Newer developments in immunohistology.

The development of sensitive reagents and detection systems, together with the introduction of heat-induced antigen retrieval, has rapidly entrenched immunohistology as an indispensable adjunct to routine histological examination, contributing to diagnosis, prognosis and treatment. New antibodies continue to be produced and new applications for "old" antibodies are described. The production of antibodies enabling the detection of genetic abnormalities, including mutations, gene amplifications and specific chromosomal translocations associated with novel chimeric proteins, promises to yield further insights into the genesis and behaviour of tumours. The ability to stain for target molecules that regulate tumour growth and proliferation is essential for selecting tumours for treatment with monoclonal antibodies. The mechanism of antigen retrieval remains debated. The absence of optimal controls continues to hinder standardisation of immunostaining and invalidates current attempts at quantification of immunostaining.

FAT, E-cadherin, beta catenin, HER 2/neu, Ki67 immuno-expression, and histological grade in intrahepatic cholangiocarcinoma.

AIM: To identify surrogate prognostic markers in intrahepatic cholangiocarcinoma (IHCC). METHODS: Thirty one cases of IHCC were graded and immunostained for FAT, Ki67, E-cadherin, beta catenin, and HER 2/neu. RESULTS: Twenty two cases were high grade and 27 had high Ki67 counts. Strong membranous staining of HER 2/neu was found in 10 tumours and reduced membranous E-cadherin and beta catenin in 19 and 18 tumours, respectively. Nuclear localisation of beta catenin was identified in five tumours and 22 showed weak cytoplasmic staining of FAT. Strong HER 2/neu and weak FAT immuno-expression were significantly correlated with high histological grade (p=0.01) and high Ki67 index (p=0.03). Upregulation of HER 2/neu was also significantly associated with nuclear localisation of beta catenin (p=0.01). Reduced membranous beta catenin was significantly related to reduced membranous E-cadherin (p=0.03), weak staining for FAT (p=0.01), and nuclear translocation of beta catenin (p=0.04). CONCLUSIONS: Reduced immuno-expression of E-cadherin and FAT at their normal membranous location may be potential prognostic markers, and the overexpression of HER 2/neu and beta catenin nuclear translocation may have a role in cholangiocarcinogenesis.

Nesidioblastosis, islet cell hyperplasia, and adenomatosis in a case of metastasizing insulinoma: contribution to the genesis of the islets of Langerhans.

An unusual case of insulinoma is described in a 40-yr-old woman whose resected pancreas showed multiple adenomas with lymph node metastasis, associated hyperplasia of islets, and nesidioblastosis. The association of nesidioblastosis with this continuum of proliferative changes adds further evidence for a ductular origin of islet cell tumors.

Dual staining of lymphocyte membrane antigens with colloidal gold and biotinylated horseradish peroxidase.

Double immunoenzymatic labelling procedures for the localization of antigens on cells in tissue sections using horseradish peroxidase (HRP) and alkaline phosphatase have been described previously, but mainly for detecting antigens on different cells. With this type of staining when two antigens are present on the same cell, an optimal colour combination that shows a high contrast between the basic colour of each enzyme substrate product is difficult to achieve and the interpretation of their mixed colour intermediate is subjective. We present a method for the simultaneous demonstration of two antigens on the same cell. The method can be used to label either single cells in suspension, or cells in paraffin fixed tissue, using a combination of a particulate label, colloidal immunogold-silver, and an enzymatic label HRP-DAB. The method is easy to perform and utilises commercially available staining kits.

Malignant transformation in a thymic cyst.

Thymic cysts have not been previously reported to show malignant transformation. This is the first documented case of well-differentiated papillary squamous cell carcinoma arising in a preexisting thymic cyst. The tumor was treated by surgical excision with no evidence of metastasis 6 1/2 years later.

Preferential epidermotropism in adult t-cell leukemia-lymphoma.

Three patients with systemic T-cell leukemia-lymphoma presented with generalized pruritic skin rash which was clinically nondiagnostic. Biopsies revealed infiltration of the superficial dermis by atypical lymphoid cells with epidermal exocytosis (Pautrier abscesses) producing a pattern of histologic involvement indistinguishable from that of mycosis fungoides and Sézary syndrome. This striking similarity suggests that the phenomenon of preferential epidermotropism is not confined to mycosis fungoides and Sézary syndrome, but may be a property shared by other subpopulations of T-lymphocytes.

Nucleolar organizer regions relate to growth fractions in human breast carcinoma.

An argyrophil stain for nucleolar organizer regions (NORs) has recently been applied to paraffin sections of human tissues. This report describes a positive relationship between the mean numbers of AgNOR sites per nucleus and tumor growth fraction, as determined by immunostaining with the monoclonal antibody Ki-67, in 83 malignant breast tumors (P less than .01). This relationship supports recent suggestions that the NOR count may reflect cell synthetic activity and hence, proliferation. AgNOR counts correlated inversely with immunocytochemically assessed estrogen receptor content (P less than .002), but there was no relationship between the AgNOR count and primary tumor size, histologic grade, axillary node status, or patient age. A significant difference (P less than .00001) was found between the AgNOR counts in 64 benign breast lesions (mean, 2.05) and 85 malignant breast neoplasms (mean, 5.46). The limitations of the silver staining technique and the problems of reproducibility in AgNOR counting are detailed.

Silver staining of nucleolar organizer regions in malignant melanoma and melanotic nevi.

The recently described method of staining nucleolar organizer regions (NOR) with colloidal silver nitrate was applied to the paraffin sections of five junctional nevi, 13 compound nevi, seven Spitz nevi, nine cellular blue nevi, 11 dysplastic nevi, seven malignant lentigines, 12 superficial spreading melanomas, and 14 secondary melanomas. There was a significant difference between the pooled silver-NOR (AgNOR) numbers of the 45 benign lesions (mean, 1.22; SD, 0.51) and the 33 melanomas (mean, 9.18; SD, 4.05) by t test analysis (P less than .01). The difference was striking enough to be recognized on casual microscopic examination, suggesting that AgNOR staining may be a useful technique to help separate melanocytic nevi from malignant melanomas.

The mucosal changes and pathogenesis of pneumatosis cystoides intestinalis.

The clinical and pathologic findings in 11 patients with pneumatosis cystoides intestinalis (PCI) are described. Symptoms were variable but localized to the gastrointestinal tract. Only one patient had obstructive airway disease. Although gas cysts of varying sizes were present in the colonic wall in all cases, distinctive changes were also observed in the mucosa. Architectural disturbances were seen in 11 of 13 specimens, and changes mimicking those of inflammatory bowel disease, including cryptitis, crypt abscesses, chronic inflammation, and granulomas, were present. In addition, crypt dilation and partial crypt rupture, with formation of intramucosal cysts, were seen in association with clusters of small gas cysts in the lamina propria. The transgression of gas cysts through the muscularis mucosae and the presence of larger giant cell-lined cysts in the submucosa suggested entry of gas generated in the colonic lumen or within inflamed crypts from the mucosal aspect. Other postulated pathogeneses of PCI are discussed.

Granulomatous mediastinitis due to rhizopus species.

Phycomycosis is seen most frequently in association with states of immunologic disturbance or hormonal imbalance. Several characteristic sites of infection with Phycomycetes are recognized, but mediastinal involvement has not been previously reported. A case of mediastinal granuloma due to Rhizopus species is described, and comparisons are made with other infections of similar histologic appearance.

Biologic markers in ductal carcinoma in situ and concurrent infiltrating carcinoma. A comparison of eight contemporary grading systems.

The relevance of 8 contemporary classification and grading systems for ductal carcinoma in situ (DCIS) of the breast was examined in 100 tumors by comparing DCIS grade with grade of the concurrent infiltrating ductal carcinoma (IDC). Besides tumor size and nodal status, the immunohistochemical parameters in both lesions were compared, including estrogen receptor, progesterone receptor, c-erbB-2 protein, E-cadherin, vimentin, Ki-67 (MIB1), and p27. Nuclear grading of DCIS alone or in combination with architectural pattern and necrosis showed the best correlation with grade of the invasive component. There also was a positive correlation between every biologic marker expressed in DCIS and in the concurrent IDC, supporting a clonal relationship. Biologic markers varied between the different grades of DCIS. DCIS is heterogeneous, and the progression of DCIS to IDC may be from low-grade DCIS to low-grade IDC and high-grade DCIS to high-grade IDC. This concept is different from the conventional model held for intraepithelial neoplasia in the cervix, vulva, vagina, and skin, in which there is increasing severity of in situ atypia (dysplasia) before the development of stromal invasion.

Effect of desiccation on the ultrastructural appearances of Acinetobacter baumannii and Acinetobacter lwoffii.

An Acinetobacter baumannii isolate survived desiccation beyond 30 days and an Acinetobacter lwoffii isolate up to 21 days. For both species, desiccation resulted in a significant increase in the proportion of round cells (A baumannii, 40% to 80%; A lwoffii, 51% to 63%) and a significant decrease in rod shaped cells (A baumannii, 58% to 13%; A lwoffii, 46% to 34%). Electronmicroscopic examination showed that there was also a corresponding significant increase in the cell wall thickness (A baumannii, up to 53%; A lwoffii, up to 26%). Desiccated A baumannii cells became more electron-dense and had significantly thicker cell walls (x1.3) than those of A lwoffii. Cell wall structures of A baumannii strains with different abilities to resist desiccation deserve further study.

Brugia malayi in a cervical smear.

A microfilaria, identified as that of Brugia malayi, was found in a routine cervical smear from a healthy, 38-year-old female Malaysian aborigine.

Vasculitis of peripheral nerve and skeletal muscle: clinicopathological correlation and immunopathic mechanisms.

In order to elucidate the pathogenesis of vasculitis in muscle and nerve a clinicopathological study was performed in 23 patients. The patients could be classified into three groups: I multisystem vasculitis (n = 4); II multisystem disease with vasculitis detectable in nerve, muscle, or both (n = 11); and III localised disease with vasculitis in nerve, muscle, or both (n = 8). Ten of 23 patients had vasculitis in both muscle and nerve biopsies; 6 from groups I and II, and 4 from group III indicating that vasculitis confined to muscle and nerve represents a distinct clinicopathological entity. The neuromuscular manifestations in the 23 patients were sensori-motor polyneuropathy (n = 9), mononeuritis multiplex (n = 6), mononeuropathy (n = 3), proximal myopathy (n = 4), and muscle tenderness without weakness (n = 1). Sensori-motor polyneuropathy was found in 8/11 patients from group II, and mononeuritis multiplex in 5/8 patients from group III. Immunotypic analysis, and immunofluorescence patterns indicate that the pathogenesis of vasculitis in muscle and nerve is heterogeneous and suggest that cell mediated and immune complex mechanisms predominate and may be operative in all three patient groups, with an antibody-mediated humoral mechanism in some patients with paraproteinaemic states (group II).

Marchiafava-Bignami disease in a non-alcoholic Indian male.

Marchiafava-Bignami disease, a rare affliction of alcoholic males, is described in a severely malnourished Malaysian Indian male who took no alcohol. It is the second report of the disease in an Asian and represents one of the few cases which have occurred in non-alcoholics. Besides the pathognomonic demyelination of the central portion of the corpus callosum, there were striking demyelinative plaques in the subcortical white matter. In addition, neuropathological features of Wernicke's disease were found suggesting that severe malnutrition with thiamine deficiency was probably the cause of his demise.

Segmental biliary ectasia and congenital hepatic fibrosis in a patient with chromosomal abnormality.

An unusal case of intrahepatic biliary ectasia and congenital hepatic fibrosis confined to one segment of the liver is described in a young woman who died from a ruptured splenic artery aneurysm in the third trimester of pregnancy. The possible mechanisms for production of the severe portal hypertension in this patient and the association of an aneurysm in the splenic artery are discussed. The patients karyotype was 45, XX, -D, -D +t (13q 15q).

A critique of some contemporary classifications of non-Hodgkin's lymphoma. Which one should we now use?

Major contemporary classifications of non-Hodgkin's lymphoma are discussed and compared with the recently released Working Formulation prepared by an international panel of expert pathologists. The Working Formulation was constructed according to prognosis and represents a compromise of existing terminologies. It has many weaknesses and appears to be unsuitable for use by pathologists as the sole classification for non-Hodgkin's lymphoma; however, it serves as an important means of translation between existing classifications. As all the major systems have been shown to have prognostic relevance, it is suggested that we continue to use the classification of our choice, preferably one which is familiar to our clinical colleagues, and that the terminology of the Working Formulation be employed as an alternative diagnosis in parentheses.

The relevance of ultrastructural examination in the classification of primary lung tumours.

Biopsies from 50 primary lung tumours were classified according to the World Health Organisation's Histological Typing of Lung Tumours. They were also subjected to electron microscopic examination. Comparison of the diagnoses made by these separate methods showed that many poorly differentiated squamous cell carcinomas had been incorrectly classified. Agreement was good in the diagnosis of adenocarcinoma whilst ultrastructural examination of small anaplastic carcinomas disclosed a neuroendocrine tumour with a combination of squamous and glandular elements. Large cell anaplastic carcinoma proved to be a 'waste-basket' containing tumours which displayed ultrastructural characteristics of poorly differentiated squamous cell carcinoma, poorly differentiated adenocarcinoma or neuroendocrine carcinoma. Electron microscopy was also valuable in characterization of other pulmonary tumours whose identity could not be resolved at the light microscopic level. Ultrastructural examination may provide a better understanding of the histogenesis and derivation of lung tumours, as well as their behaviour and therapeutic response.

Microwave fixation and rapid processing in a large throughput histopathology laboratory.

Technical details of a method of microwave fixation and rapid processing are described. Fresh biopsy specimens are sampled and trimmed into 2 mm thick blocks and irradiated to a temperature of 68 degrees C. They are then processed through cycles of absolute alcohol (75 min), xylene (50 min) and wax (50 min). It is possible, in this manner, to run several 'short' cycles of about 3 hrs each during the working day so that stained sections are available on the same day as receipt of the specimens. Endoscopic biopsies are processed through a shorter cycle comprising 30 min of absolute alcohol (4 changes), 20 min of xylene (3 changes) and 20 min of wax (2 changes). For convenience we also employ an overnight cycle whereby microwave-fixed blocks are processed through several changes of absolute alcohol, xylene and wax without the use of formalin. This method of processing not only removes the use of noxious and potentially toxic formalin but also allows rapid preparation of good quality diagnostic sections with superior antigen preservation compared to formaldehyde fixation.

Antibody probes in the diagnosis of anaplastic tumours. I. Malignant round cell tumours.

The introduction of hybridoma technology has rapidly expanded the scope of immunohistological analysis in the identification of anaplastic malignant tumours. High affinity antibodies now allow the detection of a wide range of tissue-specific antigens so that anaplastic tumours can be accurately classified without the need for costly and often unrewarding, time-consuming ancillary investigations such as electron microscopy, cell culture and chromosomal studies. This review examines the application of a panel of commercially available antibodies of proven specificity, chosen to allow separation of the various entities considered in the differential diagnosis of anaplastic round cell tumours. The specifications of the antibodies are discussed and details of selected cases are provided to illustrate the role of such tissue-specific antibodies as diagnostic probes.