Electron Microscopy In Diagnosis Research Articles

Potential of diagnostic electron microscopy of calcification, pathological neovascularization and elastolysis in combination with phenotyping of cell populations in large arteries

Aim. To determine the potential of diagnostic electron microscopy of intraplaque processes (severity of lipid damage, fibrous cap thickness and condition, severity of pathological neovascularization, presence, nature and severity of calcification, ratio and distribution of various cell populations).Material and methods. The study objects were plaques removed during endar­terectomy from the human carotid artery and segments of the human internal mammary artery removed during coronary bypass surgery. Whole specimens were subjected to chemical fixation, staining with heavy metal salts, embedding in epoxy resin followed by layer-by-layer grinding, polishing, contrasting, visualization using back-scattered electron scanning electron microscopy and three-dimensional reconstruction with color mapping (modified EM-BSEM).Results. The use of a modified EM-BSEM made it possible to: 1) visualize the fibrous cap thickness and assess the extracellular matrix; 2) analyze the neointimal lipid distribution; 3) perform three-dimensional reconstruction and analyze the microenvironment of calcifications of various sizes; 4) visualize endothelial cells, defects in interendothelial contacts and the basement membrane of neointimal capillaries with their subsequent three-dimensional reconstruction; 5) perform an analysis of age-dependent defects in the basement membrane and internal elastic membrane of the internal mammary artery. The resolution of the obtained images was significantly superior to intravascular imaging methods (intravascular ultrasound and optical coherence tomography), allowing additional assessment of capillary fluidity, the degree of calcification encapsulation and the condition of elastic fibers. Three-dimensional reconstruction of calcifications, neointimal capillaries and elastic fibers made it possible to assess their spatial density and heterogeneity. Simultaneously with the identification and assessment of these histological structures, objective phenotyping of cell populations was performed, which made it possible to isolate macrophages and foam cells, vascular smooth muscle cells, fibroblasts and endothelial cells in atherosclerotic plaques and automatically identify them through color mapping determined by their electronic contrast distribution signatures.Conclusion. The modified EM-BSEM method allows for universal electron microscopic diagnosis of atherosclerotic and elastolytic lesions of large arteries with high information content about vascular remodeling and high accuracy. Electronic contrast distribution signatures unique for each cell population indicate the possibility of their automated phenotyping using specialized neural network algorithms.

Hydroxychloroquine-induced cardiomyopathy: a novel case highlighting the role of electron microscopy in diagnosis

Hydroxychloroquine-induced cardiomyopathy is a rare adverse reaction with serious and potentially fatal outcomes. We report a 56-year-old female who presented in acute right heart failure with NYHA class III symptoms, ascites, raised JVP pressure, and peripheral oedema. Significant medical history included SLE (diagnosed in her late 20s, well controlled on hydroxychloroquine), CKD, and poorly controlled hypertension. Extensive investigations included transthoracic ECHO, cardiac MRI, cardiac amyloidosis scan, and right heart catheter.

Spectrum and Distribution of Biopsy-proven Kidney Diseases: A 12-year Survey of a Single Center in Iran.

Data about the prevalence of biopsy-proven kidney diseases in Iran are rare, and none of the previous studies used electron microscopy for diagnosis. This study aimed to analyze the prevalence of biopsy-proven kidney diseases in Iran's primary referral center. To the best of our knowledge, this is the most extensive study carried out in Iran. Reports of kidney biopsy samples from patients referred to our center in 2007-2018 were reviewed for demographic data, clinical presentation, and final diagnosis. Statistical analyses were performed. Among the 3455 samples received, 2975 were analyzed. Nephrotic syndrome (39%) was the most common cause of biopsy, followed by subnephrotic proteinuria (18%), hematuria in association with proteinuria (15%), renal failure (9%), isolated hematuria (6%), and lupus nephritis (LN) (4%). The most common diagnoses were membranous glomerulonephritis (17.9%), focal segmental glomerulosclerosis (FSGS) (15.9%), LN (13.7%), minimal histopathological findings (unsampled FSGS vs. minimal change disease, 12.1%), Immunoglobin A nephropathy (IgAN) (6.5%) and Alport syndrome (6.1%). NS and proteinuria were the most common indications for a kidney biopsy. IgAN and LN were the most common causes of primary and secondary glomerulonephritis, presenting with hematuria and proteinuria, respectively. Although membranous glomerulonephritis was the most common disease, it has been replaced by FSGS in recent years.

A novel technique of STEM observation of TEM section using LVSEM and application of pathological diagnosis of renal biopsy.

The usefulness of the transmission electron microscope (TEM) for pathological diagnosis is apparent. However, high operating costs and other disadvantages have limited the ability to maintain and operate a TEM. In recent years, a general-purpose benchtop low-vacuum scanning electron microscope (LVSEM), which is inexpensive and easy to operate, has been developed and is expected to be applied in electron microscopic pathological diagnosis. To date, we have previously observed TEM ultrathin sections of Immunoglobulin A (IgA) nephropathy with a benchtop LVSEM using an ultra variable-pressure detector (UVD) and a newly developed holder for observing scanning transmission electron microscope (STEM) images (UVD-STEM holder) and compared the images with those obtained with typical TEM observations. We reported the results in the 53rd Annual Meeting of the Japanese Society for Clinical Molecular Morphology and the 64th Symposium of The Japanese Society of Microscopy and discussed the validity of the methods in the pathological diagnosis of IgA nephropathy and other renal diseases. As a result, we demonstrated the potential for pathological diagnosis using benchtop LVSEM. In this study, we similarly examined typical kidney diseases such as membranous nephropathy, lupus nephritis and amyloidosis. We could obtain sufficient data for the pathological diagnosis of IgA nephropathy, membranous nephropathy and lupus nephritis. However, it is difficult to detect amyloid fibres that are characteristic of amyloidosis. The development of this method is expected to expand the possibilities for pathological diagnosis using electron microscopy, including its application to other diseases.

Digital Measurement of the Diameter of the Collagen Fibers by Transmission Electron Microscopy for Diagnosis of Ehlers-Danlos Syndrome-Like in Small Animals

Background: Ehlers-Danlos-Like Syndrome (EDS) is a rare disease in small animals, whose diagnosis is based on the clinical findings and histopathological examination. The definitive diagnosis may require transmission electron microscopy. Despite this, the ultrastructural changes are poorly described in the literature. The aim of the present study is to describe the ultrastructural findings of collagen fibers and fibroblasts present in the dermis of two animals with EDS, and to evaluate the digital measurement of the diameter of the collagen fibers by transmission electron microscopic images.Cases: Two animals were evaluated with EDS by transmission electron microscopy. The first animal was an 1-year-old mixed breed female cat, due to spontaneous skin laceration, increased skin elasticity and an extensibility index corresponding to 25%. The second animal evaluated was a 5-month-old Golden Retriever female dog due to articular hypermobility, increased skin elasticity and an extensibility index corresponding to 16.6%. After a skin biopsy of the interscapular and lumbar regions, the samples were fixed in formalin 10% and glutaraldehyde for, respectively, histopathological examination by HE staining, and transmission electron microscopy. The histopathology of the affected cat revealed collagen fibers shortened and sometimes fragmented. The histopathology of the affected dog revealed disarranged and more eosinophilic staining collagen fibers. The collagen fibers were also of unequal sizes, shortened and slightly undulate. At the transmission electron microscopy of the affected cat was evidenced a greater spacing of the collagen fibers of variable diameters. Further this, the fibroblasts showed elongated nuclei with heterochromatic regions, which was surrounded externally by scant cytoplasm. The cytoplasm showed elongated and discrete organelles. At the transmission electron microscopy of the affected dog was evidenced a greater spacing of the collagen fibers of variable diameters. Further this, fibroblasts exhibited intense cytoplasmic vacuolization with similar appearance to that found in the dying process by autophagy. In addition, the images obtained by transmission electron microscopy were submitted to digital analysis to measure the diameter of the collagen fibers using the software Image J. For this purpose, it was obtained the average of the diameter of 10 collagen fibers in cross-section into four quadrants of 1μm each. The digital analysis of collagen fibers revealed significant alterations in the ultrastructure of collagen. In addition, it was verified cellular changes, such as the large amount of intracytoplasmic vesicles and the small amount of collagen fibers dispersed around the cells.Discussion: Microscopic abnormalities visualized by HE staining in this present study were compatible with the literature Transmission electron microscopy is fundamental to confirm the suspicion, since it revealed dermal alterations in the ultrastructure of collagen and fibroblasts. These findings indicate possible failure mechanisms of secretion and release of cellular products, as well as collagen. The digital measurement of the diameter of the collagen fibers contributed to the confirmation of the disease, since it was made randomly and reduced the subjectivity inherent of the evaluator. However, there are no studies using this method to allocate a range of significance for the diameter of collagen fibers that may be considered suggestive for the syndrome in small animals.

Value of electron microscopy in the pathological diagnosis of native kidney biopsies in children.

Pediatric native kidney diseases are common worldwide. The pathological diagnosis of kidney lesions is crucial for clinical treatment and prognosis. The aim of the current study was therefore to evaluate the value of electron microscopy (EM) to the final diagnosis of native kidney biopsies in children. A retrospective evaluation of 855 pediatric kidney biopsies obtained from the Department of Pediatrics in Peking University First Hospital between November 2010 and December 2017 was performed to assess the contribution of EM to the final diagnosis. The role of EM in the final diagnosis was determined to be crucial in 300 cases (35.1%), important in 280 cases (32.7%), and auxiliary in 275 cases (32.2%). EM is considered most valuable in a large percentage of glomerular diseases, mainly including minimal change disease, early-stage membranous nephropathy, postinfectious glomerulonephritis, Alport syndrome, thin basement membrane nephropathy, and thrombotic microangiopathy. EM also provided helpful diagnostic information in cases of focal segmental glomerulosclerosis, lupus nephritis, IgA nephropathy, and IgA vasculitis (Henoch-Schonlein purpura nephritis). Additionally, EM was crucial in 90.0% of cases of subtle pathological changes observed with light microscopy (LM) and immunofluorescence (IF) and in 69.3% of the IF-negative specimens. Patients with nephrotic syndrome or hematuria also benefit from ultrastructural examination. The present study demonstrated the crucial or important role of EM in the diagnosis of a majority of native kidney biopsies in children. The application of EM should be integrated together with LM and IF as a routine method of assessing pediatric kidney specimens. Graphical abstract.

Scraping cytology and scanning electron microscopy in diagnosis and therapy of corneal ulcer by mycobacterium infection

Scraping cytology and scanning electron microscopy in diagnosis and therapy of corneal ulcer by mycobacterium infection

A light and scanning electron microscopic diagnosis of leaf epidermal morphology and its systematic implications in Dryopteridaceae: Investigating 12 Pakistani taxa

Dryopteris and Polystichum are the 2 complex taxonomic genera of Dryopteridaceae. The comparative foliar epidermal anatomy of 12 species of both genera from Pakistan were studied using standard protocols of light microscopy (LM) and scanning electron microscopy (SEM). The objective of which was systematic comparison and investigation to elucidate the taxonomic importance of foliar micromorphology, which may be useful to taxonomists for identifying complex Dryopteridaceae taxa. Principal component analysis and UPGMA clustering analysis were performed to test the validity of leaf anatomical features as method of separating species and genera. The foliar epidermal anatomy described here is a good source of taxonomic characters in both groups that can help genera and species delimitation. This is the first report on leaf micromorphology in most of these species. Observation of foliar anatomy showed that stomata are only present on the abaxial surface; i.e., leaves of all species are hypostomatic. The shapes of epidermal cells in all studied species are irregular. The anticlinal walls are strongly lobed, irregular wavy and elongated wavy. However, substantial variation in epidermal cell size and other stomatal features were observed on both upper and lower surfaces in all investigated species. Two types of stomata were observed in all studied species. The presence of polocytic stomata in Dryopteris and staurocytic stomata in Polystichum are the important characters for the segregation of these genera. Elongate elliptic stomatal shape, narrow kidney shaped guard cells and broad elliptic shaped stomatal pores are diagnostic for all five species of Dryopteris selected. On the other hand, size and number of epidermal cells, lobes per cell, stomatal size, subsidiary cell size, stomatal pore size and stomatal index are the key features for species differentiation in Polystichum. An identification key was developed in order to apply the foliar anatomical characters in the discrimination of the species studied.

Inherited epidermolysis bullosa

To summarize an update on epidermolysis bullosa as a polymorphic group of inherited diseases with a failure of epidermal-dermal integrity. Emphasis is placed on the role of transmission electron microscopy in diagnosis and search directions for new types of the abnormality and its molecular markers. Despite numerous mutations in the genes encoding the components of desmosomes and epithelial basement membrane, the stereotyped manifestations of pathological processes in the group of epidermolysis bullosa have been identified. The paper gives a positive result of cell and gene therapies used by European scientists in the treatment of a 7-year-old child with borderline epidermolysis bullosa, which opens up new prospects for patients with butterfly disease that has long been considered fatal.

Morphological features of congenital dyserythropoietic anemia type I: The role of electron microscopy in diagnosis.

Congenital dyserythropoietic anemias are rare blood disorders characterized by congenital anemia and a wide range of morphological and functional abnormalities of erythroid precursors. To analyze the relative frequency of both light microscopic (LM) and electron microscopic (EM) morphological features of erythroblasts in a large group of patients with molecular proven congenital dyserythropoietic anemia type I (CDAI). We retrospectively evaluated the LM and EM of bone marrow (BM) erythroblasts in 35 patients with CDAI. Thirty-four patients carried the CDAN1 Arg1042Trp founder mutation and one the p.Pro1130Leu mutation. BM slides of 24 patients were available for LM examination. EM studies were performed in all 35 patients. On LM, marked erythroid hyperplasia, binuclear erythroblasts, and various non-specific dyserythropoietic features were documented in every case; internuclear chromatin bridges were detected in 19 patients (79%). In all, EM of erythroblasts revealed a spongy appearance of heterochromatin, a widening of nuclear pores, and invagination of cytoplasm into the nuclear region. EM studies revealed high morphological frequency of specific ultrastructural changes in erythroblasts which facilitate prompt diagnosis of CDAI. Due to low specificity of BM LM findings, when BM EM is unavailable diagnostic approach should also include other inherited anemias.

Pathology of Podocytopathies Causing Nephrotic Syndrome in Children.

Nephrotic syndrome (NS) in children includes a diverse group of diseases that range from genetic diseases without any immunological defects to causes that are primarily due to immunological effects. Recent advances in molecular and genomic studies have resulted in a plethora of genetic defects that have been localized to the podocyte, the basic structure that is instrumental in normal filtration process. Although the disease can manifest from birth and into adulthood, the primary focus of this review would be to describe the novel genes and pathology of primary podocyte defects that cause NS in children. This review will restrict itself to the pathology of congenital NS, minimal change disease (MCD), and its variants and focal segmental glomerulosclerosis (FSGS). The two major types of congenital NS are Finnish type characterized by dilated sausage shaped tubules morphologically and diffuse mesangial sclerosis characterized by glomerulosclerosis. MCD has usually normal appearing biopsy features on light microscopy and needs electron microscopy for diagnosis, whereas FSGS in contrast has classic segmental sclerosing lesions identified in different portions of the glomeruli and tubular atrophy. This review summarizes the pathological characteristics of these conditions and also delves into the various genetic defects that have been described as the cause of these primary podocytopathies. Other secondary causes of NS in children, such as membranoproliferative and membranous glomerulonephritis, will not be covered in this review.

PM 7/126 (1) Electron microscopy in diagnosis of plant viruses

Specific scopeThis Standard describes the use of transmission electron microscopy (TEM) for the detection of virus particles in plant sap.Specific approval and amendmentApproved as an EPPO Standard in 2015‐09.

原発性線毛運動不全症―最近の進歩

Primary ciliary dyskinesia (PCD) is a hereditary disorder with structural and/or functional impairment of the cilia of the whole body. PCD is characterized by chronic sinusitis, bronchiectasis, male infertility and ectopic pregnancy due to impaired ciliary motility. The prevalence of PCD is estimated at 1 in 20,000 live births. Cases with situs inversus are termed “Kartagener’s syndrome” and usually these diagnoses are not difficult to make. However, in cases without situs inversus, the diagnosis can be very difficult. As such, it is most likely that PCD without situs inversus is underdiagnosed at the present time. The diagnosis of PCD is made when the patients have characteristic clinical features and (1) structural abnormalities of the cilia observed with electron microscopy and/or (2) presence of mutations in one of the genes associated with PCD. Electron microscopic diagnosis is not easy because ultrastructural ciliary defects can also be found in a few cilia among the healthy populations. Measuring levels of nasal nitric oxide (NO) concentration is a very useful tool in screening for PCD, because low NO values are a feature of PCD. Hand-held devices using tidal breathing are as useful as stationary devices. The sensitivity and specificity of nasal NO levels of <250 ppb for diagnosing PCD were 97% and 90%, respectively. Some other features can help to diagnose PCD. Frontal sinuses and sphenoid sinuses are hypoplastic. Having a combination of rhinosinusitis, middle ear and lung diseases increases the probability of PCD. The effectiveness of endonasal sinus surgery to treat rhinosinusitis associated with PCD is controversial.

Glomerular filtration barrier in pediatric idiopathic nephrotic syndrome

Nephrotic syndrome (NS) is a common proteinuric disorder with defect in the perm-selectivity of the glomerular filtration barrier (GFB). Ultrastructural morphometric evaluation of the GFB in pediatric NS has been attempted in only a few studies. This study was aimed at qualitative and quantitative evaluation of the alterations involving the GFB in pediatric idiopathic NS with an attempt to correlate these alterations with the clinico-laboratory data. For this study, renal biopsies from nine patients with NS and two children with interstitial nephritis were included. Relevant clinical and laboratory data, including degree of 24-h proteinuria and renal function tests, were recorded. Renal biopsies were reviewed for morphologic and electron microscopic diagnosis. Ultrastructural morphometry of the GFB was performed using image analysis software. The age at onset of NS, duration of illness, presence of hypertension, and renal function tests were comparable between the group of patients with minimal change disease (MCD) and those with mesangioproliferative glomerulonephritis (mesPGN)/focal segmental glomerulosclerosis (FSGS). However, the latter group showed higher 24-h proteinuria compared with the group with MCD. Among the detected ultra-structural changes, glomerular basement membrane thickness and foot process width were significantly different between the MCD and the mesPGN/FSGS groups. The slit pore diameter in the glomeruli showed a positive correlation with the degree of proteinuria. We conclude that our study demonstrated remarkable differences in certain parameters and the glomerular ultrastructural alterations in the various categories of NS. These differences might underlie the observed variation in response of these entities to various therapies.

Ultrastructural Aspects of Enterocyte Defects in Infancy and Childhood

Although there has been substantial progress in the identification of diarrheal diseases in infancy and childhood, electron microscopy may be still required for establishing diagnosis, staging, and response to therapy. This review describes severe conditions in which histopathologic examination alone cannot provide a firm diagnosis needed for therapeutic decisions. Microvillus inclusion disease, in its several variants, typifies this category. In certain forms of congenital disorders of glycosylation with gastrointestinal involvement, electron microscopic diagnosis is helpful. Among disorders due to abnormal immune-mediated reactions, celiac disease and cow's milk protein intolerance show fine structural changes of both diagnostic and staging value. Likewise, protein-losing enteropathies, including lymphangectasia, reveal information on the nature and extent of intestinal involvement.

Cytochemical, immunophenotypic and ultrastructural characterization of acute leukemias: A prospective study of fifty cases

Cytochemistry and immunophenotyping are established methods in the diagnosis of most leukemias but the role of electron microscopy in diagnosis, apart from understanding the cellular morphology is less studied. We present here 50 cases of acute leukemias that were studied for morphology, conventional cytochemistry, immunophenotyping and transmission electron microscopy (TEM), including ultrastructural cytochemistry using myeloperoxidase (MPO) and platelet peroxidase activity. TEM morphology using ultrastructural cytochemistry helped in definitive typing in one mixed lineage leukemia case, one AML-M5b, one AML-M6b and one microgranular variant of APML. Thus, ultrastructural studies may be useful in accurate diagnosis of biphenotypic leukemia and further classification of acute leukemias. Also, in cases with hypercellular marrow and with associated myelofibrosis, where the marrow aspirate gives low cell count, ultrastructural studies are a valuable aid to arriving at an accurate diagnosis.

Use of Transmission Electron Microscopy in Diagnosis of Acute Leukemias: A Prospective Study of Fifty Cases.

Ultrastructural studies have contributed substantially to the understanding of the cellular morphology of the acute leukemias. We present here 50 cases (aged 3-55 years, M: F-32: 18) of acute leukemias, which were studied for morphology, conventional cyto chemistry, immunophenotyping, and transmission electron microscopy (TEM) including ultrastructural cytochemistry using myeloperoxidase (MPO) and platelet peroxidase activity. TEM morphology using ultrastructural cytochemistry of MPO helped diagnose three cases of acute myeloid leukemia with minimal myeloid differentiation (AML M0). The blasts showed large round nuclei, 1–2 nucleoli, chromatin with peripheral condensation and abundant mitochondria. Of total of 5 cases of acute promyelocytic leukemias (APML), all had strong Sudan Black, MPO, dual esterase positivity; one case was non-specific esterase positive and sensitive to fluoride. On TEM, this unusual case was identified to be microgranular variant of APML. TEM morphology and ultrastructural cytochemistry using platelet peroxidase helped diagnose 3 cases of AML M7 (acute megakaryocytic leukemia), 2 cases of acute biphenotypic leukemias and also in differentiating one case of acute proerythroblastic leukemia (AML M6b) from 3 cases of AML M6a or acute erythroleukemia. Thus, TEM is helpful in differentiating further the subgroups of AML-M5 and AML-M6, in identifying the microgranular variant of APML, and in confirming the diagnosis of AML-M0 and biphenotypic leukemia. Also, in cases with very hypercellular marrow and with associated myelofibrosis, where the bone marrow aspiration gives low cell count, TEM and ultrastructural cytochemistry are a valuable aid to arriving at a accurate diagnosis.Characteristics of acute leukemia cases (n=50)FAB SubtypeMorphology, Conventional Cytochemistry and ImmunophenotypingTransmission Electron MicroscopyAML-M03 (?)3AML-M1and M21111AML-M34, 1(?)5: Hypergranular-4, hypogranular-1AML-M455AML-M555a-4, 5b-1AML-M646a-3 (erythroleukemia), 6b-1AML-M733Biphenotypic2 (?)2ALL-L188ALL-L244(?) = doubt in diagnosis

Ten-year experience using a plastic, disposable curette for the diagnosis of primary ciliary dyskinesia

Primary ciliary dyskinesia (PCD) results in impaired mucociliary clearance. Patients with this disorder develop chronic sinopulmonary disease with recurrent sinusitis, otitis media, nasal polyposis, pneumonia, and, ultimately, bronchiectasis. Other associated findings of dysfunctional ciliary activity include situs inversus, dextrocardia, and infertility. To describe our 10-year experience using a small, plastic, disposable curette to perform a screening procedure for cilia function and to collect samples for electron microscopy. In the past 10 years, we screened infants and children with severe chronic sinusitis and other chronic recurrent upper respiratory tract problems for PCD by using a plastic, disposable curette to collect tissue samples from the nasal mucosa. Samples were placed in sterile saline and examined under light microscopy for the presence of cilia. Failure to note ciliary movement prompted another examination 1 month later. If no functional cilia were noted at the follow-up examination, a specimen was obtained and sent for electron microscopy. We identified 7 patients with PCD; 2 had situs inversus totalis. Average age at diagnosis was 3 years. The most common symptom at presentation was frequent upper respiratory tract infections with severe otitis media (7 patients) and sinusitis (5 patients). Recurrent pneumonia was present in 6 patients. Dynein arm deficiency was the most common electron microscopic diagnosis. Evaluating children for PCD by using a plastic, disposable curette is a relatively simple procedure that could be used by allergists in practice. Primary ciliary dyskinesia occurs frequently enough that physicians should consider it as part of the differential diagnosis in evaluating children with recurrent, severe sinopulmonary infections.

Immunohistochemistry and Electron Microscopy in Diagnosis of Neoplasms

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Light microscopic and electron microscopic diagnosis of gastrointestinal opportunistic infections in HIV-positive patients

The surgical pathologist is expected to recognise gastrointestinal opportunistic infections in biopsies from HIV-positive patients, and patients immunocompromised iatrogenically by cancer therapy, steroid treatment and transplantation immunosuppression regimes. This review article presents the diagnostic features in gastrointestinal biopsies of microsporidia, cyclospora, isospora, cryptosporidia, mycobacteria, adenovirus, enteropathogenic bacteria, cryptococcus and leishmania. All of these infections have been diagnosed at our hospital in Sydney, Australia, since the AIDS epidemic began. A protocol for the examination and assessment of these gastrointestinal biopsies is presented and discussed.