Diagnostic Electron Microscopy: A Practical Guide to Interpretation and Technique

Acknowledgements and Dedication

All three editors wish to thank the many individuals who have helped to make this volume possible. Firstly, they would like to express their appreciation to all the authors for their hard work and generosity in sharing their professional experience, as well as all the ‘behind-the-scenes’ staff and colleagues without whom this book could not have been produced.

John Stirling thanks the staff of the Centre for Ultrastructural Pathology, SA Pathology, Adelaide, for their support and photographic contributions—especially Alvis Jaunzems and Jeffrey Swift—and Dr Sophia Otto of the Department of Surgical Pathology, SA Pathology, for her advice and for proofreading.

Alan Curry acknowledges the contributions to his work of the pathologists, particularly Dr Helen Denley and Dr Lorna McWilliam, and technical staff of the Manchester Royal Infirmary, as well as two inspirational organisations—the Public Health Laboratory Service Electron Microscopy network and the Manchester Electron Microscope Society.

Brian Eyden wishes to thank all of the Pathology Department staff at the Christie NHS Foundation Trust (Manchester), without whose technical and light microscopic input the interpretation of tumour ultrastructure would be compromised, if not, in some instances, impossible.

Secondly, the editors wish to recognise the support and encouragement of their families in this endeavour. John Stirling thanks his partner, Jill, and expresses a special appreciation of his teachers and mentors, particularly Alec Macfarlane who helped him achieve his dream of a career in biology and Andrew Dorey who introduced him to electron microscopy and the wonders of cell ultrastructure. Alan Curry thanks his wife, Collette (particularly for her exceptional computer skills), and Brian Eyden thanks his wife, Freda, for understanding the needs of a writing scientist.

Finally, the editors dedicate this book to diagnostic electron microscopists—wherever they may be—who continue to make uncertain diagnoses more precise as a result of their labours, which, in turn, help clinicians to treat their patients better, the ultimate purpose of our work.

List of Contributors

Joseph Alroy, Department of Pathology, Tufts University Cumming's School of Veterinary Medicine, Grafton, Massachusetts, United States and Department of Pathology and Laboratory Medicine, Tufts Medical Center and Tufts University School of Medicine, Boston, Massachusetts, United States

John Brealey, Centre for Ultrastructural Pathology, Surgical Pathology—SA Pathology (RAH), Adelaide, Australia

Hilary Christensen, Program in Cell Biology, The Hospital for Sick Children, Toronto, Ontario, Canada

Alan Curry, Health Protection Agency, Clinical Services Building, Manchester Royal Infirmary, Manchester, United Kingdom

Elizabeth Curtis, Muscle Biopsy Service/Electron Microscope Unit, Department of Cellular Pathology, Queen Elizabeth Hospital Birmingham, Birmingham, United Kingdom

Gary Paul Edwards, Chelford Barn, Stowmarket, Suffolk, United Kingdom

Brian Eyden, Department of Histopathology, Christie NHS Foundation Trust, Manchester, United Kingdom

Pierre Filion, Electron Microscopy Section, Division of Anatomical Pathology, PathWest Laboratory Medicine, QE II Medical Centre, Nedlands, Australia

A. Hadjisavvas, Department of Electron Microscopy/Molecular Pathology, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus

Trinh Hermanns-Lê, Department of Dermatopathology, University Hospital of Liège, Liège, Belgium

Walter H.A. Kahr, Division of Haematology/Oncology, Program in Cell Biology, The Hospital for Sick Children, Toronto, Ontario, Canada and Departments of Paediatrics and Biochemistry, University of Toronto, Toronto, Ontario, Canada

Rosalind King, Institute of Neurology, University College London, London, United Kingdom

K. Kyriacou, Department of Electron Microscopy/Molecular Pathology, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus

M. Nearchou, Department of Electron Microscopy/Molecular Pathology, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus

Rolf Pfannl, Department of Pathology and Laboratory Medicine, Tufts Medical Center and Tufts University School of Medicine, Boston, Massachusetts, United States

Gérald E. Piérard, Department of Dermatopathology, University Hospital of Liège, Liège, Belgium

Claudine Piérard-Franchimont, Department of Dermapathology, University Hospital of Liège, Liège, Belgium

Marie-Annick Reginster, Department of Dermatopathology, University Hospital of Liège, Liège, Belgium

Victor L. Roggli, Department of Pathology, Duke University Medical Center, Durham, North Carolina, United States

Yong-xin Ru, Institute of Haematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, China

Josef A. Schroeder, Zentrales EM-Labor, Institut für Pathologie, Klinikum der Universität Regensburg, Regensburg, Germany

Caroline Sewry, Wolfson Centre for Inherited Neuromuscular Diseases, RJAH Orthopaedic Hospital, Oswestry, United Kingdom and Dubowitz Neuromuscular Centre, Institute of Child Health and Great Ormond Street Hospital, London, United Kingdom

John W. Stirling, Centre for Ultrastructural Pathology, IMVS—SA Pathology, Adelaide, Australia

Angelo A. Ucci, Department of Pathology and Laboratory Medicine, Tufts Medical Center and Tufts University School of Medicine, Boston, Massachusetts, United States

P. Yiallouros, Cyprus International Institute, Cyprus University of Technology, Limassol, Cyprus

Preface–Introduction

John W. Stirling, Alan Curry and Brian Eyden

Diagnostic Electron Microscopy

Science progresses as a result of a variety of factors. Critical to progress, however, is the invention and availability of appropriate tools and techniques that can completely transform our ability to investigate and understand the world around us—without such tools our ability to investigate even basic phenomena would be severely restricted. One such ‘transformational’ technology is the electron microscope. Although transmission electron microscopy (TEM) is now taken for granted, its application to the biological and medical sciences in the late 1950s and early 1960s ranks as one of the single most important factors that has impacted on our knowledge in biology and medicine. The resolving power of the transmission electron microscope (∼0.2 nm as compared with the light microscope with a resolution of ∼200 nm) made two important things possible for the first time, these being the visualisation of: (1) cell organelles and cytoplasmic structures at the macromolecular level (both useful indicators of cell differentiation) and; (2) viruses and microorganisms in general. Thus, TEM gave us new fundamental insights into cell structure and function, histogenesis and differentiation, and, following from this, our understanding of disease and disease processes.

TEM was quickly taken up as a diagnostic tool. In the clinical setting, electron microscopy has been used to improve diagnostic precision and confidence in many fields, including renal disease, neuromuscular disease, microbiology (particularly virology), tumour pathology, skin diseases, industrial diseases, haematology, metabolic storage diseases and conditions involving abnormalities of cilia and sperm. A number of encyclopaedic atlases of normal and pathological tissues quickly followed the introduction of electron microscopy and the medical literature contains many articles describing diagnostic applications of TEM in a wide range of conditions and specialist areas. Diagnostic TEM reached a zenith during the 1980s; however, since then, the introduction of new methodologies (particularly molecular techniques and affinity labelling systems) has reduced the need for TEM, particularly in tumour diagnosis. Despite this, TEM continues to play a significant and important role in pathology, and techniques continue to develop and improve. For example, the introduction of microwave processing and digital cameras has transformed tissue processing and screening so that ‘same-day’ reporting is easily achieved.

The purpose and use of TEM

The purpose of TEM is to diagnose disease based on the ultrastructural features of the tissue. These features include:

1. The presence (or sometimes the absence) of specific or characteristic cellular structures or organelles that indicate cell differentiation

2. The general ultrastructural architecture, including the identity, location and morphology of specific structural features that may be associated with pathology, or indicate disease.

In general, the use of TEM will be predetermined either as a stand-alone protocol (e.g., CADASIL) or as part of a broad integrated diagnostic strategy (e.g., renal biopsies). However, TEM can also be applied on an ad hoc basis whenever there is a chance it will give an improved diagnosis (and therefore better patient care). The general criteria indicating the use of TEM may be summarised simply as follows:

1. When it provides useful (complementary) structural, functional or compositional information in respect to diagnosis, differential diagnoses or disease staging

2. When only atypical features or minor abnormalities are visible by light microscopy despite clear clinical evidence of disease (e.g. some renal diseases)

3. When affinity labelling results are equivocal (e.g. renal disease and tumours)

4. When there is no realistic alternative diagnostic technique or a ‘simple’ test is not available or feasible (e.g. genetic diseases with multiple mutations such as CADASIL and primary ciliary dyskinesia)

5. The investigation and diagnosis of new diseases and microorganisms

6. When it is time and/or cost effective in respect to alternative techniques.

The aim and purpose of this book

The prime aim and purpose of this book is to summarise the current interpretational applications of TEM in diagnostic pathology. In this respect, we have not attempted to reproduce previous encyclopaedic texts but to provide what we regard as a working guide to the main, or most useful, applications of the technique given the limited space available in a text of this size. In addition, we have also included practical topics of concern to laboratory scientists, including brief guides to traditional tissue and microbiological preparation techniques, microwave processing, digital imaging and measurement uncertainty.

Chapter 1

Renal Disease

John W. Stirling¹ and Alan Curry²

¹Centre for Ultrastructural Pathology, IMVS—SA Pathology Adelaide Australia

²Health Protection Agency, Clinical Sciences Building Manchester Royal Infirmary, Manchester United Kingdom

1.1 The Role of Transmission Electron Microscopy (TEM) in Renal Diagnostics

The ultrastructural examination of renal biopsies has made a significant contribution to our understanding of renal disease and is fundamental to accurate diagnosis. For overall tissue evaluation, light microscopy (LM), immunolabelling and transmission electron microscopy (TEM) are generally combined as an integrated protocol. LM is used to make an assessment of overall tissue morphology and to identify the major pathological processes present. Immunolabelling (preferably using immunofluorescence or by the immunoperoxidase technique) is used to determine the composition and location of glomerular immune deposits. Local practices vary, but an antibody panel can contain antibodies directed against IgG, IgA, IgM, complement (C3, C1q and sometimes C4), κ and λ light chains and albumin. TEM can play a major role when LM and immunolabelling findings are normal, only mildly atypical or equivocal and difficult to interpret, particularly in respect to conditions where there may be similar LM or immunolabelling findings. Thus, the technique is particularly useful in the setting of familial disease where the structural abnormalities in the glomerular basement membrane (GBM) cannot be resolved by LM (e.g. Alport's syndrome). TEM can also provide critical information not revealed by the other methodologies to identify underlying primary disease and unexpected concomitant disease. Similarly with immunolabelling, the full classification and staging of deposits require ultrastructural analysis. Some transplant biopsies can also benefit from ultrastructural evaluation (see Chapter 2); however, TEM rarely contributes to the diagnosis of tubular, vascular or interstitial disease. Overall, ultrastructural screening is essential; it can change the diagnosis in ∼25% of cases and provides ‘useful’ information in ∼66% of cases (Pearson et al., 1994; Elhefnawy, 2011).

1.2 Ultrastructural Evaluation and Interpretation

Examination of glomeruli (and other areas, if necessary) should be thorough and systematic with all components being evaluated for possibly significant features or changes. During screening, a range of representative images should be taken. These should include low-power images to show overall glomerular morphology, plus a representative selection of higher power images to show the specific and critical diagnostic features. In some instances, it may also be important to show that certain features are, in fact, absent (e.g. deposits) or normal (e.g. foot processes). The principal elements that should be examined are (i) the location, size and morphology of immune-related deposits and other inclusions; (ii) the thickness, overall morphology and texture of the GBM; (iii) the size and morphology of the mesangial matrix and (iv) the number and morphology of the cellular components of the glomerulus (Stirling et al., 2000). Sclerotic glomeruli should be avoided, and only well-preserved functional (or significantly functional) glomeruli should be examined. It is also important to ensure that the glomeruli screened are representative of the LM findings: this means that, ideally, the choice of glomeruli to be screened (from semithin sections) should be done in collaboration with the reporting pathologist. Finally, it should be stressed that screening should be unbiased, although some knowledge of the pathology and immunolabelling results may be useful if the features expected are minor or uncommon. The vascular pole should be avoided during ultrastructural evaluation as it may contain misleading nonpathologic deposits, and likewise Bowman's capsule which has no real diagnostic value, although the presence of crescents can be confirmed.

Following evaluation, representative images and findings should be communicated to the reporting pathologist, the latter verbally or in a concise written report. If the initial evaluation does not correspond with the LM evaluation (e.g. the electron microscopy (EM) samples only a tiny fraction of the available tissue), then the specimen should be re-examined or additional glomeruli observed to increase diagnostic confidence.

A critical question is ‘How many glomeruli should be examined, and for how long?’ Unfortunately, there is no definitive answer to this dilemma except to say that enough tissue should be examined to answer the diagnostic question posed and to ensure that no additional or unexpected pathology is present. A single glomerulus (or even part of one) may be adequate in respect to diffuse disease and/or when the glomerulus screened is typical of the disease process identified by LM. In contrast, several glomeruli, or possibly glomeruli from different blocks, may be required to capture the full range of pathological changes in focal disease. Perhaps the final word on this issue is to say that the tissue must be screened thoroughly; it is bad practice to stop screening once the features that were expected have been located because additional findings that affect the accuracy of the diagnosis may be missed.

1.3 The Normal Glomerulus

The glomerulus (Figure 1.1) is composed of a tuft of branching capillaries that originate from the afferent arteriole at the vascular pole to form a series of lobules (segments) that ultimately rejoin at the vascular pole and exit the glomerulus via the efferent arteriole. At the core of each lobule is the mesangium which supports the capillary loops; capillary loops are lined by endothelial cells (Figure 1.1). The mesangial matrix principally consists of collagen IV and is populated by mesangial cells (usually 1–3 in normal mesangium) plus a small number of immune-competent cells and rare transient cells of the monocyte–macrophage lineage (Sterzel et al., 1982). The entire capillary tuft is enclosed within Bowman's capsule, the inner aspect of which is lined by a thin layer of epithelial cells (the parietal epithelial cells); a second inner population of epithelial cells (the visceral epithelial cells or podocytes) is closely associated with the capillary tufts, and extensions of these cells form the foot processes (pedicels) that cover the outer aspect of the capillary walls (Figure 1.1). The podocytes are the sole source of the collagen IV α3, α4 and α5 subtypes that form the bulk of the GBM (Abrahamson et al., 2009), and the foot processes play a major role in ultrafiltration and the maintenance of the filtration barrier. As a result, podocyte dysfunction plays a major role in a wide range of glomerular diseases (Wiggins, 2007; Haraldsson, Nystrom and Deen, 2008). Opposite the vascular pole, Bowman's capsule is continuous with the proximal tubule which drains filtrate from the glomerulus (the urinary pole). Overall, filtration is said to be a function of size, shape and charge selection, although the nature and contribution of charge selection are debated (Harvey et al., 2007; Haraldsson, Nystrom and Deen, 2008; Goldberg et al., 2009). The capillary wall as a whole is responsible for the filtration process, and it appears that the capillary endothelium, the GBM and the podocyte foot processes must all be intact for normal filtration to occur (Patrakka and Tryggvason, 2010).

Figure 1.1 Detail of a normal glomerulus. The capillary loops are supported by the mesangium (M). Mesangial cells with nuclei (MC); capillary lumens (L); urinary space (U); podocyte (P) (epithelial cell) and foot processes (FP). Here, the overall width of Overall, the glomerular basement membrane (GBM) averages ∼380 nm in width. Loops are lined with fenestrated endothelial cells (E). Bar = 5 μm.

1.3.1 The Glomerular Basement Membrane

The GBM (Figure 1.1) is made of three layers: (i) the lamina rara interna, the electron-lucent layer immediately adjacent to the endothelium; (ii) the lamina densa, the central layer and (iii) the lamina rara externa, the outer electron-lucent area immediately adjacent to the foot processes. The lamina densa makes up the bulk of the GBM and is its main structural element; it has a felt-like fibrillar construction, and knowledge of its molecular makeup is helpful in understanding and interpreting familial and autoimmune disease. The principal component is collagen IV, which consists of six subtypes (α1–α6) (Patrakka and Tryggvason, 2010). In the developing kidney, the GBM is initially formed of the α1 and α2 subtypes with the α3, α4 and α5 subtypes forming later (the additional subtype, α6, is restricted to Bowman's capsule and some tubular basement membranes) (Harvey et al., 1998; Miner, 1998). In the mature kidney, the α1 and α2 subtypes are restricted to a narrow band immediately adjacent to the capillary endothelium; the α3, α4 and α5 subtypes form the remaining bulk of the GBM (extending out to the foot processes). The core of the mesangial matrix is composed of the α1 and α2 subtypes (continuous with the inner aspect of the GBM), while the outer peripheral layer is made up of the α3, α4 and α5 subtypes (continuous with the outer layer of the GBM) (Butkowski et al., 1989; Harvey et al., 1998; Miner, 1998). The α3, α4 and α5 subtypes are essential for the maintenance of normal glomerular function, and mutations in the genes for these subtypes are responsible for the various forms of membrane-related hereditary nephritis. The structural abnormalities of the GBM in hereditary disease are caused by the absence of the α3 and α5 subtypes, because without either of these, the membrane fails to form correctly (Kalluri et al., 1997; LeBleu et al., 2010; Miller et al., 2010). The α3 subtype has been identified as the Goodpasture epitope (Saus et al., 1988). However, it appears that both the α3 and α5 subtypes are targeted in anti-GBM disease, while in Alport's post-transplantation nephritis, only the α5 subtype is involved (Pedchenko et al., 2010).

1.4 Ultrastructural Diagnostic Features

1.4.1 Deposits: General Features

Immune-related material accumulates as discrete or linear deposits of finely granular electron-dense material within or adjacent to the GBM and/or mesangium in several diseases. Deposits may also be ‘organised’ as tubules and fibrils of various diameters, as crystals and as whorls with a fingerprint-like appearance (Herrera and Turbat-Herrera, 2010). The identity and content of specific deposits vary and must be confirmed by immunolabelling.

Note that scattered deposits may sometimes be an incidental finding with no obvious pathological or diagnostic relevance. Approximately 4–16% of normal individuals have mesangial IgA deposits (without IgG or C3) (Coppo, Feehally and Glassock, 2010), and small numbers of discrete deposits are occasionally seen in individuals with naturally high levels of antigenic challenge.

1.4.2 Granular and Amorphous Deposits

1.4.2.1 Subepithelial Deposits

Subepithelial deposits are finely granular, medium-density deposits located on the outer surface of the GBM and the mesangium. Foot processes that lie over the surface of the deposit are generally effaced.

Large oval or dome-like deposits (humps). True humps are not usually associated with a GBM reaction (spikes). Seen typically in post-infectious glomerulonephritis (PIGN) (Figure 1.14).

Flat or nodular deposits. There may be an associated GBM reaction with ‘spikes’ of new membrane forming adjacent to the deposits, a process that may ultimately lead to the deposits becoming incorporated into the GBM (chain-link appearance by LM). Seen typically in stage I membranous glomerulonephritis (Figure 1.10).

1.4.2.2 Intramembranous Discrete Deposits

These are finely granular, medium-density deposits that lie completely within the lamina densa. The material may be uniform in appearance, or patchy and irregular. Resorption of deposits results in irregular electron-lucent areas surrounded by thickened GBM; badly damaged membrane may become laminated and similar in appearance to the ‘basket weave’ pattern seen in Alport's syndrome. Seen typically in stage III membranous glomerulonephritis (Figure 1.12).

1.4.2.3 Intramembranous Linear Deposits

Linear transformation: a uniform dense amorphous transformation of the GBM that results in a dark, ‘ribbon-like’ appearance. The material is essentially sited within the subendothelial layer of GBM matrix and may penetrate the GBM for some distance. Seen typically in mesangiocapillary glomerulonephritis (MCGN) type II (dense deposit disease (DDD)) (Figure 1.17). A similar effect may be seen around the periphery of loops when subepithelial deposits merge to form a continuous or semicontinuous band.

Medium-density linear deposits of finely granular or powdery material within the GBM. Seen typically in κ light-chain disease (Figure 1.21).

1.4.2.4 Subendothelial Deposits

These are linear or plaque-like, finely granular, medium-density deposits located between the inner aspect of the GBM and the capillary endothelium. Large deposits may be visible by LM as nodular hyaline ‘thrombi’ or as ‘wire-loop’ capillary wall thickening. Seen typically in MCGN type I (Figure 1.16).

1.4.2.5 Mesangial Deposits

Mesangial: finely granular, medium-density deposits within the central mesangial matrix. Seen typically in IgA disease (Figure 1.15).

Paramesangial: finely granular, medium-density deposits around the outer periphery of the mesangial matrix, especially at the junction of the capillary loop and the mesangium. Seen typically in IgA disease (Figure 1.15).

1.4.3 Organised Deposits: Fibrils and Tubules

Many normal and pathological fibrils are found in the glomerulus, and the deposition of proteins, such as monoclonal immunoglobulins or their light-chain or heavy-chain subunits, can produce several glomerular diseases (see reviews by Furness, 2004; Basnayake et al., 2011). The accurate identification of fibrils can be problematic, and distinguishing between normal and pathological types may require the correlation of ultrastructural, immunolabelling and LM-staining characteristics (Table 1.1). A number of algorithms have been published to aid in the diagnosis of renal diseases containing organised deposits (Figure 1.2) (see Ivanyi and Degrell, 2004; Herrera and Turbat-Herrera, 2010).

Figure 1.2 Algorithm for diagnosis of organised deposits. An algorithm to aid in the diagnosis of diseases with organised deposits (based on Ivanyi and Degrell, 2004; Herrera and Turbat-Herrera, 2010). Measurements given are fibril and tubule diameters (see Table 1.1 for source references). This strategy may be combined with silver methenamine staining to further define matrix-derived components (Herrera and Turbat-Herrera, 2010).

Table 1.1 Characteristics of immune and non-immune glomerular fibrils.

1.4.3.1 Amyloid

Amyloid is composed of insoluble fine fibrils made up of low-molecular-weight proteins of various types in a β-pleated sheet conformation (Dember, 2006; Vowles, 2008). Amyloid deposits may form anywhere in the glomerulus (and other tissues) as extracellular nonbranching fibrils of indeterminate length and without cross-striations (Table 1.1; Figure 1.22). Immunofluorescence can sometimes indicate the possibility of amyloid deposition (dull green colouration), but this can be difficult to differentiate from diabetic changes (increase in matrix).

1.4.3.2 Non-amyloid Fibrils and Tubules

Both fibrils and tubules of immune-related material may be found in glomeruli; they may be randomly orientated or organised in parallel arrays. Reported diameters for both fibrils and tubules vary greatly, presumably because of their individual physiochemical makeup, the range of cases sampled and variability in laboratory processing regimes (Table 1.1). Fingerprint deposits (parallel or linear arrays of fibrils—usually curved in a fingerprint-like pattern; Figure 1.20) may also be found, particularly in systemic lupus erythematosus (SLE) and cryoglobulinaemia. There is some dispute as to whether diseases featuring immune fibrils (fibrillary glomerulonephritis; Figure 1.23) and those featuring tubules (immunotactoid glomerulopathy; Figure 1.24) should be treated together or separately (Schwartz, Korbet and Lewis, 2002; Ivanyi and Degrell, 2004; Herrera and Turbat-Herrera, 2010). Here, they are treated as separate entities as there are important clinical differences between them, that is, immunotactoid is normally associated with a lymphoproliferative disorder, but typically, fibrillary glomerulonephritis is not.

1.4.4 Nonspecific Fibrils

A variety of nonspecific fibrils that may be confused with immune deposits have been found in glomeruli (see Herrera and Turbat-Herrera, 2010; and review by Coleman and Seymour, 1992). Some normal matrix components may be more prominent in pathological conditions. Mesangial microfibrils may be particularly well developed in MCGN and diabetic glomerulosclerosis (Table 1.1). Fibrillar collagen is found within the GBM in nail–patella syndrome (Coleman and Seymour, 1992).

1.4.5 General and Nonspecific Inclusions and Deposits

Several nonspecific inclusions such as microparticles (small electron-dense granules), fibrils and membrane-like material may be found within the GBM and mesangial matrix—mostly with doubtful or unknown diagnostic significance. Most common are accumulations of spherical particles and vesicles (often referred to as inclusions or virus-like particles): these may be found in the mesangium but are often seen in a subepithelial location, especially in the angle (or ‘notch’) between two adjoining capillary loops. Microparticles are sometimes seen in areas of laminated GBM in Alport's syndrome (Figure 1.5); similar granules are also found in thickened loops in diabetic sclerosis. Local accumulations of moderately electron-dense material similar to immune-related deposits may be observed, most commonly in a subendothelial location. In the absence of positive immunolabelling, these may relate to the insudation of plasma proteins and/or the development of ‘fibrin’ caps.

1.4.6 Fibrin

Fibrin may be present in numerous diseases in almost any location in the glomerulus. It may be found as irregular, angular or needle-like accumulations of amorphous material of medium electron density, or it may show cross-striations with a characteristic periodicity. The periodicity of normal fibrin is reported as 22.5 nm (Standeven, Ariëns and Grant, 2005); however, periodicities of ∼19–35 nm are reported in pathological tissues in general (Ghadially, 1988). Fibrin is easily distinguished from fibrillar collagen which has an axial banding periodicity of 64–68 nm.

1.4.7 Tubuloreticular Bodies (Tubuloreticular Inclusions)

Tubuloreticular bodies (TRBs) are small clusters of fine anastomosing tubules that arise within the cisternae of the rough endoplasmic reticulum; in the glomerulus, they are most commonly found in endothelial cells (Figure 1.18b). The formation of TRBs has been linked to α- and ß-interferon activity (Hammar et al., 1992). In renal disease, TRBs are most commonly associated with SLE (Figure 1.18b) (and collagen vascular diseases in general), with viral infections (especially HIV/AIDS and hepatitis B) and as a result of α-interferon treatment (Hammar et al., 1992; Haas et al., 2000; Haas, 2007; Yang et al., 2009b). However, TRBs are not specific to these conditions as they have also been found in renal transplants and have been linked to diabetes, lymphoma and Helicobacter pylori infection (Yang et al., 2009b). The presence of TRBs in idiopathic conditions can act as an indicator of underlying systemic disease, and their presence should prompt additional investigations (Yang et al., 2009b).

1.4.8 The Glomerular Basement Membrane

1.4.8.1 GBM Width

Reported measurements for adult GBM vary greatly, but mean widths generally fall in the range of 300–400 nm (Figure 1.1) (see Coleman et al., 1986; review by Dische, 1992; Bonsib, 2007). The GBM of children is reported as being slightly thinner up to age 9 (Morita et al., 1988). A review by Marquez et al. (1999) found that in addition to the variability in values reported for GBM width in adults, there are conflicting data regarding age- and sex-related differences: some studies indicate that the GBM in males and females is similar, but in others, males are said to have slightly thicker GBM than females. Similarly, some studies suggest that the GBM continues to increase in width with age, whereas others do not. Overall, this variation is presumed to reflect the type of tissue used to obtain ‘normal’ data, the differing morphometric techniques employed and the variation inherent in tissue processing and choice of reagents (Marquez et al., 1999; Edwards et al., 2009). With these variations in mind, it is clear that statements about GBM width must be treated with caution. For measurements to be meaningful, a set of normal values must be established based on local processing methodology.

1.4.8.2 Thickness, Texture and General Morphology

Thickness: the GBM may be:

Thin: the membrane may be evenly thinned to ∼20–30% of normal width (±100 nm) (Figure 1.6).

Thickened: the membrane may be evenly thickened up to four times normal width or more (±1000 nm or more) (Figure 1.7).

Irregular: the membrane may be highly irregular in thickness, although the overall mean may be close to normal (Figure 1.5).

Texture: the GBM may appear to be split or laminated. Lamination may be irregular with a ‘basket weave pattern’ (Figure 1.5). Small areas of irregular lamination combined with poorly defined electron-lucent areas may be caused by the resorption of deposits. Faint circumferential lamination is sometimes seen in thickened loops in diabetic glomerulosclerosis.

Folding: loops may become folded or show concertina-like wrinkling as a result of ischaemic collapse. Folds may consolidate to form a thickened area of GBM in which the original folds remain visible. Similar folding may be seen around the periphery of the mesangium (Figure 1.4).

Double contouring (interposition): the GBM becomes duplicated. The duplication is caused by the interposition of mesangial and inflammatory cells, matrix and (in some cases) deposits between the original GBM and the endothelium with the eventual formation of a new inner (luminal) layer of basement membrane material (‘tram-tracking’ by LM) (Figure 1.16).

Subendothelial widening: the endothelium and the GBM may become separated with the formation of a subendothelial space filled with flocculent material (Figure 1.9) or, more rarely, microfibrils and cellular elements from the blood.

Gaps: small breaks are sometimes seen in the GBM. Despite speculation that these are linked to haematuria, breaks are rare, even in cases of macroscopic haematuria.

1.4.9 The Mesangial Matrix

The principal pathological change seen in the mesangium is enlargement of the extracellular matrix, a process that may lead to the obliteration of part or all of the glomerulus (sclerosis). Matrix expansion may be accompanied by an increase in cellularity, immune-related deposits and several nonspecific inclusions such as microfibrils and granular and/or vesicular material. Rarely, the mesangium may be dissolved or attenuated (mesangiolysis) so that the capillary loops are able to fuse and expand (Morita and Churg, 1983).

1.4.10 Cellular Components of the Glomerulus

1.4.10.1 Capillary Endothelium

Endothelial swelling and hypercellularity may occur in many conditions. Significant numbers of TRBs may be found in several conditions and may contribute to the identification and understanding of underlying pathology (Yang et al., 2009b).

1.4.10.2 Epithelium

1.4.10.2.1 Visceral Epithelium (Podocytes)

In proteinuric diseases, the normal structure and arrangement of the podocyte foot processes are often lost: this occurs when the actin cytoskeleton is reorganised, and the processes merge to form a continuous or semicontinuous cytoplasmic layer (foot process effacement, obliteration or fusion) (D'Agati, 2008; Haraldsson, Nystrom and Deen, 2008). Numerous cytoplasmic inclusions are found in podocytes, most notably lysosomes, lipid vesicles and protein droplets. Abnormal lysosomes may be present because of an inherited storage disorder such as Fabry's disease or as a result of drug treatment (e.g. amiodarone and gold therapy). Podocytes may develop numerous long microvilli in a process known as microvillous transformation (Figure 1.3).

Figure 1.3 Minimal change disease. Foot processes are almost completely effaced (foot process effacement), (FPE). The podocyte (P) contains several vesicles, some of which contain lipid (V); there is also significant microvillous transformation (MV). The glomerular basement membrane (GBM) is slightly thin (190 nm in the thinnest areas). L = capillary lumen. Bar = 5 μm.

1.4.10.2.2 Parietal Epithelium

The parietal epithelium may proliferate to form cellular ‘crescents’ that range from small groups of cells to large masses that completely surround the capillary tuft. Fibrin and inflammatory cells such as polymorphs and macrophages may be intermingled with the proliferating cells; in older crescents, fibroblasts and myofibroblasts may also be present. Crescents are nonspecific and a marker of significant glomerular damage; some degree of scarring always remains. At the urinary pole, the junction of the columnar epithelium of the proximal tubule and the parietal epithelial cells is normally sharply marked. Rarely, the tubular epithelium encroaches into the glomerulus, replacing the parietal epithelium in the immediate vicinity of the tubule (termed tubularisation).

1.4.10.3 Mesangial Cells

Mesangial cells proliferate in a number of conditions; this may be accompanied by matrix expansion.

1.4.11 The Capillary Lumen

The capillary lumen may be partially or wholly occluded (stenosed) because of the presence (or effect) of immune-related deposits, insudate, mesangial interposition, endothelial swelling, infiltrating inflammatory cells, capillary wall collapse and/or GBM thickening or folding. Fibrin tactoids and platelets may also be present. On rare occasions, tubular epithelial cells and cell fragments may be found within capillary loops (and the urinary space)—presumably a biopsy artefact.

1.5 The Ultrastructural Pathology of the Major Glomerular Diseases

1.5.1 Diseases without, or with Only Minor, Structural GBM Changes

1.5.1.1 Minimal Change Disease (Minimal Change Nephrotic Syndrome or Lipoid Nephrosis)

Minimal change disease (MCD) is a diagnosis based on morphological features, clinical presentation (nephrotic syndrome) and response to therapy (steroid responsive). Primary disease appears to