Apoptosis is a major cause of so-called "caseous necrosis" in mycobacterial granulomas in HIV-infected patients.

AIM: To demonstrate that so-called "caseous necrosis" is the result of apoptosis and investigate the association of B and T cells, and macrophages with the granulomas and their relationship to some apoptosis-related proteins. METHODS: Cervical lymph node biopsy specimens from 55 HIV-infected Thai patients with caseating granulomas, confluent caseating granulomas, sarcoid-like granulomas, foamy macrophage response, pseudo-inflammatory tumour response or non-specific lymphoid hyperplasia were examined histologically and for apoptosis by immunostaining for caspase 3 and TUNEL. Classic tuberculoid caseating granulomas in cervical lymph node and lungs from non-HIV-infected patients were also stained with caspase 3. RESULTS: All areas of caseous necrosis frequently displayed extensive apoptosis that readily accounted for the so-called "necrosis". Small foci of apoptosis were present in the other reaction patterns and fibrotic granulomas often showed residual apoptosis. The extent of apoptosis was inversely related to the numbers of identifiable acid-fast bacilli; all epithelioid macrophages revealed strong immunoexpression of the pro-apoptotic proteins Bax and Fas, whereas the anti-apoptotic protein Bcl-2 was not present. Apoptosis occurred in CD68+ macrophages and CD3+ CD8+ T cells; all nodes were deficient of CD4+ cells. CD8+ T cells were intimately related to the apoptotic foci, suggesting a role in the process, particularly in the absence of CD4+ cells. In non-HIV-infected cases, similar extensive apoptosis was confirmed with caspase 3. CONCLUSIONS: So-called "caseous necrosis" is shown for the first time to be the result of apoptosis. In the absence of CD4+ cells the findings negate many of the postulated mechanisms of apoptosis in the murine model and have implications for the treatment of mycobacterial infections.

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.

Digital photography in anatomical pathology.

Digital imaging has made major inroads into the routine practice of anatomical pathology and replaces photographic prints and Kodachromes for reporting and conference purposes. More advanced systems coupled to computers allow greater versatility and speed of turnaround as well as lower costs of incorporating macroscopic and microscopic pictures into pathology reports and publications. Digital images allow transmission to remote sites via the Internet for consultation, quality assurance and educational purposes, and can be stored on and disseminated by CD-ROM. Total slide digitisation is now a reality and will replace glass slides to a large extent. Three-dimensional images of gross specimens can be assembled and posted on websites for interactive educational programmes. There are also applications in research, allowing more objective and automated quantitation of a variety of morphological and immunohistological parameters. Early reports indicate that medical vision systems are a reality and can provide for automated computer-generated histopathological diagnosis and quality assurance.

Significance and assessment of margin status in ductal carcinoma in situ of the breast.

This article reviews practical and theoretical aspects of margin assessment for surgically excised ductal carcinoma in situ of the breast. Different methods of assessing surgical margins are discussed, including selected tangential sections, the margin shaving, and cavity peel methods. Criteria for margin adequacy and the relevance of margin status in the selection of cases for breast conservation are discussed in the context of other important risk factors for local failure.

Synoptic/checklist reporting of breast biopsies: has the time come?

Narrative descriptive reporting has been the traditional format employed in surgical pathology for almost as long as its inception as a specialty. While the descriptive prose has served us well in the past, its accuracy and readability is variable. Descriptions of color, shape, and texture are often subjective. Surgical pathologists are trained observers, but there are inherent differences in reporting style, and descriptive prowess depends on language skills and vocabulary. These differences are reflected in reports generated by pathologists in the same laboratory and may even be more evident in reports from different laboratories and across nations using the English language. The reproducibility of morphologic descriptions is thus a matter of some concern.

Capsaicin-evoked CGRP release from rat buccal mucosa: development of a model system for studying trigeminal mechanisms of neurogenic inflammation.

Many of the physiological hallmarks associated with neurogenic inflammatory processes in cutaneous tissues are similarly present within orofacial structures. Such attributes include the dependence upon capsaicin-sensitive sensory neurons and the involvement of certain inflammatory mediators derived therein, including calcitonin gene-related peptide (CGRP). However, there are also important differences between the trigeminal and spinal nervous systems, and the potential contributions of neurogenic processes to inflammatory disease within the trigeminal system have yet to be fully elucidated. We present here a model system that affords the ability to study mechanisms regulating the efferent functions of peptidergic terminals that may subserve neurogenic inflammation within the oral cavity. Freshly dissected buccal mucosa tissue from adult, male, Sprague-Dawley rats was placed into chambers and superfused with oxygenated, Krebs buffer. Serial aliquots of the egressing superfusate were acquired and analysed by radioimmunoassay for immunoreactive CGRP (iCGRP). Addition of the selective excitotoxin, capsaicin (10-300 microm), to the superfusion buffer resulted in a significant, concentration-dependent increase in superfusate levels of iCGRP. Similarly, release of iCGRP from the buccal mucosa could also be evoked by a depolarizing concentration of potassium chloride (50 mm) or by the calcium ionophore A23187 (1 microm). The specific, capsaicin receptor antagonist, capsazepine (300 microm), completely abolished the capsaicin-evoked release of iCGRP while having no effect whatsoever on the potassium-evoked release. Moreover, capsaicin-evoked release was dependent upon the presence of extracellular calcium ions and was significantly, though incompletely, attenuated by neonatal capsaicin denervation. Collectively, these data indicate that the evoked neurosecretion of iCGRP in response to capsaicin occurs via a vanilloid receptor-mediated, exocytotic mechanism. The model system described here should greatly facilitate future investigations designed to identify and characterize the stimuli that regulate the release of CGRP or other neurosecretory substances in isolated tissues. This system may also be used to elucidate the role of these mediators in the aetiology of inflammatory processes within the trigeminal field of innervation.

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.

The prognostic dilemma of nodal micrometastases in breast carcinoma.

The presence of axillary lymph node metastasis in patients with breast cancer is a major prognostic factor and also determines the use of adjuvant chemotherapy. Micrometastasis has been arbitrary defined as deposits of < 2 mm dimension. Earlier studies of micrometastases failed to demonstrate prognostic relevance. However, when larger numbers of patients were followed up for longer periods, micrometastasis was shown to be a significantly poor prognostic parameter with patients having a survival rate similar to those with macrometastasis or nodal disease. There are no compelling reasons to retain the term "micrometastasis" in the light of these findings and our understanding of tumor biology. Routine histological examination of axillary lymph nodes is a notoriously inaccurate method for the detection of metastases. When serial or multilevel sectioning and/or immunohistochemical staining for cytokeratin were employed, detection rates increased by as much as 33%. Reverse transcriptase-polymerase chain reaction and Southern blotting for CK19 may be a more accurate method of examination. However, there are inherent technical problems associated with this method, and the recent finding of a pseudogene with great homology to CK19 in normal peripheral blood nucleated cells further emphasises the need for caution in this approach. It is not cost-effective to employ serial sectioning and immunohistochemistry when examining the axillary contents. However, the introduction of sentinel-node biopsy may allow detailed examination of the single node most likely to harbour a metastatic tumor.

An advanced digital image-capture computer system for gross specimens: a substitute for gross description.

The description of macroscopic appearances of surgically excised specimens together with the sites of specimen sampling form an important component in the documentation of a histopathology specimen. Unfortunately, accuracy of description depends on the vocabulary and descriptive prowess of the pathologist which means that the result can be variable. Transcription of the dictated word also takes time and involves typists. We developed a user-friendly, high-resolution image capture system that will supplement word description of macroscopic specimens and has the potential to replace it completely. It also provides the opportunity of revisiting traditional methods of documenting specimens with words, allowing the production of more relevant and user-friendly reports containing information relevant to clinical management and supplemented by high-resolution digital images. The accompanying Windows-based software has capabilities of generating the entire histopathology report and allows rapid retrospective searches through any one of several common search parameters. The stored images form a powerful database for teaching and research. Connection to remote transmission facilities allows teleconferencing and telepathology consultations.

Proliferating ductules are a diagnostic discriminator for intrahepatic cholangiocarcinoma in FNA biopsies.

The histologic distinction of cholangiocarcinoma (CC) from metastatic carcinoma and hepatocellular carcinoma (HCC) is difficult. In particular, the distinction of CC from metastatic carcinoma on morphologic features alone is not possible and is dependent on the identification of an extrahepatic primary carcinoma. The proliferative response to many types of liver injury is characterized by a proliferation of either hepatocyte ductular clusters (HDC) or biliary ductular clusters (BDC). This study examined the presence of such ductular reactions in fine needle aspiration biopsies of 20 consecutive cases each of CC and HCC, and compared the findings to those of 20 cases of hepatic metastases from a wide variety of sites. All 18 cases of CC with adequate smears showed ductular proliferation of either HDC or BDC type, the latter being more common; in 13 cases, there were more than 10 ductular clusters per smear. In contrast, only one case of metastatic carcinoma displayed so many ductular clusters, this being a case with multiple hepatic deposits. Five cases of HCC showed more than 10 clusters. The presence of more than 10 ductular clusters associated with malignant cells is a useful discriminator to separate CC from metastatic carcinoma.

Immunolocalization of the fodrin, E-cadherin, and beta-catenin adhesion complex in infiltrating ductal carcinoma of the breast-comparison with an in vitro model.

Fodrin, E-cadherin, and beta-catenin immunolocalization was studied in 54 cases of infiltrating ductal carcinoma of the breast and compared with an in vitro model in order to study the dynamic relationship between these components of an adhesion complex. In low-grade tumours, the staining patterns were similar for both fodrin and E-cadherin, with localization of these proteins to the cell membranes. beta-Catenin showed reduced membrane staining compared with non-neoplastic epithelium. High-grade tumours displayed strong membranous as well as cytoplasmic immunolocalization of fodrin, while E-cadherin staining was fragmented or lost from the membranes, with only occasional weak intracellular staining. beta-Catenin showed fragmented membrane staining and cytoplasmic accumulation. In addition, nuclear staining of beta-catenin was occasionally observed. In a v-src-transformed MDCK cell line, following 15min of src activation, beta-catenin began to detach from the cell membrane and localize to the cytoplasm, while fodrin and E-cadherin remained unchanged. After 30-45min of src activation, the cells lost their cuboidal shape and began to lose cell-to-cell contact. Fodrin staining remained mostly membranous while that of E-cadherin and beta-catenin was fragmented and spiky. After 60min of src activation, fodrin localized completely in the cell cytoplasm, while E-cadherin and beta-catenin were partly cytoplasmic with fragmented and spiky membranous staining. Occasionally, beta-catenin was seen in the nucleus. Both in vivo and in vitro findings clearly demonstrated a disruption of the E-cadherin/beta-catenin/fodrin/cytoskeleton linkage concomitant with the loss of cell-to-cell adhesion and change in cell shape, from epithelioid to a fibroblastoid phenotype. Membranous localization of E-cadherin showed a positive correlation with oestrogen and progesterone expression, whereas loss of membranous E-cadherin and cytoplasmic accumulation of fodrin was more often observed in high-grade carcinomas and showed a positive correlation with p53 expression.

Microwave procedures for electron microscopy and resin-embedded sections.

Microwaves now have well-established applications in routine light microscopy. They are employed in tissue fixation and to accelerate a wide spectrum of staining procedures. Besides producing superior preservation of cellular antigens through microwave fixation, this form of irradiation has been employed for antigen retrieval, a procedure that has been a major factor in the optimization of immunolabelling in paraffin sections and cytological preparations. A commercial tissue processor has recently been developed which employs microwaves in a markedly accelerated, one-step processing of tissue blocks, completing the procedure within a fraction of conventional times. Microwaves have also been successfully applied in a variety of molecular techniques such as in situ hybridization and polymerase chain reaction. The adoption of microwaves in electron microscopic procedures has been slower, largely because the requirement for speed in processing is not as great, except in diagnostic samples. However, as this review will show, there are equally as many innovative applications of microwaves in electron microscopy. Microwaves have been employed for rapid processing of fine needle aspiration biopsy samples, in keeping with the requirement for speed in this method of diagnosis. Ultrafast fixation of tissue samples has resulted in the better demonstration of cellular enzymes and proteins. It has been clearly shown that microwave-stimulated on grid staining in uranyl acetate and lead citrate produces more consistent results and without background precipitation. Microwaves can be used to hasten resin polymerization and exposure to microwaves results in antigen retrieval in both resin-embedded thick sections and for immuno-electron microscopy. Immunolabelling shows enhanced sensitivity and the technique is anticipated to contribute greatly to the optimization of immuno-electron microscopy. The potential for greatly accelerated preparation of samples for electron microscopy exists but is yet to be fully realized.

Hep Par 1 and selected antibodies in the immunohistological distinction of hepatocellular carcinoma from cholangiocarcinoma, combined tumours and metastatic carcinoma.

AIMS: To examine the usefulness of Hep Par 1 together with selected antibodies in the separation of hepatocellular carcinoma (HCC) from cholangiocarcinoma (CC), combined tumours (HCC-CC) and metastatic carcinoma. METHODS AND RESULTS: Antibodies to Hep Par 1, CK19, CK20 and factor XIIIa were applied to 32 HCCs, 27 CCs, five HCC-CCs and 19 metastatic carcinomas from a variety of sites. Hep Par 1 produced distinctive granular staining of all benign hepatocytes and stained 30 HCCs in a heterogeneous manner, irrespective of the degree of differentiation. While labelling all cases of combined HCC-CC, the antibody also stained the mucus-secreting cells of four cases of pure CC. Anti-CK19 produced distinctive staining of bile ducts and CC but also decorated four HCCs and 10 metastatic tumours. Factor XIIIa was not found in normal, reactive or neoplastic hepatocytes. CK20 was found in some cases of HCC and CC and in all cases of metastatic carcinomas from the colon. CONCLUSIONS: Hep Par 1 was a sensitive marker of hepatocytes but its variable staining in HCC may produce false negative results in small biopsies and it was occasionally found in CC. The highest diagnostic yield was obtained when anti-Hep Par 1, CK19 and CK20 were used in a panel. Factor XIIIa staining has no role in the diagnosis of liver cancers.

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.

A simple index to predict prognosis independent of axillary node information in breast cancer.

BACKGROUND: Since the course of breast cancer is often unpredictable, we wished to develop a model using characteristics of the primary tumour alone to predict prognosis. METHODS: Several tumour features were determined, and after a median follow-up duration of 65 months, multivariate analysis identified tumour size and grade, oestrogen receptor concentration, axillary lymph node metastasis and tumour cell proliferation fraction (MIB-1 count) as being independently associated with increases in risk for both relapse and death from breast cancer. A prognostic model was constructed using tumour size and grade, oestrogen receptor concentration and MIB-1 count only. A score of 1 for each was given to tumour size > 20 mm, tumour grade 2 or 3, oestrogen receptor concentration < 10 fmol/mg cytosol protein and MIB-1 count > 9%. Five groups established by assigning a combined score of 0, 1, 2, 3 or 4 for each patient were analysed for their associations with disease-free and overall survivals. RESULTS: This preliminary model predicted 5-year survival rates of 97, 91, 85, 68 and 50% for the five groups. The model was further simplified by excluding tumour grade from the analysis. The revised model identified four risk groups with predicted 5-year survival rates of 91, 86, 66 and 52%. This model, the Adelaide prognostic index, was also able to identify four risk groups in both node-negative and node-positive patients. CONCLUSIONS: The Adelaide prognostic index can be used to predict prognosis even in the absence of axillary lymph node information.

Immunostaining of estrogen receptor, progesterone receptor, MIB1 antigen, and c-erbB-2 oncoprotein in cytologic specimens: a simplified method with formalin fixation.

An effective but simple fixation protocol for the immunocytochemical staining of cytologic smears for estrogen and progesterone receptors, the Ki-67 antigen (using MIB1 antibody), and c-erbB-2 protein is described. One hundred twenty-seven smears from a variety of malignant and benign breast lesions showed good preservation of antigenicity when subjected to the following fixation protocol: Freshly made smears were air-dried for 20 min to 14 h at 22 degrees C before immersing in 10% buffered formalin for 2-14 h. Immunostaining followed microwave-stimulated epitope retrieval. There was strong concordance of staining with corresponding tissue sections in 15 cases of malignant tumors (ER: r = 0.7381; PR: r = 0.6684; MIB1: r = 0.7234). Immunostaining staining, when delayed for 5-10 days in about half the smears, showed no noticeable difference in reactivity, attesting to effective storage of the formalin-fixed smears at room temperature.

Microwaves in diagnostic immunohistochemistry.

Immunostaining has been shown to be clearly superior in tissues immersed in normal saline and fixed by primary exposure to microwaves (MWs) as compared to that fixed routinely in 10% buffered formalin. MWs have also been applied to dry cryostat sections and to accelerate antibody-antigen reactions in the staining of labile lymphocyte membrane antigens in such sections. MWs can also be employed to accelerate immunostaining in paraffin sections, producing significant reduction in all stages of the staining procedure. The more recent application of MWs for epitope retrieval has made an important step towards not only producing better immunostaining of routinely fixed tissues but also towards the standardisation of the immunostaining process. MW-irradiation of paraffin-embedded sections in 10 mMol citrate buffer solution produced, with few exceptions, increased intensity and extent of immunostaining of a wide variety of tissue antigens. Moreover, proteolytic enzyme digestion was not necessary in most instances and some primary antibodies could be used at higher working dilutions.