An inter-observer and intra-observer variability study on the diagnosis of lymph node biopsy specimens.

One hundred lymph node biopsy specimens were examined on two separate occasions by seven pathologists differing in experience in lymphoreticular pathology. Neither history nor immunohistochemistry was provided and the study, therefore, focused on morphological interpretation alone. The participants evaluated each case using a constructed response form in which the confidence with which they entered each response was also entered. Agreement on various points, between pathologists, between the two rounds, and with the referring centre was assessed. Whilst there was a high level of agreement over a diagnosis of benign vs. malignant and non-Hodgkin lymphoma vs. Hodgkin's disease, there was considerably less agreement over both T vs. B cell phenotype and high vs. low grade. The lack of agreement over grade, an evaluation which is usually made independent of immunohistochemistry, is particularly important, because of the relevance to selection of treatment. Proliferation markers may be more appropriate determinants of treatment choice.

Integrins and their extracellular-matrix ligands in gastric-cancer.

Extracellular matrix (ECM) proteins and their specific cellular receptors, play an important role in the regulation of epithelial morphogenesis and differentiation. Alterations in their expression and function have been found in a number of malignant tumours and these changes may help to explain their dedifferentiation and altered behaviour. In this study we have investigated expression and distribution of the epithelial beta 1 integrins (alpha 2 beta 1, alpha 3 beta 1 and alpha 6 beta 1) and their ECM ligands (fibronectin, tenascin and laminin) in normal and neoplastic tissue. An up-regulation of two isoforms of fibronectin, and tenascin was seen in tumour associated matrix compared to normal stroma. Loss or down regulation of alpha integrin chains was seen more frequently in poorly differentiated carcinomas (alpha 2 p=0.002; alpha 3 p=0.013; alpha 6 p=0.0012) irrespective of tumour type (diffuse or intestinal) than in well/moderately differentiated tumours. Cell adhesion assays revealed that the ability of gastric carcinoma cell lines to bind matrix glycoproteins correlated to their degree of differentiation. Furthermore, poorly differentiated cell lines showed a down-regulation of alpha 2 and alpha 6 integrin expression. These data indicate that architectural and cytological differentiation in gastric carcinoma relates to altered patterns of expression of matrix glycoproteins and their receptors. The traditional Lauren classification seems to reflect these differences in cell-matrix interactions. Differing patterns of expression of those molecules involved in cell-matrix interactions may prove to be a more objective and biologically more relevant means of classifying gastric cancer.

Fat necrosis presenting as obscure abdominal mass: birefringent saponified fatty acid crystalloids as a clue to diagnosis.

AIMS: To describe the birefringent saponified fatty acid crystalloids seen in pancreatic fat necrosis. METHODS: A histological review, including polarising microscopy, of three cases of subacute or subclinical acute pancreatitis was performed. Histochemical analysis using Nile blue sulphate for lipid, Holczinger's copper rubeanate for fatty acids, and Alizarin Red S for calcium was performed in one case. Scanning electron microscopy and x-ray energy dispersive spectroscopic micro-analysis were performed in two cases. Necropsy pancreatic tissue, surgical archival tissue from cases of pancreatitis, and pancreatic and adipose tissue permitted to autolyse together in the laboratory, were also examined. The autolysed tissue was also examined histochemically. Stained and unstained sections were mounted in DPX and Canada balsam. Surgical material showing traumatic fat necrosis was reviewed. RESULTS: In each of the three cases there were subtle clues to subclinical pancreatitis. In neither surgical case was the true nature of the mass apparent to the operator. Histological analysis in all cases showed ghost adipocytes containing numerous polarising crystalloids, as well as some basophilic debris. Microanalysis showed calcium but no other substantial heavy element signals. Histochemical analysis showed a labile, polar, acidic lipid and the crystalloids behaved as calcium salts of free fatty acid. The crystalloids were not seen in archival material mounted in Canada balsam. No crystalloids were seen in traumatic fat necrosis. CONCLUSIONS: Little recognised, strongly birefringent, saponified free fatty acid crystalloids occurring in pancreatic fat necrosis may survive routine processing, and can point to the origin of obscure mesenteric masses related to subclinical pancreatitis.

Pathology, histochemistry and immunocytochemistry of lesions in acute multiple sclerosis.

Twenty cases of acute or early multiple sclerosis have been examined using staining, histochemical or immunocytochemical methods. They had died within 6 months after initial clinical onset (12) or commencement of an "anatomically-remote" acute relapse (8). Plaques in these acute cases showed the following characteristics: lymphocytic perivascular infiltration, plaque hypercellularity, plaque macrophage infiltration and intra-macrophage myelin debris. In most cases of clinical duration of less than 12 weeks, some macrophages showed characteristic formaldehyde-resistant markers for haematogenous macrophages (muramidase, anti-alpha 11-antitrypsin, MAC and HAM56) but, with the exception of the last, these markers subsequently declined indicating a haematogenous origin for macrophages in the early lesion. Lymphocytes were prominent in perivascular (perivenous) regions but, except in one case, were only scanty in or at the demyelinating edge of plaques. Oligodendroglial hyperplasia, indicative of remyelinating activity, was seen at the edge of plaques in one quarter of these acute cases (7 times the rate seen in chronic lesions). Astrocytic activation was not apparent in the earliest stages but was usually seen from about 6 weeks onwards. The conclusion from these observations is that the prime inflammatory process is around blood vessels with usually only scanty initial inflammatory activity in the parenchyma of the brain. Macrophages emigrating from blood vessels digest myelin either as a response to inflammatory damage to the myelin or as a response to activation signals produced in either the perivascular region or plaque.

Inflammatory vasculitis in multiple sclerosis.

Fifty-two plaque or lesion areas were examined from 25 cases of multiple sclerosis. Twenty-four of these showed acute features, whereas the rest were more chronic in nature. The acute lesions showed lymphocytic infiltration (79%), fibrinous exudation (63%), lymphocytic meningitis (50%) and venulitis (58%). Of the chronic lesions, there were only 21% with lymphocytic infiltration, 11% with fibrinous exudates, none with meningitis, 29% with organising endovenulitis, 36% with fibrosed vein walls. The finding of a fibrinous inflammatory exudate in the acute lesion is a new observation in multiple sclerosis. Likewise, the observation of an inflammatory infiltrate confined to the vein wall (and often present at a distance from the plaque) has not been previously recorded in the disease. The chronic lesion, by contrast, showed relatively little fibrin, but there was considerable reparative thickening of the walls of the involved veins. The evidence provides new humoral and cellular evidence of an inflammatory process in multiple sclerosis which precedes or is not directly associated with the demyelinating process.

Simultaneous demonstration of connective tissue elastica and fibrin by a combined Verhoeff's elastic-Martius-scarlet-blue trichrome stain.

A method for the routine combined demonstration of elastica, connective tissue in general, and fibrin is described. Elastica, stained blue-black by Verhoeff's iron hematoxylin, is contrasted with muscle and collagen, stained red and blue or green respectively, by a modification of the Martius-scarlet-blue (MSB) trichrome for fibrin of Lendrum et al. The MSB technique selectively stains fresh, mature and old fibrin orange-yellow, red and blue respectively; the Masson trichrome does not distinguish between erythrocytes and fibrin. Nuclei are stained at the same time as the elastica. The technique takes approximately one and a half hours and is ideal for the study of connective tissue and vascular pathology, especially the necrotizing vasculitides.

Periodate oxidation of glycolipids: a borohydride-periodate-Schiff method for ganglioside demonstration in tissue sections.

Among lipids, gangliosides can be selectively stained with Schiff's reagent if the oxidizing agent (sodium metaperiodate) is sufficiently dilute to exclude all but the readily oxidized sialic acid sugars. A borohydride-periodate-Schiff (BhPS) sequence is recommended as a reliable method, convenient to perform, for the detection of the intraneuronal lipid accumulations in the ganglioside storage disorders.

Perls' ferrocyanide test for iron in the diagnosis of vasculitic neuropathy.

Preliminary observations suggested that arterial and arteriolar necrosis in vasculitis of the peripheral nerve leads to local haemorrhage and subsequent deposition of haemosiderin. This pigment is more readily recognized in the nerve by the sensitive Perls' test for iron than by relying on recognizing its yellow colour. To support the use of iron staining as an index of vasculitis, in addition to necrosis and fibrin deposition, we obtained the following results from nerve biopsy and autopsy nerve specimens: vasculitis confined to PNS = 5/6 iron positive; polyarteritis nodosa and Wegener's granulomatosis = 4/5 iron positive; systemic lupus erythematosus 2/3 iron positive; rheumatoid disease 1/1 iron positive; acute Guillain-Barré syndrome and subacute or chronic demyelinating polyradiculoneuropathy = 12/12 iron negative; and other perivascular inflammation in the PNS (without evidence of vasculitis) = 2/2 iron negative. One case of Churg-Strauss syndrome showed no changes on nerve biopsy. Iron staining was also demonstrated in the kidneys of five or six patients with polyarteritis nodosa.

Intraosseous secondary calcium salt crystal deposition: an artefact of acid decalcification.

We previously observed, in decalcificated bone specimens, intraosseous crystal deposits with morphological and analytical similarity to calcium pyrophosphate dihydrate. We have now been able, by a combination of more detailed morphological studies of these and similar cases, and by infrared spectroscopy in three cases, to show that this is, in fact deposition of the secondary calcium salts brushite and monetite, occurring as an artefact during formic acid decalcification. Our earlier postulate of bone as an additional primary crystallization site for calcium pyrophosphate dihydrate is effectively disproved. This artefact deserves wider recognition.