John W. Stirling

Histopathological and ultrastructural features of dermal telangiectasias in systemic sclerosis

Aims: To investigate the histological, ultrastructural and immunohistochemical features of the vascular lining of dermal telangiectasia, a characteristic clinical finding in scleroderma. Methods: Standard histological, electron microscopic and immunohistological techniques were used to examine dermal telangiectasias in five patients with limited scleroderma, the most common scleroderma variant in Caucasian populations. Results: The telangiectasias were dilated postcapillary venules located in the papillary and superficial reticular dermis. The vessel walls consisted of non‐fenestrated endothelial cells surrounded by a variable number of pericytes and smooth muscle cells. There were no unique ultrastructural features. Thickened collagen fibres in the reticular or deep dermis were seen in all but one patient, although in variable and generally minimal quantities. Surrounding infiltrating inflammatory cells were scarce. No enhanced endothelial staining was obtained with antibodies directed against endoglin, endothelin, E‐selectin and ICAM‐1 suggesting a resting or inactivated state. Conclusion: The immunohistological and ultrastructural features of the lining endothelium of established telangiectasias in long‐standing, limited scleroderma appear benign. It would be of interest to examine telangiectasias in the early phase of their formation. Alternatively, other explanations need to be explored in understanding the aetiopathogenesis of telangiectasia in scleroderma.

Paraproteinemic Crystalloidal Keratopathy: An Ultrastructural Study of Two Cases, Including Immunoelectron Microscopy

The ultrastructural appearances of corneal crystalloidal deposits are described in two patients with an IgG-kappa paraproteinemia of uncertain pathogenesis. The crystalloids in one patient were overwhelmingly intracellular and were found mainly in stromal keratocytes, but also in basal corneal epithelial cells and the limbal vascular endothelium. Four types of crystalloid or immunoprotein-containing granules were recognizable in this case: 1) fibrillary crystalloids with a curvilinear filamentous substructure; 2) angulated geometric crystalloids that often had a linear filamentous substructure and transverse or oblique periodicity; 3) cordlike crystalloids; and 4) lysosomelike granules with amorphous contents. Immunoelectron microscopy demonstrated that all of these structures labeled for kappa-light chains, and rectangular type 2 crystalloids showed approximately a twofold greater concentration of the colloidal gold probe than the type 1 fibrillary crystalloids. The evidence suggested development of the crystalloids within lysosomes, with a progression from the granules containing amorphous material, through fibrillary crystalloids, to the geometric structures. The circumferential distribution of the corneal deposits, as well as the presence of vascular endothelial crystalloids and reduplication of external laminae around limbal blood vessels, suggests that the crystalloids originated predominantly or entirely from the blood, with transport of immunoprotein across damaged limbal microvasculature. The abnormal vasculature may also have contributed to corneal edema, which in turn may have exacerbated corneal opacification. The crystalloidal deposits in the other case were exclusively extracellular; they were located beneath and between corneal basal epithelial cells, and predominantly as a mantle around individual keratocytes. The crystalloids in this case consisted overwhelmingly of thick-walled tubules about 40 nm in diameter that labeled for both kappa-light chains and gamma chains with the colloidal gold immunoprobe. In addition, lucent vesicles within keratocytes were found only in sections labeled for kappa-light chains and were positive. The factors that might contribute to the formation of corneal crystalloidal deposits in immunoproliferative disorders are discussed, and include: 1) an inherent propensity for crystallization of some immunoglobulins or kappa-light chains, perhaps because of abnormal molecular structure; and 2) local factors in the cornea that might promote deposition and crystallization of immunoprotein, such as temperature, pH, the water content, and extracellular matrix components.

A novel GATA1 mutation (Stop414Arg) in a family with the rare X-linked blood group Lu(a-b-) phenotype and mild macrothrombocytic thrombocytopenia.

116, 140–143. Raanani, P., Trakhtenbrot, L., Rechavi, G., Rosenthal, E., Avigdor, A., Brok-Simoni, F., Leiba, M., Amariglio, N., Nagler, A. & Ben-Bassat, I. (2005) Philadelphia-chromosome-positive Tlymphoblastic leukemia: acute leukemia or chronic myelogenous leukemia blastic crisis. Acta Haematologica, 113, 181–189. Vardiman, J.W., Thiele, J., Arber, D.A., Brunning, R.D., Borowitz, M.J., Porwit, A., Harris, N.L., Le Beau, M.M., Hellstrom-Lindberg, E., Tefferi, A. & Bloomfield, C.D. (2009) The 2008 revision of the world health organization (WHO) classification of myeloid neoplasms and acute leukemia: rationale and important changes. Blood, 114, 937–951.

Unfixed tissue for electron immunocytochemistry: a simple preparation method for colloidal gold localization of sensitive epitopes using ethanediol dehydration

SummaryA quick, simple protocol is described for the preparation of tissue for electron immunocytochemistry without the use of fixatives or deleterious solvents. Fresh, normal human colon was rapidly dehydrated in ethanediol (ethylene glycol) then embedded directly in low-acid glycol methacrylate. Using both mono-and polyclonal antibodies, in conjunction with colloidal gold probes, a range of intra-and extracellular epitopes were localized; these epitopes included lysozyme, chromogranin, desmin and collagen IV. Overall, the tissue compared well with material fixed in glutaraldehyde, partially dehydrated and embedded in LR White acrylic resin. Ultrastructural detail was good and was further enhanced, without affecting probe density and epitope localization, by the addition of 1% tannic acid or 1% uranyl acetate to the dehydrant. The technique is applicable to a wide range of tissues, allowing excellent antigen retention which might prove useful for the immunolocalization of sensitive epitopes.

Quality Standards for Diagnostic Electron Microscopy

Transmission electron microscopy (TEM) continues to play a critical role in diagnostics; [1] however, due to a combination of economic and staffing issues, diagnostic TEM is currently at a significant crossroads. In some countries rationalization of TEM facilities is resulting in laboratory closures and dedicated electron microscopists are retiring; both are processes that will result in a serious loss of skill, knowledge, and experience. Furthermore, the staffing profile of many units is changing; the scientific and technical staff responsible for diagnostic TEM are increasingly likely to be multiskilled rather than TEM specialists. In such an environment, attention to quality to maintain standards and ensure a rapid and reliable diagnostic service is essential. To ensure quality, two strategies are available. First, it is essential to underpin good practice with clear guidelines that define ‘‘quality standards’’ for laboratory operations (processes and outcomes). Second, it is advisable to participate in an external quality assurance program (EQAP) that audits laboratory processes and outputs; ideally the EQAP should also function as an educational tool. The second strategy has been addressed; two EQAPs for general diagnostic transmission electron microscopy (TEM) are currently available. The Royal College of Pathologists Australasia (RCPA), through the RCPA Quality Assurance Programs Pty Ltd (RCPA QAP), successfully initiated an internationally accredited (ILAC G13) program in 2006. Also, in the United Kingdom (UK) the Association of Clinical Electron Microscopists (ACEM) has initiated an informal quality assurance program for ACEM members. In fact, it is the first strategy that requires additional action. Quality control in the histopathology laboratory, much of which applies to TEM, has been described [2] and a range of activities that support quality practices in TEM facilities are in place. Some TEM laboratories have quality processes for viral screening and many facilities have standard operating procedures (SOPs). Indeed, in some countries (such as Australia and the UK) ‘‘controlled’’ manuals describing SOPs and a record of processing details for auditing purposes are mandatory for laboratory accreditation. Laboratory accreditation also requires the documentation of staff training, another process that supports quality practice when used to define the competence, range, and level of duties permitted for each member of staff. Finally, while a laboratory might not have ‘‘published’’ quality standards, it must be said Received 17 July 2007; accepted 2 July 2007.

Spindle and epithelioid (histiocytoid) haemangioendothelioma of cervical lymph nodes

Sir, Primary vascular tumours of lymph nodes are extremely rare with the exception of AIDS-related Kaposi’s sarcoma. Spindle and epithelioid haemangioendothelioma is a vascular lesion that is considered to be a variant of epithelioid haemangioendothelioma. The lesion is an extremely rare entity that has been reported to occur in inguinal lymph nodes and, in one case, in the spleen. Here, we report a case that arose in cervical lymph nodes. A 30-year-old previously well man presented in May 2000 with left cervical lymphadenopathy of uncertain duration. Physical examination showed mobile left cervical lymph nodes, the largest of which was 40 mm. No lesion was found in the head and neck region apart from the lymphadenopathy. An excisional biopsy was performed on the largest lymph node and a diagnosis of spindle and epithelioid haemangioendothelioma was made following detailed immunohistochemical work-up and review by experts in the field. Clinical and radiological staging only showed disease in the left neck. The patient was treated with radiotherapy, with resolution of the lymphadenopathy. No further treatment was given. The patient was last reviewed in October 2002 and was disease free. The gross specimen comprised a single lymph node measuring 40 mm in maximum dimension. The cut surface of the lymph node showed an eccentric firm and pink area involving approximately 75% of the cut surface, surrounded by a rim of grey-tan, apparently uninvolved, lymphoid tissue. Microscopically, the lymph node showed a discrete, expansile mass pushing the residual follicular architecture (Fig. 1 left inset). The nodular proliferation consisted predominantly of spindle cells disposed in intersecting fascicles and characterised by abundant eosinophilic cytoplasm, elongated nuclei with finely dispersed chromatin and conspicuous nucleoli. A second population of cells, interspersed amongst the spindle cells, had an epithelioid or histiocytoid appearance with oval nuclei, finely dispersed chromatin and prominent nucleoli (Fig. 1). Rare mitotic figures were noted (Fig. 1 right inset). Large numbers of eosinophils were present throughout the lesion. Focally, the stroma within the nodule was sclerotic with keloid-like collagen bands. Vacuolated cells were not observed, and vessel formation was not a feature of the tumour. Immunohistochemically, the only positive marker that stained both the epithelioid and the spindle components of the neoplasm was the vascular marker CD31, with more intense membranous, and to a lesser extent cytoplasmic, staining in the epithelioid tumour cells than the spindle cells (Fig. 2). Staining for AE1/AE3 and CAM 5.2, S100 and HMB45, CD34, Ulex europaeus, factor VIII, CD68, CD21, CD20, CD3, desmin, smooth muscle actin, and neurofilament proteins (NFP) was negative. Scattered cells were CD68 positive, most likely representing admixed histiocytes. Electron microscopy of reprocessed, formalin-fixed, wax-embedded tissue showed neoplastic cells arranged in small groups and surrounded by an incomplete basal Fig. 1 The spindle-shaped neoplastic cells form fascicles with interspersed eosinophils (H&E, original magnification, 6400). The neoplasm forms an eosinophilic mass that is well-delineated from surrounding residual nodal tissue (left inset, H&E, substage image). Mitotic figures are rare (right inset, H&E, original magnification, 6400).

Deep microsporidial keratitis after keratoconjunctivitis

[Extract] Microsporidia are intracellular pathogens of animals andhumans. Although not hyphal, a recent study suggests theyare fungi descended from an ancestral zygomycete. Theyhave a unique method for invading susceptible tissue: an internal coiled tube everts rapidly and sporoplasm moves into the cell as if through a hypodermic needle. In the human eye, microsporidial infection presents as keratoconjunctivitis or stromal keratitis. The former has been reported in HIV+ patients because of Encephalitozoon and in immunocompetent patients because of several genera. Stromal infection is seen in immunocompetent patients and is caused mostly by Vittaforma corneae. We report a case in which keratoconjunctivitis preceded deep stromal infection in a patient with a low neutrophil count.

Glomerular Basement Membrane Thinning in a Patient with Hematuria and Hemoptysis Mimicking Goodpasture's Syndrome

Ultrastructural morphometric studies of glomerular basement membrane (GBM) thickness are described in two renal biopsy specimens from a patient who presented with hemoptysis and hematuria mimicking Goodpastures syndrome. Significant GBM abnormality, with attenuation as the main lesion, identified in a biopsy specimen taken during active clinical disease appeared to have resolved in a second biopsy specimen taken during the recovery phase. There was no evidence of glomerulonephritis. Concurrent lung biopsy studies showed focal alveolar-capillary wall basal lamina changes of uncertain diagnostic significance. These observations suggest the alternative possibilities that GBM attenuation may be either an acquired consequence of systemic disease or may be part of an hitherto unrecognized primary multisystem abnormality of basal lamina affecting, in this case, glomerular and pulmonary laminae, resulting in hematuria and hemoptysis. The morphometric studies in this case indicate that simple-mean measurements of GBM thickness are inadequate alone for the quantitative study of this lamina because significant inter- and intraglomerular membrane variation, if irregularly distributed, can remain undetected.

Diagnostic Electron Microscopy - A Practical Guide to Interpretation and Technique: Stirling/Diagnostic Electron Microscopy - A Practical Guide to Interpretation and Technique

Diagnostic Electron Microscopy: A Practical Guide to Interpretation and Technique summarises the current interpretational applications of TEM in diagnostic pathology. This concise and accessible volume provides a working guide to the main, or most useful, applications of the technique including practical topics of concern to laboratory scientists, brief guides to traditional tissue and microbiological preparation techniques, microwave processing, digital imaging and measurement uncertainty.

Unusual Organelles in an Epithelioid Angiosarcoma

We recently encountered unusual organelles in a tumor from a 75-year-old white British female presenting with a scalp lesion which histologically was a poorly differentiated malignant neoplasm (Fig. 1). On the basis of histology and immunohistochemistry [positive staining for factor Vlll-related antigen (Fig. 2), JC70A (CD31),1 and Ulex europaeus agglutinin 1] the tumor was diagnosed as an epithelioid angiosarcoma. An unexpected finding was that some unambiguous tumor cells were positive for S100 protein. Occasional S100 protein-positive dendritic cells, interpreted as reactive Langerhans cells, were also present. To confirm a confidently held diagnosis, but one which was nevertheless uncertain because of the S100 protein staining, electron microscopy was conducted. Not unexpectedly, Weibel-Palade bodies were not identified,2 but the rough endoplasmic reticulum, polyribosomes, aggregates of haphazardly arranged intermediate filaments, and a well developed lamina were seen as being consistent with epitheli...