts of interest.

REFERENCES AND RECOMMENDED READING Papers of particular interest, published within the annual period of review, have been highlighted as: & of special interest && of outstanding interest 1. Haas M. A reevaluation of routine electron microscopy in the examination of native renal biopsies. J Am Soc Nephrol 1997; 8:70–76. 2. Haas M. Electron microscopy in renal biopsy interpretation-when and why we still need it. US Nephrol 2007; 1:19–22. 3. Walker PD. The renal biopsy. Arch Pathol Lab Med 2009; 133:181–188. 4. El-Zoghby ZM, Stegall MD, Lager DJ, et al. Identifying specific causes of kidney allograft loss. Am J Transplant 2009; 9:527–535. 5. Sellares J, de Freitas DG, Mengel M, et al. Understanding the causes of kidney transplant failure: the dominant role of antibody-mediated rejection and nonadherence. Am J Transplant 2012; 12:388–399. 6. Pullman JM, Ferrario F, Nast CC. Actual practices in nephropathology: a survey and comparison with best practices. Adv Anat Pathol 2007; 14:132–140. 7. Chailimpamontree W, Dmitrienko S, Li G, et al. Probability, predictors, and prognosis of posttransplantation glomerulonephritis. J Am Soc Nephrol 2009; 20:843–851. 8. Briganti EM, Russ GR, McNeil JJ, et al. Risk of renal allograft loss from recurrent glomerulonephritis. N Engl J Med 2002; 347:103–109. 9. Van Stralen KJ, Verrina E, Belingheri M, et al. Impact of graft loss & among kidney diseases with a high risk of posttransplant recurrence in the paediatric population. Nephrol Dial Transplant 2013; 28:1031 – 1038. European collaborative effort establishing graft loss rates due to recurrence per disease, including rare diseases, in a large cohort of paediatric renal transplant patients. This confirms high rates of graft loss in patients with dense deposit disease, membranoproliferative glomerulonephritis type 1 and FSGS. 10. Otsuka Y, Takeda A, Horike K, et al. A recurrent fibronectin glomerulopathy in a renal transplant patient: a case report. Clin Transplant 2012; 26 (Suppl 24): 58–63. 11. Ponticelli C. Recurrence of focal segmental glomerular sclerosis (FSGS) after renal transplantation. Nephrol Dial Transplant 2010; 25:25–31. 12. Kowalewska J. Pathology of recurrent diseases in kidney allografts: membranous nephropathy and focal segmental glomerulosclerosis. Curr Opin Organ Transplant 2013; 18:313–318. 13. Cheong HI, Han HW, Park HW, et al. Early recurrent nephrotic syndrome after renal transplantation in children with focal segmental glomerulosclerosis. Nephrol Dial Transplant 2000; 15:78–81. 14. Alachkar N, Wei C, Arend LJ, et al. Podocyte effacement closely links to suPAR levels at time of posttransplantation focal segmental glomerulosclerosis occurrence and improves with therapy. Transplantation 2013; 96: 649–656. 15. Gallon L, Leventhal J, Skaro A, et al. Resolution of recurrent focal segmental glomerulosclerosis after retransplantation. N Engl J Med 2012; 366: 1648–1649.

16. Chang JW, Pardo V, Sageshima J, et al. Podocyte foot process effacement in postreperfusion allograft biopsies correlates with early recurrence of proteinuria in focal segmental glomerulosclerosis. Transplantation 2012; 93: 1238–1244. 17. Noris M, Remuzzi G. Thrombotic microangiopathy after kidney transplantation. Am J Transplant 2010; 10:1517–1523. 18. Nadasdy T. Thrombotic microangiopathy in renal allografts: the diagnostic challenge. Curr Opin Organ Transplant 2014; 19:283–292. 19. Noone D, Al-Matrafi J, Tinckam K, et al. Antibody mediated rejection associated with complement factor h-related protein 3/1 deficiency successfully treated with eculizumab. Am J Transplant 2012; 12:2546–2553. 20. Golgert WA, Appel GB, Hariharan S. Recurrent glomerulonephritis after renal transplantation: an unsolved problem. Clin J Am Soc Nephrol 2008; 3:800–807. 21. Vernon KA, Gale DP, de Jorge EG, et al. Recurrence of complement factor Hrelated protein 5 nephropathy in a renal transplant. Am J Transplant 2011; 11:152–155. 22. Zand L, Lorenz EC, Cosio FG, et al. Clinical findings, pathology, and out& comes of C3GN after kidney transplantation. J Am Soc Nephrol 2014; 25:1110–1117. A detailed report of 14 cases of recurrent C3 glomerulonephritis, including electron microscopy on most cases. 23. Porter KA, Dossetor JB, Marchioro TL, et al. Human renal transplants. I. Glomerular changes. Lab Invest 1967; 16:153–181. 24. Hamburger J, Crosnier J, Dormont J. Observations in patients with a well tolerated homotransplanted kidney: possibility of a new secondary disease. Ann N Y Acad Sci 1964; 120:558–577. 25. Hamburger J, Crosnier J, Dormont J. Experience with 45 renal homotransplantations in man. Lancet 1965; 1:985–992. 26. Zollinger HU, Moppert J, Thiel G, Rohr HP. Morphology and pathogenesis of glomerulopathy in cadaver kidney allografts treated with antilymphocyte globulin. Curr Top Pathol 1973; 57:1–48. 27. Monga G, Mazzucco G, Novara R, Reale L. Intertubular capillary changes in kidney allografts: an ultrastructural study in patients with transplant glomerulopathy. Ultrastruct Pathol 1990; 14:201–209. 28. Ivanyi B, Kemeny E, Szederkenyi E, et al. The value of electron microscopy in the diagnosis of chronic renal allograft rejection. Mod Pathol 2001; 14:1200–1208. 29. Hsu HC, Suzuki Y, Churg J, Grishman E. Ultrastructure of transplant glomerulopathy. Histopathology 1980; 4:351–367. 30. Haas M, Sis B, Racusen LC, et al. Banff 2013 meeting report: inclusion of && C4d-negative antibody-mediated rejection and antibody-associated arterial lesions. Am J Transplant 2014; 14:272–283. This official report from the 12th Banff Conference on Allograft Pathology provides details on the diagnostic criteria of C4d-negative acute/active and chronic, active ABMR. Refer to Table 2 of this manuscript, footnotes 8 and 9, for details on the criteria requiring electron microscopy. 31. Ivanyi B, Fahmy H, Brown H, et al. Peritubular capillaries in chronic renal allograft rejection: a quantitative ultrastructural study. Hum Pathol 2000; 31:1129–1138. 32. Ivanyi B. Transplant capillaropathy and transplant glomerulopathy: ultrastructural markers of chronic renal allograft rejection. Nephrol Dial Transplant 2003; 18:655–660. 33. Liapis G, Singh HK, Derebail VK, et al. Diagnostic significance of peritubular capillary basement membrane multilaminations in kidney allografts: old concepts revisited. Transplantation 2012; 94:620–629. 34. Drachenberg CB, Steinberger E, Hoehn-Saric E, et al. Specificity of intertubular capillary changes: comparative ultrastructural studies in renal allografts and native kidneys. Ultrastruct Pathol 1997; 21:227–233. 35. Takeuchi O, Oikawa T, Koyama K, et al. Multilayering of peritubular capillary is a specific diagnostic criterion for immunologic chronic rejection: does a humoral factor contribute to the pathogenesis of peritubular capillary lesions in chronic rejection? Transplant Proc 2000; 32:306–307. 36. Gough J, Yilmaz A, Miskulin D, et al. Peritubular capillary basement membrane reduplication in allografts and native kidney disease: a clinicopathologic study of 278 consecutive renal specimens. Transplantation 2001; 71:1390–1393. 37. Regele H, Bohmig GA, Habicht A, et al. Capillary deposition of complement split product C4d in renal allografts is associated with basement membrane injury in peritubular and glomerular capillaries: a contribution of humoral immunity to chronic allograft rejection. J Am Soc Nephrol 2002; 13: 2371–2380. 38. Kiyici H, Demirhan B, Ozdemir BH, et al. Significance of peritubular capillary basement membrane multilamellation in diagnosis of chronic allograft nephropathy. Transplant Proc 2003; 35:2643–2644. 39. Vongwiwatana A, Gourishankar S, Campbell PM, et al. Peritubular capillary changes and C4d deposits are associated with transplant glomerulopathy but not IgA nephropathy. Am J Transplant 2004; 4:124–129. 40. Wavamunno MD, O’Connell PJ, Vitalone M, et al. Transplant glomerulopathy: ultrastructural abnormalities occur early in longitudinal analysis of protocol biopsies. Am J Transplant 2007; 7:2757–2768. 41. Sis B, Campbell PM, Mueller T, et al. Transplant glomerulopathy, late antibody-mediated rejection and the ABCD tetrad in kidney allograft biopsies for cause. Am J Transplant 2007; 7:1743–1752.




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Pathology 42. Aita K, Yamaguchi Y, Horita S, et al. Thickening of the peritubular capillary basement membrane is a useful diagnostic marker of chronic rejection in renal allografts. Am J Transplant 2007; 7:923–929. 43. Lerut E, Naesens M, Kuypers DR, et al. Subclinical peritubular capillaritis at 3 months is associated with chronic rejection at 1 year. Transplantation 2007; 83:1416–1422. 44. Hidalgo LG, Campbell PM, Sis B, et al. De novo donor-specific antibody at the time of kidney transplant biopsy associates with microvascular pathology and late graft failure. Am J Transplant 2009; 9:2532–2541. 45. Einecke G, Sis B, Reeve J, et al. Antibody-mediated microcirculation injury is the major cause of late kidney transplant failure. Am J Transplant 2009; 9:2520–2531. 46. Einecke G, Reeve J, Sis B, et al. A molecular classifier for predicting future graft loss in late kidney transplant biopsies. J Clin Invest 2010; 120:1862– 1872. 47. Sis B, Einecke G, Chang J, et al. Cluster analysis of lesions in nonselected kidney transplant biopsies: microcirculation changes, tubulointerstitial inflammation and scarring. Am J Transplant 2010; 10:421–430. 48. Haas M, Mirocha J. Early ultrastructural changes in renal allografts: correlation with antibody-mediated rejection and transplant glomerulopathy. Am J Transplant 2011; 11:2123–2131. 49. Ivanyi B, Kemeny E, Rago P, et al. Peritubular capillary basement membrane changes in chronic renal allograft rejection. Virchows Archiv 2011; 459: 321–330. 50. Roufosse CA, Shore I, Moss J, et al. Peritubular capillary basement membrane multilayering on electron microscopy: a useful marker of early chronic antibody-mediated damage. Transplantation 2012; 94:269–274. 51. Bro¨cker V, Pfaffenbach A, Habicht A, et al. Beyond C4d: the ultrastructural & appearances of endothelium in ABO-incompatible renal allografts. Nephrol Dial Transplant 2013; 28:3101–3109. The authors investigated the ultrastructural correlate of C4d positivity in biopsies from both ABOi and ABO-compatible transplant recipients. ABOi C4d-positive graft biopsies mostly showed normal endothelium, possibly illustrating the phenomenon of ‘accommodation’, whereas the presence of anti-HLA antibodies was associated with endothelial changes in glomeruli and peritubular capillaries, probably indicating a degree of ABMR. 52. Martinez MA, Gil YR. Value of a simple method to assess chronic rejection in renal allograft on electron microscopy. Ultrastruct Pathol 2013; 37:449–451. 53. Cosio FG, Gloor JM, Sethi S, Stegall MD. Transplant glomerulopathy. Am J Transplant 2008; 8:492–496.

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54. Zhang X, Reed EF. Effect of antibodies on endothelium. Am J Transplant 2009; 9:2459–2465. 55. Sis B, Jhangri GS, Bunnag S, et al. Endothelial gene expression in kidney transplants with alloantibody indicates antibody-mediated damage despite lack of C4d staining. Am J Transplant 2009; 9:2312–2323. 56. Drachenberg CB, Papadimitriou JC. Endothelial injury in renal antibody& mediated allograft rejection: a schematic view based on pathogenesis. Transplantation 2013; 95:1073–1083. This article provides a detailed pathophysiological exploration of the effect of antibodies on graft endothelium, using electron microscopy pictures to illustrate endothelial lytic and nonlytic damage, as well as chronic remodelling of the glomeruli. 57. Papadimitriou JC, Drachenberg CB, Ramos E, et al. Antibody-mediated & allograft rejection: morphologic spectrum and serologic correlations in surveillance and for cause biopsies. Transplantation 2013; 95:128–136. This is a detailed report of histological, serological and immunohistochemical (C4d) features of ABMR, including electron microscopy, in 1101 transplant biopsies, including for cause biopsies and surveillance biopsies. The authors find that electron microscopy features of ABMR correlate with DSA and could provide an opportunity for the diagnosis of early ABMR-related microvascular pathology, including subclinical forms. 58. Maryniak RK, First MR, Weiss MA. Transplant glomerulopathy: evolution of morphologically distinct changes. Kidney Int 1985; 27:799–806. 59. Stegall MD, Diwan T, Raghavaiah S, et al. Terminal complement inhibition decreases antibody-mediated rejection in sensitized renal transplant recipients. Am J Transplant 2011; 11:2405–2413. 60. Ellis CL, Gupta G, Racusen LC, Arend LJ. The significance of tubuloreticular inclusions (TRIs) in allograft kidney biopsies. Mod Pathol 2012; S2:398A. 61. Willicombe M, Roufosse C, Moran L, et al. Significance of tubuloreticular inclusions in renal allografts. Am J Transplant 2014; 14:529. 62. Liptak P, Kemeny E, Morvay Z, et al. Peritubular capillary damage in acute humoral rejection: an ultrastructural study on human renal allografts. Am J Transplant 2005; 5:2870–2876. 63. Herrera GA, Isaac J, Turbat-Herrera EA. Role of electron microscopy in transplant renal pathology. Ultrastruct Pathol 1997; 21:481–498. 64. Boonyapredee M, Moore J. Electron microscopy in determining the etiology of kidney allograft dysfunction. Transplant Proc 2012; 44:2992–2996. 65. Stegall MD, Gaston RS, Cosio FG, Matas A. Through a glass darkly: seeking clarity in preventing late kidney transplant failure. J Am Soc Nephrol 2015; 26:20–29.



Value of a simple method to assess chronic rejection in renal allograft on electron microscopy.

An improved method for renal allograft biopsy.

C4d immunoreactivity of intraoperative zero-hour biopsy in renal allograft.

Electron microscopy of single detached glomerulus in needle biopsy tissue.

Molecular diagnostics in renal allograft biopsy interpretation: potential and pitfalls.

Scanning electron microscopy of renal vascular casts.

Utility of renal allograft biopsy: An audit of 80 allograft biopsies.

Refsum's disease: electron microscopy of an iris biopsy.

Evaluation of flowcytometric crossmatching in renal allograft recipients.

Renal pseudoaneurysm after core-needle biopsy of renal allograft successfully managed with superselective embolization.

The role of biopsy in incidental renal tumours.

Fascicular nerve allograft evaluation. Part 2: comparison with whole-nerve allograft by light microscopy.

Chronic reduction of renal mass: glomerular morphometry by electron microscopy.

Impact of specimen adequacy on the assessment of renal allograft biopsy specimens.

Electron microscopy of nephropathia epidemica. Renal tubular basement membrane.

Rapid processing of renal glomeruli for electron microscopy.

Early detection of osseointegration using scanning electron microscopy and the interfacial biopsy chamber: a pilot study.

Current Safety of Renal Allograft Biopsy With Indication in Adult Recipients: An Observational Study.

Evaluation of percutaneous kidney biopsy in advanced renal failure.

Use of electron microscopy in examination of faeces and rectal and jejunal biopsy specimens.

Percutaneous injection of acrylic glue into renal allograft pseudoaneurysm for control of intractable post-biopsy hematuria.

Prognostic value for immediate function of one-hour renal allograft biopsy.

Salvage of bleeding renal allograft following biopsy, with suture technique: a case report.

Fluorescence microscopy as an alternative to electron microscopy for microscale dispersion evaluation of organic-inorganic composites.