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Aims —To perform a toxicological analysis of deaths involving methadone and to determine the fatal concentration of methadone in such deaths. Methods— Deaths in which methadone was mentioned in the cause of death were identified. Deaths were divided into those associated with methadone only and deaths in which the cause of death was a combination of methadone and other drugs. Toxicological findings in these deaths were analysed and compared with previously published data. Results —One hundred and eleven cases were analysed. In 55 cases, methadone poisoning was given as the sole cause of death. Fifty victims were adults, age range 17–51 years (median, 23), with five victims under 14 years of age. The mean methadone concentration in the adult deaths was 584 μg/litre (median, 435; range, 84–2700). In 56 cases, age range 15–49 years, (median, 28), death was ascribed to a combination of methadone and other drugs. The mean methadone concentration in these deaths was 576 μg/litre (median, 294; range, 49–2440). In 26 cases, multiple site sampling was performed. This revealed that there could be a 100% discrepancy between methadone concentrations, and other drugs, in samples collected in different sites in the same body. Conclusions —There is an overlap between quoted therapeutic methadone concentrations and methadone concentrations seen in fatalities. However, those dying from methadone poisoning might not be the same as those in a methadone programme. A degree of caution must be exercised in determining a fatal concentration because of the phenomenon of postmortem redistribution. Pathologists and toxicologists need to examine all the available postmortem findings in identifying the cause of death.

Background —Increased dietary calcium might reduce colorectal cancer risk, possibly by reduction of colonic epithelial hyperproliferation, but not all studies have demonstrated this. Little is known about the effects of calcium on colonic apoptosis. Aim —To quantify the effects of increasing calcium on apoptosis and cell proliferation in normal murine colonic crypt epithelium. Methods —Twenty one day old male C57Bl/6 mice were fed either control AIN-76 diet (0.5% calcium wt/wt; n = 10) or the same supplemented with calcium carbonate (1.0% calcium; n = 10) for 12 weeks. Apoptotic cells in proximal and distal segments were counted and expressed as an apoptotic index (AI: frequency of apoptosis/100 longitudinal crypts). The bromodeoxyuridine (BrdU) labelling index was also determined. Differences were analysed by the student9s t test. Results —In control animals, the AI was significantly higher in the caecum/proximal colon (mean, 28.6; SEM, 2.0) compared with the distal colon (mean, 19.9; SEM, 1.8; p = 0.004). In the calcium treated group, the AI in the caecum/proximal colon (mean, 30.6; SEM, 1.7) was similar to controls (p = 0.71) but the AI in the distal colon was significantly greater (mean, 32.6; SEM, 1.8; p = 0.001) than in control mice and was raised to values similar to those in the proximal colon. Calcium was also associated with reduced crypt cellularity and, in the proximal colon, a downward shift in the crypt position at which apoptosis occurred. There were no significant differences in the BrdU labelling index between groups or between proximal and distal colonic segments in each group. Conclusions— Increased dietary calcium is associated with the induction of apoptosis in normal mouse distal colonic epithelium without affecting cell proliferation. This might contribute to its putative chemopreventive role in colorectal carcinogenesis. Whether this effect is direct or indirect requires further study.

Aims —To compare blood drug concentrations during life with postmortem drug concentrations measured from a peripheral site and a central site. Methods —Coroner9s cases from October 1990 to July 1997 were reviewed. Six cases had data on both antemortem and postmortem blood drug concentrations. The postmortem to antemortem ratio was compared with the postmortem central to peripheral ratio, using cardiac blood as a central site and femoral blood as a peripheral site. Results —Drugs that have a high postmortem central to peripheral ratio; that is, drugs that exhibit considerable postmortem redistribution, also have high postmortem to antemortem ratios. Conclusions —A large degree of error can arise from attempting to estimate antemortem drug concentrations and the ingested dose from postmortem measurements. The chosen site and technique for postmortem blood sampling can greatly influence the concentration of drug measured.

Aims—To investigate the sensitivity of immunohistochemical (IHC) assays for oestrogen receptors (ER) and progesterone receptors (PR) achieved by laboratories on breast tumours fixed and processed in their own department, and to compare this with the degree of sensitivity they achieve on tumours circulated as part of an external quality assessment (EQA) programme. Methods—On 10 occasions between April 1994 and June 1998, histological sections from breast cancers showing various degrees of expression of ER and PR were circulated for IHC staining to laboratories participating in the UK national external quality assessment scheme for immunocytochemistry (UK NEQAS-ICC). The staining of these tumours, in addition to that of tumours fixed and processed in the participants own laboratories (in house tumours), was assessed by a panel of four assessors, using the established UK NEQAS-ICC scoring system. For a selected assessment run, the degree of expression of participants in house tumours was evaluated by means of the semiquantitative quick score method. Results—Although the scores awarded for the staining of in house tumours were generally higher than those awarded for the staining of UK NEQAS tumours, there was also a significant positive correlation between the two sets of scores. Using the quick score method of evaluation for one of the assessment runs, 47% of in house tumours were classified as having a high degree of ER expression. Of the remaining cases, a significant proportion initially classified as having only low or medium expression of ER were found to have higher expression when stained by the organising laboratory. The UK NEQAS-ICC centre's routine assay for hormonal receptors was found to be 90–100% efficient in achieving optimal demonstration of breast tumours from over 150 different laboratories. Conclusions—The significant positive correlation between the results obtained on the UK NEQAS tumours and the in house tumours provides evidence for the view that results achieved on EQA material are accurate indicators of in house laboratory performance. Although most laboratories adequately detected tumours with high receptor expression, a large proportion of in house tumours classified initially by participants' staining as being of low or medium ER expression had a higher degree of expression when stained by the UK NEQAS-ICC centre. The efficiency of the organising centre's routine IHC method for ER and PR in optimally demonstrating participants in house breast tumours shows that variations in fixation and tissue preparation are not limiting factors preventing a different laboratory achieving optimal demonstration. Key Words: immunohistochemistry • oestrogen receptors • progesterone receptors • external quality assessment

Aim —The status of vaginal lactobacillary flora, an indicator of possible genital infection and pregnancy complications, can be assessed on wet mount or Gram stained specimens. The former is quick, the latter more routine. The accuracy of the two preparative techniques to detect normal vaginal lactobacillary microflora was compared for 646 patients. The effect of delay in transport medium before Gram staining was also investigated. Methods —Patients presented with infectious vaginitis or for a routine prenatal visit. After placement of a speculum, duplicate smears were taken from the upper vaginal vault and examined fresh or after Gram staining. Lactobacillary grades from both methods were compared with lactate concentration in vaginal rinses. In a subgroup of 238 patients, Gram staining was performed both on fresh smears and those that had been transported in Stuart's growth medium. Results —Higher lactobacillary grades (more disrupted flora) were diagnosed 2.9 times more frequently on Gram stained specimens than on wet mounts (p < 0.0001), a difference even more pronounced after transport in Stuart's medium (relative risk, 4.2; p < 0.0001). Lactobacillary grades assessed on wet mounts correlated better with vaginal lactate concentration than those assessed on Gram stains. Conclusions —Easier recognition of lactobacillary morphotypes on wet mounts than on Gram stains might result from the loss of lactobacilli by the process of fixation or Gram staining. Wet mount microscopy of vaginal smears for assessment of lactobacillary grades, rather than Gram staining, is strongly recommended.

To investigate the clinicopathological differences among gastric low grade MALT lymphomas (low MALT), large B cell lymphomas with low grade components (secondary high grade MALT lymphomas, high MALT), and diffuse large B cell lymphomas without low grade features (primary high grade MALT lymphomas, DLL).Clinicopathological and morphological characters of 126 gastric lymphoma cases were studied: 82 cases of low MALT lymphoma including 40 that were surgically resected, 17 cases of high MALT lymphoma including 13 surgically resected, and 27 cases of DLL including 12 surgically resected.Age ranges were as follows: low MALT lymphoma, 34 to 85 years (mean 59.9); high MALT lymphoma, 53 to 88 years (mean 68.5); DLL, 29 to 83 years (mean 62.3). The average age for low and high MALT lymphomas was significantly different (p0.05), but there were no differences in other comparisons. There was a female predominance of low MALT lymphoma patients (female to male ratio, 47/35), while for high MALT patients the ratio was almost even (8/9), and for DLL patients there was a male predominance (11/16). Examination of surgically resected material showed that MALT lymphomas had a wider distribution in the gastric wall than DLL.The findings suggest that at least some of the high grade gastric lymphomas, especially in patients younger than the fifth decade, do not originate from high grade transformation of low MALT lymphomas. It seems to take about one decade at least for high grade transformation of low MALT lymphomas.

Aims—To assess the relation between the grade and the status of follow up cytology, the completeness of loop excision biopsies with cervical intraepithelial neoplasia (CIN), and the findings at follow up cytology, as well as the differences between complete and incomplete exclusion, using the odds ratio. Treatment failure was assessed. Methods—1600 women with CIN (290 CIN1, 304 CIN2, 1006 CIN3) were followed for a minimum of six months and a maximum of 10 years. A database was created and comparisons performed. The mean age of the patients was 37 years. Results—Excision was complete in over 84% of loops. Residual disease and recurrence of high grade dyskaryosis was more common in women with CIN 3 than CIN 2 or 1. No high grade dyskaryosis was seen in the fifth follow up smear in patients with CIN 1 and CIN 2. Residual, recurrent, and persistent disease was most common in patients with incompletely excised CIN at ectocervical and endocervical margins and deep margins of resection than in patients with completely excised CIN. The odds ratios were significantly higher in the women who had incomplete excision of CIN at ectocervical, endocervical, both ecto- and endocervical, and deep margins of resection compared with those with apparent complete excision of CIN lesions. One patient developed invasive squamous cell carcinoma 44 months after loop excision which showed CIN 3 invading endocervical crypts and extending to both ectocervical and endocervical margins of resection. Conclusions—At long term follow up, patients with CIN who have residual disease are at increased risk of persistent disease and should therefore be followed up regularly with cytology and colposcopy. The findings support national policy of returning women with treated CIN of any grade to normal recall after five years except for cases of CIN3 where excision was incomplete or equivocal. In these cases follow up with annual smear for 10 years is recommended. Key Words: cervical cytology • cervical intraepithelial neoplasia

Aims—To assess the expression of SIX5 (a homeobox gene) mRNA in surface coelomic epithelium, endocervical epithelium, Fallopian tube epithelium, and benign, borderline, and malignant epithelial ovarian tumours. Methods—10 normal premenopausal ovaries, 10 normal Fallopian tubes, 10 normal cervices, 10 normal postmenopausal ovaries, 10 benign epithelial ovarian tumours, 10 malignant epithelial ovarian tumours, and 40 borderline epithelial ovarian tumours were studied retrospectively. The tissues had been fixed in formalin and embedded in paraffin wax. The tumours had previously been typed into mucinous, serous, or mixed tumours and assigned to the borderline category according to the FIGO/WHO criteria. Expression was assessed by in situ binding of SIX5 specific sense and antisense riboprobes. Hybridisation of the riboprobes was detected using a standard immunohistochemical technique and the results correlated with expression in the normal epithelium of the endocervix, Fallopian tube, surface coelomic epithelium, and ovarian tumours. Results—Expression of SIX5 mRNA was demonstrated in normal Fallopian tube epithelium and normal endocervical epithelium. SIX5 mRNA was not detected in normal ovarian epithelial tissue at any of the times studied during the menstrual cycle. Expression of SIX5 was not shown in benign epithelial ovarian tumours or in any of the malignant epithelial ovarian tumours. In 31 of 37 borderline epithelial ovarian tumours (84%), SIX5 expression was found in the epithelial cells. Conclusions—SIX5 expression is present in the normal epithelium throughout most of the female reproductive tract, suggesting it may have a role in maintaining epithelial differentiation in these tissues. SIX5 expression appears to be restricted to borderline epithelial ovarian tumours and may be a marker of epithelial differentiation in these tumours; thus borderline ovarian tumours may not be part of a continuum of disease between benign and malignant epithelial ovarian tumours. Further investigation of expression of SIX5 may clarify the molecular processes that promote differentiation of the ovarian surface epithelium. Key Words: ovarian epithelial tumours • SIX5 • homeobox genes

Background—Apoptosis, or programmed cell death, can be induced by radiotherapy. The extent of apoptosis in a tumour before treatment may have important implications for response to radiotherapy and long term survival. Aim—To examine the extent of apoptosis in tumour tissue from patients with squamous carcinoma of the cervix before radiotherapy, and to correlate this with response to treatment and prognosis. Methods—The percentage of apoptotic cells was assessed in 146 carcinomas of the cervix from patients scheduled to receive radiotherapy. The CAS 200 static image analysis system was used to count the number of tumour nuclei per high power field, while the numbers of apoptotic cells in the same field were visualised simultaneously on the image analyser and recorded manually. Results—The median apoptotic level was 0.73%. Patients were divided into two groups around the median. There was no statistically significant difference in outcome between the two groups as determined by long term survival following radiotherapy. Conclusions—The CAS 200 static image analyser system can be used to assist in the rapid semiautomated assessment of apoptosis in conventionally prepared tissue. The results suggest that the apoptotic state of a tumour before treatment is of no value in predicting response to radiotherapy and subsequent prognosis. Tumour stage, size, and BrdU labelling index, as a measure of proliferation rate, remain the most important prognostic factors in terms of predicting local tumour control. Key Words: apoptosis • uterine cervix • squamous cell carcinoma

Aim—To investigate the correlation between immunohistochemical and biochemical steroid receptor analyses by measurement of oestrogen, progesterone, and androgen receptor status in ovarian neoplasia. Methods—Tissue samples were obtained from 27 ovarian neoplasms, including two borderline tumours. Immunohistochemical staining of the tissue slides was scored semiquantitatively, incorporating the intensity and percentage of positive staining (histoscore). Tumours with a histoscore of 10 or more were considered steroid receptor positive. The epithelial and stromal fractions of the tumours were analysed separately. To study the uniformity of receptor expression throughout a tumour, up to four samples were analysed. Results—Immunohistochemical histoscores of the oestrogen receptor in the epithelial fractions were significantly correlated with the biochemical oestrogen receptor values (r = 0.408). Androgen receptor status in the epithelial fraction was correlated with that in the stromal fraction (r = 0.741), while androgen receptor histoscores in the epithelial fraction correlated with the biochemical assay values (r = 0.463). On biochemical analysis, 17 of the 27 ovarian tumours were oestrogen receptor positive and seven were progesterone receptor positive. On immunohistochemical analysis, eight tumours were oestrogen receptor positive and two were progesterone receptor positive. Biochemical analysis showed that 14 of the 26 tumours were slightly androgen receptor positive (10–50 fmol/mg protein), while all the others were negative. On immunohistochemical analysis, seven of the 26 tumours were androgen receptor positive. When two or more specimens from one tumour were analysed, marked differences in steroid status were found, especially in progesterone receptor and androgen receptor expression. Some parts of a tumour were steroid receptor positive, while other parts were negative owing to heterogeneity of expression. Conclusions—Immunohistochemical and biochemical analysis of steroid receptors in ovarian tumours correlated weakly or not at all. Heterogeneity of expression within a tumour and the presence of progesterone and androgen receptors in the stromal fraction partly accounted for this observation. Biochemical and immunohistochemical androgen receptor status was much lower than in previous reports. Key Words: steroid receptors • ovarian neoplasia • immunohistochemical analysis • biochemical analysis

Aim—To study the distribution of Hodgkin's lymphoma in South African children and report the incidence of Epstein-Barr virus (EBV) as regards age, race, sex, and histological subtype; to investigate whether EBV is relevant to survival. Methods—Immunohistochemistry (IHC) and in situ hybridisation (ISH) to detect EBV were performed on 47 South African children with classical Hodgkin's lymphoma, ranging in age from 3 to 14 years and coming from different ethnic backgrounds. The correlation between the presence of the virus and clinical outcome was assessed. Results—The nodular sclerosing subtype predominated, comprising 89% of cases; the remaining 11% were of the mixed cellularity subtype. EBV was present in 68%. Full clinical data were available for 36 cases; EBV positive patients presented with less aggressive symptoms at diagnosis and had a significantly longer median survival than EBV negative patients. Conclusions—The distribution of EBV in South African childhood Hodgkin's lymphoma follows a pattern intermediate to that of industrialised and non-industrialised countries. Furthermore, our data suggest that there is an association between poor prognosis and the non-detection of EBV products in South African childhood Hodgkin's lymphoma. Key Words: children • Epstein-Barr virus • Hodgkin's lymphoma

Aims—To analyse the cost-effectiveness of three strategies for examining temporal artery biopsies based on data from cases examined over the past 10 years. Methods—Of a total of 172 temporal artery biopsies, five were unsuitable for further analysis, 47 had already had levels cut, and 120 had levels cut as part of the study. All the biopsies were examined blind before and after levels. A tree with eventual diagnostic outcomes for different strategies was constructed and economic and sensitivity analyses performed. Welcan units were used to assess technical workload. Results—Only one of the 132 initially normal cases and two of 14 diagnosed with periarterial lymphocytic infiltration (PALI) revealed giant cell arteritis after examining the tissue at multiple levels. Fifteen cases (8.9%) showed PALI not previously observed. The marginal cost for each extra case of giant cell arteritis detected was 83.5 Welcan units for a strategy of routine levels on all sections, and 21 Welcan units for a strategy of only cutting levels if PALI was present on the initial section. These costs were sensitive to the frequency of giant cell arteritis in cases with PALI and to the relative extra cost of moving from cutting single section to routine levels. Conclusions—Routinely examining a temporal artery biopsy at multiple levels does not increase the diagnostic yield of the test, although selective further examination may be indicated in some cases. The significance of PALI is uncertain. The cost-benefit of the different strategies in terms of clinical decision making revolve around the perceived risk inherent in not making a diagnosis of giant cell arteritis. Key Words: temporal artery biopsy • economic analysis • decision analysis

Background—MUC5AC is a secreted mucin aberrantly expressed by polypoid colorectal adenomas. It has been hypothesised that the “normal” surrounding colorectal mucosa expresses MUC5AC as a field change phenomenon that can be used to predict adenoma recurrence following resection. Aim—To determine if there is a field change of de novo MUC5AC expression in histologically normal rectal mucosa adjacent to villous and tubulovillous adenomas, and thus whether MUC5AC expression can be used as a marker of early tumour recurrence. Methods—In a prospective cohort study paired mucosal biopsies of adenomatous and macroscopically “normal” mucosa were obtained from 11 patients with villous and 11 patients with tubulovillous adenomas who underwent primary resection for purpose of cure. The tissues were studied to determine MUC5AC gene expression by immunohistochemistry and in situ hybridisation. Patients were followed up by flexible sigmoidoscopy to detect the presence of early local recurrence. Results—10 villous adenomas showed mature MUC5AC glycoprotein and all 11 expressed MUC5AC mRNA. Five tubulovillous adenomas showed mature MUC5AC glycoprotein and 10 expressed MUC5AC mRNA. Neoexpression of the MUC5AC mucin gene was not detected in any of the mucosal biopsies taken adjacent to either villous or tubulovillous adenomas, even in three patients with early, locally recurrent disease. Conclusions—Aberrant MUC5AC gene expression is not a “field change” in the colorectal mucosa in patients with rectal adenomas and therefore cannot be used to predict local recurrence of villous and tubulovillous adenomas.

Aim—To define the biological nature and malignant potential of interval cancers presenting to a breast unit within the NHS breast screening programme. Methods—112 interval cancers were compared with matched, screen detected and symptomatic cancers in terms of their radiographic, histopathological, and immunohistochemical features. Results—Interval cancers, strictly defined, showed no characteristic radiographic pattern. In terms of size, vascular invasion, lymph node status, and prognosis they were intermediate between screen detected and symptomatic cancers. Within the interval cancers there was an excess of grade 1 and grade 3 tumours, and lesions with a high Ki67 index but immunohistochemistry otherwise failed to discriminate between the three groups. Inclusion of data from false negative "interval cancers" did not significantly alter the results. Conclusions—Interval cancers are more aggressive than screen detected cancers but in general less aggressive than symptomatic cancers. However, within a heterogeneous group, occasional interval cancers are exceptionally malignant. Key Words: breast cancer screening • interval cancer

Aims —To investigate interlaboratory variance in the immunohistochemical (IHC) detection of oestrogen receptors so as to determine the rate of false negatives, which could adversely influence the decision to give adjuvant tamoxifen treatment. Methods —To ensure that similar results are obtained by different institutions, 200 laboratories from 26 countries have joined the UK national external quality assessment scheme for immunocytochemistry (NEQAS-ICC). Histological sections from breast cancers having low, medium, and high levels of oestrogen receptor expression were sent to each of the laboratories for immunohistochemical staining. The results obtained were evaluated for the sensitivity of detection, first by estimating threshold values of 1% and 10% of stained tumour cells, and second by the Quick score method, by a panel of four assessors judging individual sections independently on a single blind basis. The results were also evaluated using participants' own threshold values. Results —Over 80% of laboratories were able to demonstrate oestrogen receptor positivity on the medium and high expressing tumours, but only 37% of laboratories scored adequately on the low expressing tumour. Approximately one third of laboratories failed to register any positive staining in this tumour, while one third showed only minimal positivity. Conclusions —There is considerable interlaboratory variability, especially in relation to the detection of breast cancers with low oestrogen receptor positivity, with a false negative rate of between 30% and 60%. This variability appears to be caused by minor differences in methodology that may be rectified by fine adjustment of overall technique.

Aim —To determine whether HIV is spreading from injecting drug users and homosexual/bisexual males into lower risk heterosexual populations in Glasgow, Scotland, and to pilot a method of monitoring HIV prevalence which involves testing routine biochemistry specimens. Methods —An unlinked anonymous HIV testing study of hospital patients and general practice attenders was conducted during January 1992 to December 1997. Testing was performed on routine biochemistry specimens from patients aged 16–49 years attending two hospitals with catchment areas covering the north and the east of the city. Results —78 260 specimens were tested in the study period and no patient objected to their samples being tested anonymously. HIV prevalence rates among male and female subjects were 0.63% and 0.01%, respectively; the large difference in prevalence resulted, in part, from the inclusion of HIV infected haemophiliac patients who attended one of the hospitals. Prevalence among male general practice patients ranged between 0.1% and 0.2%, while that for male patients attending surgical or surgically related specialties was 0.1%. Conclusions —The prevalence data indicate that HIV has not seeded from the high risk groups into the wider heterosexual population, and that the risk of a surgeon acquiring HIV occupationally is extremely low in a city which has an HIV prevalence similar to or greater than that seen in most other parts of the United Kingdom. Large numbers of residual specimens from busy biochemistry laboratories can be processed for unlinked anonymous testing without interfering with the laboratories9 routine functions. This survey approach might be best suited to monitoring HIV trends in developing countries with relatively high prevalence rates and where transmission is principally heterosexual.

Aims—To use laboratory data and liver biopsies, prospectively obtained from hepatitis B surface antigen (HBsAg) and anti hepatitis B e antigen (anti-HBe) positive patients, for the assessment of: (1) the relation between biopsy length/number of portal tracts and sampling error; (2) the relation between the severity of piecemeal necrosis and the new grading terminology (minimal, mild, moderate, and severe chronic hepatitis); and (3) liver pathology, which has not been studied in patients with this specific serological profile. Methods—The study group (n = 174) included 104 patients with normal aminotransferase concentrations and no cases with clinically apparent cirrhosis. The specimen length and number of portal tracts were measured at light microscopy examination. Sampling error analysis was related to the discrepancies between aminotransferase concentrations versus histological grade. Detailed histological scorings were undertaken by the reference pathologist and compared with laboratory and hepatitis B virus (HBV) DNA precore sequence data. Results—Sampling error seemed to be a constant feature, even for biopsies ≥ 20 mm, but increased dramatically in biopsies < 5 mm long and/or containing less than four portal tracts. Between 25% and 30% of biopsies, graded as "mild" or "moderate" activity showed features of moderate and severe piecemeal necrosis, respectively. Ten per cent of the patients with normal aminotransferase values had stage III–IV hepatic fibrosis, and 20% had piecemeal necrosis. Only cytoplasmic, not nuclear, core antigen expression was a strong predictor of high hepatitis B viraemia. There was no association between precore stop codon mutations, grade/stage of liver disease, and hepatitis B core antigen (HBcAg) expression. Conclusions—The specimen available for light microscopical examination should be > 5 mm long and should contain more than four portal tracts. In addition, the new grading terminology might give the clinician an inappropriately mild impression of the severity of piecemeal necrosis. Furthermore, even in the presence of normal aminotransferase concentrations, considerable liver pathology can be found in 10–20% of HBsAg and anti-HBe positive individuals; such pathology is not associated with the occurrence of precore stop codon mutations. Key Words: liver pathology • chronic hepatitis B virus infection • anti-hepatitis B antigen e positive • core antigen expression • serum hepatitis B virus DNA • hepatitis grading • sampling error

Aim—Despite the fact that gastric carcinoma continues to be one of the most common cancers world wide, only dysplasia in flat mucosa and adenomas have been shown to evolve into invasive carcinoma. The aim of this paper is to report a novel histological phenotype of gastric adenoma with early invasive growth. Material and results—The patient presented with gastric complaints. A barium examination revealed an ulcerated tumour in the corpus, apparently infiltrating the gastric wall. The endoscopic examination showed a pediculated protruding tumour in the greater curvature. Punch biopsies were reported as invasive adenocarcinoma. Because of the poor condition of the patient, a partial gastrectomy was performed. The histological examination revealed elongated fronds with lateral crenated, saw tooth-like notches as a result of scalloped epithelial indentations. Areas with high grade dysplasia, with carcinoma in situ, and invasive carcinoma at the tip of the adenoma were demonstrated. The pedicle of the protruding neoplasia “emerged” from a non-protruding serrated adenoma. Conclusions—The protruding serrated neoplasia had apparently evolved from a non-protruding serrated gastric adenoma. This appears to be the first case of gastric serrated neoplasia in the literature. (J Clin Pathol 2001;54:849‐853)

To compare prospectively the efficacy of the Vero, LLC-MK2, MDCK, Hep-2, and MRC-5 cell lines in the isolation of the mumps virus from clinical samples by means of the shell vial method.During an epidemic outbreak of parotiditis 48 clinical samples (saliva swabs and CSF) were studied. Two vials of the Vero, LLC-MK2, MDCK, MRC-5, and Hep-2 cell lines were inoculated with 0.2 ml of the samples by the shell vial assay. The vials were incubated at 36 degrees C for two and five days. The vials were then fixed with acetone at -20 degrees C for 10 minutes and stained by a monoclonal antibody against mumps virus by means of an indirect immunofluorescence assay.The mumps virus was isolated from 36 samples. The Vero and LLC-MK2 cell lines showed a 100% isolation capacity, MDCK showed 77.7%, MRC-5 showed 44.4%, and Hep-2 showed 22.2%. The Vero and LLC-MK2 lines were significantly different to the other cell lines (p0.001). The sensitivity for the Vero and LLC-MK2 lines at two and five days of incubation was identical (100%). The values obtained in the study of the quantitative isolation capacity (positive isolation with5 infectious foci) were 94.4% for Vero, 97.2% for LLC-MK2, 5.5% for MDCK, 5.5% for Hep-2, and 0% for MRC-5.The Vero and LLC-MK2 cell lines are equally efficient at two and five days incubation for the isolation of the mumps virus from clinical samples, and the use of the shell vial method considerably shortens the time of aetiological diagnosis with higher specificity.

Aims—To determine serum salicylic acid concentrations in non-vegetarians and vegetarians not taking salicylate drugs, and to compare these concentrations with those found in patients taking aspirin, 75 mg daily. Methods—Serum samples were obtained from vegetarians (n = 37) and non-vegetarians (n = 39) not taking salicylate drugs. Non-vegetarians and vegetarians were recruited from the community and from a Buddhist monastery, respectively, in Dumfries and Galloway, Scotland. Patients (n = 14) taking aspirin (75 mg daily) were recruited from the Dumfries diabetic clinic. Serum salicylic acid concentrations were determined using a high performance liquid chromatography method with electrochemical detection. Results—Salicylic acid was detected in every serum sample analysed. Higher serum concentrations of salicylic acid were found in vegetarians than non-vegetarians: median concentrations of 0.11 (range, 0.04–2.47) µmol/litre and 0.07 (range, 0.02–0.20) µmol/litre, respectively; the median of the difference was 0.05 µmol/litre (95% confidence interval for difference, 0.03 to 0.08; p < 0.0001). The median serum concentration of salicylic acid in patients taking aspirin (75 mg daily) was 10.03 (range, 0.23–25.40) µmol/litre, which was significantly higher than that found in non-vegetarians and vegetarians. There was overlap in serum salicylic acid concentrations between the vegetarians and patients taking aspirin. Conclusions—Salicylic acid, a non-steroidal anti-inflammatory drug, is present in fruits and vegetables and is found in higher concentrations in vegetarians than non-vegetarians. This suggests that a diet rich in fruits and vegetables contributes to the presence of salicylic acid in vivo. There is overlap between the serum concentrations of salicylic acid in vegetarians and patients taking aspirin, 75 mg daily. These findings may explain, in part, the health promoting effects of dietary fruits and vegetables. Key Words: salicylic acid • salicylates • diet • low dose aspirin

Background/Aims—The serodiagnosis of infection with Salmonella typhi, using the Widal agglutination assay, relies on patients’ antibodies to the O=9,12 lipopolysaccharide (LPS) antigens, H=d flagellar antigens, and the Vi capsular antigens. A Vi agglutination titre of > 1/40 has traditionally been regarded as indicative of recent infection with S typhi.In this study, 91 sera were used to assess the reliability of the Widal agglutination assay based on antibodies to the Vi antigens. Methods—The Widal agglutination assay was carried out using protocols established by the Central Public Health Laboratory, Colindale. Antibodies to the Vi capsular antigen were detected using a standard preparation of S typhi, ViI Bhatnagar variant strain (S typhi, ViI). Sera used in the study comprised 73 from patients who were culture positive for S typhi, 10 from patients who were culture positive for other species of Salmonella not expressing a Vi antigen (namely, S javiana, S enteritidis, S typhimurium, S stanley, S saint paul, S bareilly ,o r S mbandaka), and eight from healthy blood donors. Results—Agglutination titres of > 1/40 were detected to S typhi ViI in 69 of 73 sera from patients with typhoid, although 27 of these also agglutinated an unrelated control antigen.The Widal assay also detected significant amounts of agglutinating antibodies to S. typhi ViI in all eight control sera and seven sera from patients infected with S bareilly, S enteritidis, S javiana, S mbandaka, S saint paul, and S stanley. Conclusions—Agglutinating antibodies to the Vi antigen can be detected by the Widal assay, but even with the appropriate control antigens the results were unreliable. The serodiagnosis of infections with S typhi should be based on the detection of antibodies to both the O=9,12 LPS antigen and the H=d flagellar antigen by immunoblotting, and should not use the Vi antigen-based Widal assay. Conclusions should be made in the light of patients’ clinical details and any knowledge of previous immunisation for typhoid. (J Clin Pathol 2000;53:851‐853)

Aim—To evaluate whether the assessment of multiple sections from retrieved nodes yields an increased number of metastases compared with the number that would be detected by the commonly applied method of microscopy of a single section of lymph node only. Methods—A prospective study of 72 colorectal carcinoma resection specimens. Lymph node sampling was based on the current guidelines for the detection of breast cancer metastases in axillary nodes. Lymph nodes up to approximately 5 mm in maximum extent were processed in entirety, without prior sectioning, and assessed histologically at three levels; larger lymph nodes were processed in entirety as multiple sections and histologically assessed at one level. Results—From a total of 72 carcinomas, eight were Dukes's A, 26 were Dukes's B, and 38 were Dukes's C. The mean and median numbers of nodes identified were 13 and 12, respectively (range, three to 44). Of the Dukes's C cases, four contained lymph node metastases identified by our method that might have gone undetected by the current, generally applied method. In one case, this led to the detection of the only nodal metastasis present and therefore "upstaged" the tumour from Dukes's B to C. On average, six extra tissue blocks were processed for each case in applying this method. Conclusion—The assessment of multiple sections of lymph nodes from colorectal specimens leads to the detection of only a small number of additional nodal metastases. The method involves increased workload for pathologists and laboratory staff. Key Words: colorectal carcinoma • lymph node • metastases

Background—Cytomegalovirus (CMV) is the prototype member of the s-herpesvirinae, which can cause multiple organ dysfunction in the immunocompromised host. Human herpesvirus 6 (HHV-6) and HHV-7 are newer members of the s-herpesvirinae that can cause febrile illness in young children and are also possible pathogens in the immunocompromised patient. Aim—CMV is detected in histopathological sections by visualisation of owl's eye inclusion bodies. The aim of this study was to quantify the relation between CMV, HHV-6, and HHV-7 viral loads and the presence of owl's eye inclusions in histological sections. Methods—Histopathological examination of postmortem material and recording of owl's eye inclusion bodies were performed. CMV, HHV-6, and HHV-7 were detected by qualitative and quantitative polymerase chain reaction (PCR) from the same postmortem samples. Statistical analysis of the histopathological and PCR results was performed. Results—There was a significant association between the detection of owl's eye inclusion bodies and positive CMV PCR (p < 0.001); the median CMV viral load was significantly higher in samples that were positive for owl's eye inclusions (p < 0.001). No association was found between the presence of owl's eye inclusions and HHV-6 or HHV-7 positivity. Conclusion—Histological detection of owl's eye inclusion bodies is an insensitive but highly specific method for detecting CMV organ involvement. Owl's eye inclusion bodies are not associated with HHV-6 or HHV-7 infection. Key Words: polymerase chain reaction • inclusion bodies • viral load

Aim—To investigate a possible outbreak of tuberculosis in a hostel for homeless men using IS6110 profiling, a polymerase chain reaction (PCR) based fingerprinting technique. Methods—Eight cases of tuberculosis were diagnosed in residents of the hostel over a period of 28 months. To provide epidemiological data, a heminested inverse PCR (HIP) assay targeting the insertion sequence IS6110 together with its upstream flanking region was used to fingerprint the eight isolates of M tuberculosis under investigation. Results—The HIP technique gave IS6110 profiles which showed that while three isolates were clearly distinct, the remaining five strains were indistinguishable, suggesting the latter were representatives of a single outbreak strain. Conclusions—The HIP assay proved discriminatory and facilitated repeated testing for the direct comparison of strains as more patients presented over the protracted course of this outbreak. Key Words: tuberculosis • epidemiology • molecular typing • IS6110

Aim —To use CD45RO immunohistochemistry to investigate the numbers of T lymphocytes found in sections of myocardium from a routine necropsy series, and to determine the incidence of myocarditis in this series. Methods —Myocardial sections from 163 routine hospital necropsies were stained with CD45RO and the numbers of positive lymphocytes/mm 2 were counted. The results were correlated with the H/E opinion and the clinical context of the necropsy. Results —Most (143) cases showed low numbers (0–3) of CD45RO positive lymphocytes/mm 2 . Fifteen cases showed 7–13 positive lymphocytes/mm 2 , comprising a wide variety of clinical conditions, generally with no specific cardiac pathology. Five cases showed 14 or more positive lymphocytes/mm 2 , comprising one case of active myocarditis, three cases of cardiac transplant rejection, and one post-transplant lymphoproliferative disorder, all conditions in which large numbers of lymphocytes would be expected. Conclusions —The incidence of myocarditis in our series was 0.6%. In most cases the normal myocardium has a low T lymphocyte count (0–3/mm 2 ). In some cases immunohistochemistry shows more positive cells than would have been expected on light microscopy. Immunohistochemistry is a useful and reliable means of confirming a diagnosis of myocarditis. The results support the conclusion of the 1997 ISFC task force that 14 or more lymphocytes or macrophages/mm 2 of myocardium in the appropriate clinical context is a reliable threshold for the diagnosis of chronic myocarditis.

Aim —To determine the frequency of skip lesions in an unselected series of temporal artery biopsies and compare the results with other series. Methods —The study was a retrospective review of 102 consecutive temporal artery biopsies taken in a five year period (1992–1997) in one large hospital. Results —35 cases (34.3%) showed evidence of active cranial vasculitis with pathological evidence of inflammation of the intima or media, with or without giant cells. Three of these cases (8.5%) showed apparent skip lesions: normal intima, media, and adventitia in one segment while in other segments there was clear evidence of active vasculitis. Immunocytochemical stains for leucocyte common antigen (LCA) and CD15 were helpful in identifying the absence of intimal or medial inflammatory cell infiltrates within skip lesions. Skip lesions have been described in up to 28.3% of cases in some series, while others have not found evidence of skip lesions or have identified them in a much smaller percentage of cases. Conclusions —In this series skip lesions were relatively rare, accounting for 8.5% of cases of active vasculitis. The degree of inflammation in temporal arteritis is discontinuous. Immunostaining for inflammatory cells, for example LCA and CD15, may be helpful in identifying the presence of an inflammatory cell infiltrate in skip lesion segments of the temporal artery.

Aim—Cholangiocarcinoma can be cured by surgery, but only in a minority of cases. The activation of apoptosis is a major mode of action of chemotherapy and radiotherapy, which have limited benefit in the treatment of cholangiocarcinoma. The antiapoptotic members of the Bcl-2 protein family (Bcl-2, Bcl-XL, and Mcl-1) are important inhibitors of apoptosis, but have not been investigated extensively in cholangiocarcinoma. Methods—The expression of Bcl-2, BclXL, and Mcl-1 was investigated in normal biliary epithelium (17), biliary dysplasia (three), and invasive cholangiocarcinoma (51), in addition to three human cholangiocarcinoma cell lines, by immunohistochemistry and immunofluorescence. Results—The expression of Bcl-2 was not detected in normal or malignant biliary tissue. In contrast, granular cytoplasmic Bcl-XL and Mcl-1 staining was found in 60‐100% of cells in all normal, dysplastic, and malignant specimens, including the human cell lines examined in this study. Conclusion—These findings indicate that Mcl-1 and Bcl-XL, but not Bcl-2, are involved in the survival of normal and neoplastic cells in the biliary tree. By prolonging survival through blocking apoptosis, these proteins might be reducing the eYcacy of cytotoxic anticancer treatments in cholangiocarcinoma. (J Clin Pathol 2001;54:927‐932)

Aim—To determine whether assays of carcinoembryonic antigen (CEA) and cholesterol in ascites add diagnostic value to cytology. Methods—The additional diagnostic efficacy of the biochemical assays was studied in the ascitic fluid from 130 patients, of whom 57 had peritoneal carcinomatosis. All diagnoses were verified by subsequent necropsy and/or histology. Results—CEA concentrations over 5 ng/ml indicated carcinomas, occasionally without peritoneal involvement of the tumour. However, increased values were significantly more common in cancer with peritoneal involvement (p < 0.01), giving a sensitivity of 51% and specificity of 97% for carcinomatosis. A cholesterol value exceeding 1.21 mmol/litre was found in 93% of cancers with peritoneal involvement, but it was not entirely specific (96%) for carcinomatosis. Simultaneous increases in CEA and cholesterol concentrations were specific for carcinomatosis and this combination increased the sensitivity for diagnosing carcinomatosis from 77% with cytology alone to 88%. The correct diagnosis could thus be made in five of 12 cases with inconclusive cytology. Conclusions—The measurements of both CEA and cholesterol concentrations in ascites give additional specific information about peritoneal carcinomatosis and can therefore be a useful adjunct to cytology—in particular, in inconclusive cases. Key Words: ascitic fluid • carcinoembryonic antigen • cholesterol • cytology

Aims—Comparative genomic hybridisation (CGH) is a reliable tool to gain an overview of all unbalanced chromosomal alterations within a tumour. Nevertheless, the high numbers of tumour cells required and the comparatively low resolution are drawbacks of this technique. Polymerase chain reaction (PCR) based multiplex microsatellite analysis represents a semiautomated, highly reproducible method, which requires small amounts of tumour cells. This is a comparative study of CGH and microsatellite analysis. Methods—Eighty one samples of invasive breast cancer were investigated by two sensitive multiplex PCRs containing three microsatellites each of six markers (D6S261, D11S907, D6S300, D11S927, D8S272, and D11S925), and two additional microsatellite markers located within intron 1 of the epidermal growth factor recepter gene (egfr) and p53 (p53CA). Results—At least one example of loss of heterozygosity was detectable in all breast cancer tissues.However,the overall rate of accordance between the two methods tested was only 61%. An increasing rate of the number of genetic alterations in each case was mirrored by a constantly increasing fractional allelic loss index. Conclusions—PCR based multiplex microsatellite analysis using this panel of eight microsatellite markers not only enables the characterisation of cells that have malignant potential in a high frequency of patients with breast cancer, but can also give an estimate of the degree of genetic progression. (J Clin Pathol 2001;54:836‐840)

Aims—To determine whether or not identifying recognised anatomical groupings of lymph nodes (LNs) improves LN yield in pancreatoduodenectomy resection specimens. Methods—All the pathology reports from pancreatoduodenectomy resection specimens between January 1997 and September 1999, for one specialist pathologist at the Royal Liverpool University Hospital, were examined retrospectively. The total number of LNs found in each specimen was determined and the method of identifying LNs established for each case. LNs were found using either (1) the UICC TNM anatomical groupings, termed "grouped"; (2) the Japanese Pancreatic Society classification, termed "numbered"; or (3) neither the "grouped" nor "numbered" classification, termed "non-grouped". Results—A total of 50 reports (45 neoplastic, five chronic pancreatitis) were studied, 11 with non-grouped LNs, 14 with grouped LNs, and 25 with numbered LNs, including the five inflammatory cases. A median of 7.0 LNs was found in non-grouped cases, a significantly lower number than in the grouped cases (median, 12.0; Mann-Whitney U, p < 0.039) and numbered cases (median, 17.0; p < 0.0001). There was no significant difference in the LN yield between grouped and numbered cases (p = 0.1066). LNs were found most frequently in the inferior, posterior pancreaticoduodenal, and infrapyloric regions. Conclusions—A detailed knowledge of the anatomical distribution of LNs in pancreatoduodenectomy resection specimens significantly improves LN yield. It is suggested that illustrations of LN sites in resection specimens should be included in pathology guidelines/proformas to improve LN detection and, therefore, pathological prognostic data. Key Words: audit • lymph node • pancreas

Aims—To assess the importance of tumour necrosis factor AE (TNF-AE) promoter polymorphism in relation to infection with the cytotoxin associated gene A (cagA) subtype of Helicobacter pylori within a dyspeptic Korean population. Methods—Eighty three patients with gastric disease and 113 healthy controls were studied. The DNA from gastric biopsy specimens was analysed by H pylori specific and cagA specific polymerase chain reaction (PCR). To characterise TNF-AE polymorphism at positions ˛308 and ˛238, PCR based restriction fragment length polymorphism analysis was performed. Results—Helicobacter pylori infection was closely correlated with G to A transition at position ˛308 of the TNF-AE promoter when compared with healthy controls (odds ratio (OR), 2.912; 95% confidence interval (CI), 1.082 to 7.836; p = 0.034). Although TNF-AE ˛308 polymorphism in patients with H pylori was not significantly diVerent from that in patients without H pylori, the ˛308A polymorphism was strongly associated with H pylori cagA subtype infection when compared with the polymorphism in cagA negative H pylori infection (OR, 8.757; 95% CI, 1.413 to 54.262; p = 0.019) and healthy controls (OR, 3.683; 95% CI, 1.343 to 10.101; p = 0.011). G to A genetic change at position ˛238 of the TNF-AE gene was not significantly associated with H pylori cagA subtype infection. In addition, genetic polymorphisms at both sites of the TNF-AE promoter in patients with H pylori infection did not correlate with the severity of disease. Conclusion—TNF-AE ˛308A polymorphism was significantly related to infection with the H pylori cagA subtype in Korean patients with gastric disease. (J Clin Pathol 2001;54:703‐706)

Aims— To compare the expression of immunohistochemical variables between benign and malignant components of malignant peripheral nerve sheath tumour (MPNST) arising within neurofibroma. Methods —Eight cases of MPNST arising within a neurofibroma, associated with neurofibromatosis type 1 (NF1), were studied. The areas of MPNST and neurofibroma were compared immunohistochemically with regard to the expression of proliferative activity (MIB-1), growth factors, p53, bcl-2, neural cell adhesion molecule (N-CAM), and CD34. Results —The expression of transforming growth factor β1 (TGF-β1), TGF-β receptor type II, hepatocyte growth factor α (HGF-α), c-met, p53, and N-CAM was higher in the areas of MPNST than in the neurofibromatous areas in four, five, five, eight, five, and three of the eight cases, respectively. CD34 expression was lower in the areas of MPNST than in the neurofibroma areas in three of the eight cases. Conclusions —On the basis of these findings, TGF-β1, HGF-α, and p53 might be involved in the malignant transformation of neurofibroma to MPNST.

Background/Aims —Synovial fluid examination is thought to be the pathological investigation of choice in most joint disorders, with only a few specific conditions necessitating biopsy, although no evidence based studies are available to support this belief. This study sought to investigate the validity of this assumption. Methods— One hundred and three cases in which synovial fluid aspiration and synovial biopsy had both been performed at arthroscopy were studied. The amount of diagnostically useful information produced by each investigation was assessed. Results —In most cases, both investigations provided the same amount of information and were generally equally specific or equally non-specific. Overall, the biopsy provided more information than the fluid in 29% of cases and vice versa in 18%. When only those cases in which both tests were adequate were considered, the biopsy provided more specific information than the fluid in a small number (9%) of cases, but these cases could not be predicted. Conclusion —The diagnostic usefulness of a biopsy approximates and occasionally exceeds that of a fluid. In the arthroscopic situation, the main advantage of performing both tests is that it provides a “failsafe mechanism” for the rare occasions when one of the samples is inadequate.

Aims—To evaluate in detail the extent to which step sectioning and immunohistochemical examination of sentinel lymph nodes (SNs) in patients with breast cancer reveal additional node positive patients, to arrive at a sensitive yet workable protocol for histopathological SN examination. Methods—This study comprised 86 women with one or more positive SN after a successful SN procedure for clinical stage T1–T2 invasive breast cancer. SNs were lamellated into pieces of approximately 0.5 cm in size. One initial haematoxylin and eosin (H&E) stained central cross section was made for each block. When negative, four step ribbons were cut at intervals of 250 µm. One section from each ribbon was stained with H&E, and one was used for immunohistochemistry (IHC). Results—When taking the cumulative total of detected metastases at level 5 as 100%, the percentage of SN positive patients increased from 80%, 83%, 85%, 87% to 88% in the H&E sections through levels 1 to 5, and with IHC these values were 86%, 90%, 94%, 98%, and 100%. Three of nine patients in whom metastases were detected at levels 3–5 only had metastases in the subsequent axillary lymph node dissection. Conclusions—Multiple level sectioning of SNs (five levels at 250 µm intervals) and the use of IHC detects additional metastases up to the last level. Although more levels of sectioning might increase the yield even further, this protocol ensures a reasonable workload for the pathologist with an acceptable sensitivity when compared with the published literature. Key Words: breast cancer • sentinel node • immunohistochemistry • pathology

Aims—To investigate the prognostic value of recently proposed flow cytometric S-phase fraction (SPF) variables (average SPF and SPF tertiles) compared with conventional SPF, and to compare the one with the best predictive value with the immunohistochemical Ki-67 index in breast carcinoma. Methods—A short term follow up study (median, 39.6 months) of a large series of patients (n = 306) was conducted. DNA ploidy was analysed on fresh/frozen tumour samples by flow cytometry, and the SPF was calculated from the DNA histogram using an algorithm. The Ki-67 index was assessed on paraffin wax embedded material by immunohistochemistry (cut off point, 10%). The two methods were compared by means of κ statistics, and the prognostic significance of both in relation to disease free survival (DFS) and overall survival (OS) was determined. Results—SPF and Ki-67 analysis was performed on 234 (76.5%) and 295 (96.4%) tumours, respectively. The two assessments were simultaneously available in 230 cases. All SPF variables analysed in the whole series significantly correlated with disease evolution, with the conventional median SPF (cut off point, 6.1%) showing the highest predictive value in relation to both DFS (p = 0.0001) and OS (p = 0.0003). SPF tertiles and median SPF evaluated according to DNA ploidy status had no prognostic significance. The Ki-67 index showed a trend in relation to DFS (p = 0.086) that did not reach significance, and no correlation with OS was found (p = 0.264). The comparative analysis of SPF and Ki-67 revealed some agreement between the two methods (agreement, 69.13%; κ statistic, 0.3844; p < 0.001), especially in the subgroup of diploid tumours. Conclusions—Flow cytometric SPF is a better prognosticator than the Ki-67 index, but only SPF variables applied in the whole series show potential clinical usefulness. Key Words: breast carcinoma • DNA flow cytometry • immunohistochemistry • S-phase fraction • Ki-67 • prognosis

Background/Aims—Non-melanoma skin cancers frequently harbour multiple human papillomavirus (HPV) types. A recent report suggests that a polymorphism of the p53 tumour suppressor gene that results in the substitution of a proline residue with an arginine residue at position 72 of the p53 protein might act as a risk factor in HPV associated malignancies. This study aimed to determine the following: (1) the relation between HPV infection and the development of cutaneous squamous cell carcinoma (SCC), and (2) whether there is a correlation between p53 codon 72 polymorphism and the development of SCC. Methods—Blood samples were taken from 55 patients with skin cancer (both renal transplant recipients and immunocompetent patients with skin cancer) and 115 ethnically matched volunteers. A polymerase chain reaction based assay was used to determine p53 codon 72 genotypes. In addition, 49 benign and malignant lesions from 34 of the patients with skin cancer and 20 normal human skin samples from 20 of the control volunteers were examined for HPV. Results—The proportions of p53 codon 72 genotypes found were 78% arginine homozygous, 2% proline homozygous, and 20% heterozygous among patients with skin cancer and 79% arginine homozygous, 3.5% proline homozygous, and 17.5% heterozygous among the control population. Statistical analysis showed no significant differences in the distribution of the two p53 isoforms between the patients with skin cancer and the control population. The predominant viral types detected in both the patients and the control group were EV associated HPVs, although the incidence was lower in normal skin samples than in malignant lesions or viral warts. Conclusions—These results suggest that in a Celtic population there is no correlation between the presence of HPV, the p53 codon 72 arginine polymorphism, and the development of skin cancer. Key Words: p53 codon 72 polymorphism • human papillomavirus • skin cancer

Background—The results of immunohistochemical staining are often assessed by semiquantitative scoring. However, these scoring systems are usually non-standardised and there has been little evaluation of the accuracy and reliability of this subjective assessment. Aims—To assess the accuracy of observer estimation of proportions of objects in an image. Methods—Images were generated that contained known proportions of pink squares in grids of 50 x 50 and 100 x 100 squares. Observers were shown each image for five seconds in random order and either estimated the proportion of pink squares or selected the image (from a pair of images) that contained the greater proportion of pink squares. The observers were four consultant histopathologists, seven trainee histopathologists, and six control non-histopathologists. Results—The raw estimations of proportions showed a close correlation with the real proportions, with correlation coefficients of 0.94 and 0.95 for consultant and trainee histopathologists on the 50 x 50 grids. However, the performance in the comparison task was much higher, with an almost perfect classification for grids of equal size even when the proportions only differed by 5%. Conclusions—Histopathologists can estimate proportions of objects in an image with a reasonable degree of accuracy in this abstract test system. All observers, whether histopathologists or not, can discriminate between proportions that are only 5% different in equal sized image grids. This suggests that the generation and use of carefully calibrated reference images could greatly improve the accuracy and reliability of semiquantitative scoring of immunohistochemical or any other staining. Key Words: scoring • immunohistochemistry • semiquantitative • interobserver agreement • κ statistics

Aim —To develop a new, simple, and cheap method for estimating antioxidant activity in human fluids. Methods —The assay measured the capacity of the biological fluids to inhibit the production of thiobarbituric acid reactive substances (TBARS) from sodium benzoate under the influence of the free oxygen radicals derived from Fenton's reaction. A solution of 1 mmol/litre uric acid was used as standard. Results —The following mean (SD) antioxidative activities were found (as uric acid) in the various biological fluids: serum, 2.04 (0.20) mmol/litre; urine, 176.5 (25.6) μmol/litre; cerebrospinal fluid, 95.0 (26.9) μmol/litre; aqueous humour oculi, 61.25 (9.9) μmol/litre; saliva, 838.5 (48.2) μmol/litre; tears, 247.0 (17.0) μmol/litre; ascites fluid, 270.0 (63.3) μmol/litre; kidney cyst fluid, 387.1 (28.1) μmol/litre. Small samples of the biological material were needed for the analyses: 10 μl of serum and 50–100 μl of other body fluids. In the sera of 48 healthy individuals there was a significant positive correlation between values obtained with the Randox method (as a reference method) and the new method proposed here (correlation coefficient, 0.8728; mean difference between methods

The nuclear enzyme DNA topoisomerase II has been shown to be required for chromatin condensation and chromosomal segregation during mitosis; its isoform topo II alpha is linked with active cell proliferation in mammalian cells. The aim of this study was to examine the relation of the expression of topo II alpha to the biological behaviour of conventional urinary bladder cancer.Formalin fixed, paraffin wax embedded tissue from 94 specimens of bladder urothelial cancer were immuno-histochemically stained for topo II alpha. For each case, a topo II alpha index was determined. A similar index had been determined for Ki-67, a known cell proliferation marker. Each case had also been graded, staged, and evaluated for DNA ploidy as well as for p53 and bcl-2 immunoreactivity.Raised topo II alpha expression (inor = 10% of malignant nuclei) correlated with two adverse prognosticators--high grade (p = 0.027) and invasion of the muscularis propria (p = 0.013), but with no other evaluated parameter. By multivariate survival analysis using Cox's proportional hazard model, high expression of topo II alpha was found to be predictive for worse survival (p = 0.0047). Patients' age, tumour stage, and grade were also retained as independent prognostic factors (p = 0.0349, p = 0.00005, and p = 0.0130, respectively). The negative influence of increased topo II alpha immunopositivity on patients' survival was also seen in the subgroup of patients with non-muscle invasive carcinomas (p = 0.0004), in patients with a bcl-2 negative phenotype (p = 0.0330), and in those with low Ki-67 indices (p = 0.0341).Because topo II alpha and Ki-67 failed to demonstrate a significant interrelation, they appear to be different molecules that both function at separate phases in the complex process of cellular proliferation. The assessment of increased topo II alpha immunoreactivity in specimens from urothelial carcinomas might help to select patients (particularly among those with superficial tumours) in the worse prognostic categories for new therapeutic strategies.

Background/Aims—It is not known how enteric cryptosporidiosis induces severe intestinal impairment despite minimal invasion by the parasite. The aim of this study was to analyse the histological features and locally implicated immune cells in colonic biopsies of AIDS related cryptosporidiosis. Patients/Methods—Colonic biopsies from patients with AIDS related cryptosporidiosis (n = 10, group I), patients with AIDS but without intestinal infection (n = 9, group II), and human seronegative controls (n = 9, group III) were studied. Using immunohistochemistry the infiltrating mononuclear cells were analysed in both the epithelium and lamina propria for the expression of CD3, CD8, TiA1, granzyme B, and CD68 and for glandular expression of human major histocompatibility complex DR antigen (HLA-DR). Results—Severe histological changes, resulting in abundant crypt epithelial apoptosis and inflammatory infiltrate in the lamina propria, were seen in all biopsies from group I. A significant increase of CD8+, TiA1+, and granzyme B+ T cells in the lamina propria and HLA-DR glandular expression was noted in group I compared with groups II and III. However, the number of intraepithelial lymphocytes, lamina propria CD3+ T cells, and macrophages was not significantly increased in cryptosporidiosis specimens compared with controls. Conclusion—Epithelial apoptosis mediated by granzyme B+ cytotoxic host T cells might play a major role in the development of colonic lesions in AIDS related cryptosporidiosis. Key Words: cryptosporidiosis • acquired immunodeficiency syndrome • colon • apoptosis • cytotoxicity • immunochemistry

Aims —Strong thyroglobulin immunoreactivity within sinus histiocytes in a lymph node draining a papillary thyroid carcinoma was observed in a recent case. This prompted the investigation of whether thyroglobulin immunoreactivity is common in regional lymph nodes in cases of thyroid malignancy. Methods —Eighty seven lymph nodes were studied from 21 cases of thyroid malignancy. These comprised papillary carcinoma (n = 12), follicular carcinoma (n = 4), medullary carcinoma (n = 3), and one case each of squamous and anaplastic carcinoma. Eleven cervical lymph nodes from patients with no evidence of thyroid disease were included as controls. Sections were stained with a monoclonal antibody against thyroglobulin. Results —In the cases of thyroid malignancy, 32 of 87 lymph nodes showed positive staining for thyroglobulin of histiocytes within the subcapsular and medullary sinuses. In an additional four cases, there was positive staining of lymph within lymphatic channels. Positivity was present in at least one node in 15 of 21 cases. There was no positivity in the control cases. There was no correlation between the size of the primary tumour and the presence of thyroglobulin positivity. Conclusions —Positive staining with antithyroglobulin occurs not uncommonly in sinus histiocytes in lymph nodes draining thyroid tumours. This positivity could be the result of the destruction of normal thyroid follicles, with the release of thyroglobulin, which is taken up by histiocytes, which subsequently drain to local lymph nodes. Pathologists should be aware of this phenomenon and should be careful not to interpret this as metastatic tumour.