Improved silver staining of nucleolar organiser regions in paraffin wax sections using an inverted incubation technique.

A new simple modification to the silver staining of nucleolar organiser regions (AgNORs) was devised which, by performing the incubation with the slide inverted, results in minimal undesirable background staining, a persistent problem. Inverted incubation is facilitated by the use of a commercially available plastic coverplate. This technique has several additional advantages over other published staining protocols. In particular, the method is straightforward, fast, and maintains a high degree of contrast between the background and the AgNORs.

The effect of hypoalbuminaemia, hyperbilirubinaemia and renal failure on serum fructosamine concentration in non-diabetic individuals.

We have investigated the effects of hypoalbuminaemia, hyperbilirubinaemia and renal failure on serum fructosamine concentration in 39 non-diabetic patients. All patients were hypoalbuminaemic (median serum albumin 25 g/l, range 12-34 g/l). Group 1 (n = 19) were patients with hypoalbuminaemia alone, group 2 (n = 7) with hypoalbuminaemia and impaired renal function (median serum creatinine 226 mumol/l, range 154-461 mumol/l) and group 3 (n = 13) were subjects with hypoalbuminaemia and hyperbilirubinaemia (median serum bilirubin 34 mumol/l, range 19-83 mumol/l). Serum fructosamine was significantly lower in all three groups compared to age-matched normoalbuminaemic controls, but there was no significant difference in fructosamine concentrations between the groups. There was a correlation between fructosamine concentration and serum albumin. (r = 0.82, p less than 0.001) in all three groups combined. Serum fructosamine did correlate with serum bilirubin in patients with normal renal function (r = 0.0, p less than 0.001). In patients with abnormal renal function there was no correlation between serum fructosamine and either urea (r = 0.22, ns) or creatinine (r = 0.31, ns). Albumin is the major factor affecting serum fructosamine concentrations. Moderate hyperbilirubinaemia does not affect fructosamine concentration. No difference in fructosamine concentration could be demonstrated in patients with renal failure.

Raised liver associated enzyme activity and post-prandial bile acid concentrations in sera from treated diabetic outpatients.

Aspartate aminotransferase, alanine aminotransferase and gamma-glutamyl transferase activities were measured in sera from 411 diabetic outpatients and were raised in 26 (6.4%), 34 (8.3%) and 62 (15.2%) patients, respectively. Serum total bile acid concentrations were raised in 4 patients (1%). Percentage glycated hemoglobin A1, serum fructosamine concentration and plasma glucose concentration were also measured. No relationship between the presence of raised enzyme activity and mature age, short duration of diabetic treatment regimen or glycemic control was found. Twenty-six patients with an alanine aminotransferase activity greater than 60 U/l were reviewed at 23 +/- 6.5 weeks. The activity of this enzyme had fallen to within the reference interval in 15 (58%). In the other 11 patients, its median activity was 75 U/l (range 51-181 U/l). Median gamma-glutamyl transferase activity had risen in these 11 patients from 78 U/l to 93 U/l (P less than 0.01). No statistical differences in treatment regimen or glycemic control were found between these two groups. Raised liver-associated enzyme activity in treated stabilised diabetic outpatients should therefore not be attributed to poor glycemic control or diabetic treatment regimen.

Long-term performance of the fructosamine assay.

We have investigated the long-term performance of the fructosamine assay based on secondary glycated protein standards and attempted to define the interpretation of varying degrees of increase in fructosamine concentration in comparison to haemoglobin A1 (HbA1) values both in insulin dependent (IDDM) and non-insulin dependent (NIDDM) diabetic patients. Between-batch imprecision of fructosamine over 5 months was (CV) 2.5% at 2.09 mmol/L, 2.8% at 3.52 mmol/L and 3.6% at 4.14 mmol/L. Variation of fructosamine concentration in vivo in stable diabetic patients monitored over 8-18 weeks was 2.3% to 7.1%. Fructosamine correlated with HbA1 both in IDDM (n = 110, r = 0.701, P less than 0.001) and NIDDM (n = 71, r = 0.764, P less than 0.001). Specificity and sensitivity of fructosamine for the prediction of degree of control assessed on the basis of HbA1 level (cut-off point for good vs. poor control, HbA1 = 10%) was determined. In NIDDM, specificity above 90% was achieved at a fructosamine concentration of 3.4 mmol/L with a corresponding sensitivity of 64.1%. 22.5% of patients were classified differently on the basis of fructosamine as compared to HbA1. In IDDM, specificity over 90% was achieved at 3.8% mmol/L fructosamine with a sensitivity of 35%. Discordancy rate between HbA1 and fructosamine based assessment of control was 31.8%. The assessment of diabetic control based on fructosamine may be different from that based on HbA1, particularly in IDDM. Fructosamine and HbA1 should be used as complementary rather than alternative tests.

Adenoma in the middle ear: a report of two cases.

Middle-ear adenoma has been reported only in small numbers by surgeons. The few large series reported have been presented by histopathologists. We add two cases of middle-ear adenoma to the published literature, together with pre-, per- and post-operative imaging of one case, as a demonstration of this rare clinical entity. We discuss the pathology of middle-ear adenoma, its diagnosis and treatment, and suggest ways of improving its management.

Re-evaluating silver-stained nucleolar organizer regions (AgNORs) in problematic cutaneous melanocytic lesions: a study with quantitation and pattern analysis.

Previous studies enumerating AgNORs in cutaneous melanocytic lesions have produced inconsistent results. It is probable that such inconsistencies arise from differences in fixation, staining technique, and counting strategies. Our group, having demonstrated an improved method of silver staining and having optimized counting, is now able to reconsider the role of AgNORs in evaluating borderline melanocytic lesions. Diagnostic problems similar to those encountered in routine practice have been examined. It is shown that only by counting intra- and extranucleolar AgNORs in combination with assessing the pattern of AgNOR dispersal is it possible to (1) discriminate dysplastic naevi from melanoma and (2) distinguish Spitz naevi and pigmented spindle cell naevi from melanoma. An analysis of AgNOR numbers alone results in considerable overlap between the groups studied. The value of assessing patterns of AgNOR dispersal within and outside clustered nucleolar structures is emphasized. Lesions labelled as minimal deviation melanoma (by experts in dermatopathology) were also investigated. The majority of these specimens form a distinct group lying apart from control naevi and melanomas. This finding, whilst of interest, is difficult to evaluate because of poorly defined diagnostic criteria.

Mobile laboratory unit based on the Kodak Ektachem DT system.

We evaluated the Kodak Ektachem DT system (DT60, DTE, DTSC modules), using it as a mobile laboratory unit (MLU) in different hospital settings. Imprecision of 19 assays performed with the system and correlation with routine methods in the main laboratory were assessed. The system was then transported to different departments within the hospital, where limited test profiles were offered and the time taken to produce results was recorded. It proved practicable to offer a six-test electrolyte profile to a five-bed intensive-care unit but not to an 18-bed renal unit, where more selective analysis would be required. In a low-throughput outpatient clinic (five patients per hour) it was feasible to provide a six-test on-site profile on every patient, whereas the maximum number of tests was four in a high-throughput clinic (10 patients per hour). The cost of providing a flexible extra-laboratory biochemistry service must be balanced against the benefit of having on-site results, e.g., fewer outpatient-clinic visits.

A critical evaluation of AgNOR counting in benign naevi and malignant melanoma.

There is considerable variation in the quoted mean numbers of AgNORS per nucleus for benign melanonaevi and malignant melanomas. This is partly attributable to different approaches to AgNOR counting. This study summarizes our experience in devising an optimal technique for counting AgNORs. We show that it is essential to count intra-nucleolar AgNORs in addition to those lying outside the nucleolus to obtain clear separation of naevi from melanoma. Although this seems an onerous task, we further demonstrate that a maximum of only 30 nuclei need to be counted to obtain a mean AgNOR count per nucleus which is representative of the whole lesion. This compares with the arbitrary figure of 100 nuclei chosen by most workers. Only by optimizing and standardizing all aspects of the AgNOR technique including fixation, staining, and counting will mean AgNOR counts per nucleus become a useful quick, reproducible method which can be applied to lesions which pose diagnostic problems such as borderline melanocytic lesions.

Mitotic indices, anti-PCNA immunostaining, and AgNORs in thick cutaneous melanomas displaying paradoxical behaviour.

Those melanomas which fail to behave as expected from their Breslow thickness provide interesting material for study. In an attempt to explain differences in behaviour, we have evaluated three distinct proliferative markers in 23 thick melanomas which failed to metastasize and in 20 well-matched control tumours with documented metastasis. The test group demonstrated significantly greater numbers of mitoses when expressed as an index (mitoses per 1000 cells), whilst no difference was found when the results were expressed in terms of mitoses per unit area. Tumours showing epidermal ulceration possessed higher mitotic indices than those of non-ulcerated lesions. High fractions of PCNA immunolabelling combined with low mitotic indices were observed frequently in the non-metastasizing group. This result and its possible relation to survival advantage are discussed in detail. Both AgNOR numbers and patterns failed to act as prognostic variables--indeed, AgNORs failed to correlate with the other proliferative indices, suggesting that their value as a marker of tumour growth is severely limited.

Drug therapy in patients with diabetes mellitus: an audit.

The drug therapy prescribed for 412 diabetic patients attending an outpatient clinic over a 12 week period was recorded to try and identify potential therapeutic problems. Over 90% of the patients were prescribed at least one drug (including insulin) with oral hypoglycaemic agents prescribed for 86% of non-insulin requiring diabetics. 19% of patients were prescribed more than three drugs and few patients took drug combinations. Of patients prescribed either glibenclamide or chlorpropamide, 63% were aged 65 yr or older. Despite their potential adverse clinical and biochemical effects, diuretics and beta-blockers were commonly prescribed, especially in hypertension. The prescribing of "newer" anti-hypertensive drugs, combination products in patients taking a multiple drug regimen, and the potential dangers of sulphonylureas in the elderly are three areas where alteration of prescribing habits may be of value.

The effects of a university fitness programme on health-related variables in previously sedentary males.

This study reports on the effects of a 10-week university fitness programme on health-related fitness variables. Twenty-one male exercisers, aged 37.0(10.3) years (mean(s.d.); range 21-58), and 22 male controls, aged 38.6(7.9) years (mean(s.d.); range 17-54), volunteered to take part. Two sample t-tests and 95% confidence intervals were used to determine if the exercise group demonstrated a greater average improvement than the control group and the average improvement in both groups separately. The exercise group showed a greater average improvement over the controls from Test 1 (before fitness programme) to Test 2 (after) in the following: steady-state heart rate (beats min-1) 95% confidence intervals (-7.8, -16.2); predicted VO2max (ml kg-1 min-1): 95% confidence intervals (3.2, 6.6); sit-ups (repetitions): 95% confidence intervals (3.1, 7.0); flexibility (cm): 95% confidence intervals (3.3, 6.9). There was no significant difference between the exercise group and control group in body weight, percentage body fat, blood pressure, total plasma cholesterol, high-density lipoprotein and triglycerides. The exercise programme improved aerobic fitness, local muscular endurance and flexibility. However, the increase in aerobic fitness did not coincide with beneficial changes in the coronary risk profile.

Influence of H-2 genes on growth of Mycobacterium tuberculosis in the lungs of chronically infected mice.

Mice infected by intraperitoneal injection of Mycobacterium tuberculosis were studied over a 23-week period. They showed progressive infection in the lung (with increasing microbial count and granuloma size) whereas viable bacillary counts remained largely stationary in the spleen and in the liver. The influence of H-2 genes on the progression of the lung infection was studied in four congenic strains of animals with B10 and three congenic strains of animals with BALB backgrounds. H-2k mice had significantly higher bacterial counts in the lung than H-2b mice on both B10 and BALB backgrounds, BALB. K (H-2k) mice were also more susceptible than BALB/c (H-2d) mice. Results with recombinant strains showed that bacillary counts and granulomatous infiltration were lower in the B10 (KbAbE-Db) compared with B10.A(3R) (KbAbEbDd) strain and in B10.A(4R) (KkAkE-Db) compared with B10.BR (KkAkEkDk) mice. This resistance to the late expansion of tuberculous infection in the lungs may be associated with the lack of an expressed I-E molecular or with the expression of the Db molecule.

Measurement of the immunoperoxidase staining of macrophages within liver granulomata of mice infected with Mycobacterium tuberculosis.

A quantitative image analysis technique developed for the measurement of the extent of macrophage activation and epithelioid cell differentiation was performed on mice infected experimentally with Mycobacterium tuberculosis. The granulomatous inflammatory response within the liver reached a peak at day 23 and declined by day 33. Animals of strain B10.BR (H-2k) showed an increased granuloma fraction as compared to Balb/k (H-2k) mice, thus confirming the influence of non-H2 genes in the control of granuloma formation in mice. Using a monoclonal antibody against CD11b/CD18 (Mac1;CR3), we observed two subpopulations of macrophages within the granulomata. The small, darkly staining cells at the periphery of granulomata appear to be newly recruited macrophages. Larger, paler staining cells toward the center of granulomata represent activated and mature epithelioid macrophages. Using a semiautomated image analyzer (Quantimet 970), we measured the relative numbers of these macrophage subpopulations. There were more activated macrophages (epithelioid cells) associated with the increased granuloma fraction in the B10.BR mice than in the Balb/k. However, similar numbers of newly recruited peripheral macrophages were found in both Balb/k and B10.BR strains. This technique has shown qualitative as well as quantitative differences in the granulomatous inflammatory response in this murine model of tuberculosis in strains of mice with quite different antibody repertoires to mycobacterial antigens.

Measurement of the tissue distribution of immunoperoxidase staining with polyclonal anti-BCG serum in lung granulomata of mice infected with Mycobacterium tuberculosis.

Mice inoculated with Mycobacterium tuberculosis, strain H37Rv were used as a model of human tuberculosis. The microanatomical location of immunoperoxidase staining with a polyclonal anti-BCG serum was within macrophages and appeared granular rather than delineating whole bacilli. Immunoperoxidase staining appears to demonstrate degraded mycobacterial antigens from disrupted organisms and so reflects prior turnover of bacilli. On Ziehl-Neelsen staining, intact or almost intact bacilli are seen and so the extent of this form of staining reflects the current bacillary load. Both methods have limited sensitivity, but with larger mycobacterial loads the area of immunoperoxidase stain measured on a semi-automated image analyser correlated with the numbers of bacilli observed. The immunoperoxidase method will be useful in the evaluation of residual antigen in studying the pathogenesis of experimental murine tuberculosis. In human mycobacterial granulomata, this immunohistochemical technique should provide an alternative method of estimating the extent of bacillary load: this approach may also provide evidence of mycobacterial infection from residual antigen deposits in the tissue when whole bacilli have been successfully cleared.

Morphometric analysis of Mycobacterium tuberculosis infection in mice suggests a genetic influence on the generation of the granulomatous inflammatory response.

There is evidence in natural human disease and experimental infection in mice that host genetic factors influence susceptibility to infection with Mycobacterium tuberculosis and the progress of the disease. In mouse models, both H-2 and non-H-2 genes have been implicated. In this study, four inbred strains of mice (Balb/b, Balb/k, B10, B10.BR), selected for combinations of two different H-2 haplotypes on two different non-H-2 backgrounds, were inoculated with M. tuberculosis, strain H37Rv, by intraperitoneal injection. The histological features of the granulomatous inflammatory response in the liver and lungs were investigated during the first 18 weeks of the infection. Granuloma fraction, mean granuloma area, bacillary load, and the density of acid-fast bacilli within granulomata were measured. Animals of all four strains showed the same general pattern of infection with an early, and later self-limiting, infection of the liver and delayed onset, but progressive, infection of the lung. The non-H-2 related genetic background appears to influence the morphology of the granulomatous inflammatory response. In comparison, H-2 differences appeared to be small and inconsistent.