Gene cassette PCR: sequence-independent recovery of entire genes from environmental DNA.

The vast majority of bacteria in the environment have yet to be cultured. Consequently, a major proportion of both genetic diversity within known gene families and an unknown number of novel gene families reside in these uncultured organisms. Isolation of these genes is limited by lack of sequence information. Where such sequence data exist, PCR directed at conserved sequence motifs recovers only partial genes. Here we outline a strategy for recovering complete open reading frames from environmental DNA samples. PCR assays were designed to target the 59-base element family of recombination sites that flank gene cassettes associated with integrons. Using such assays, diverse gene cassettes could be amplified from the vast majority of environmental DNA samples tested. These gene cassettes contained complete open reading frames, the majority of which were associated with ribosome binding sites. Novel genes with clear homologies to phosphotransferase, DNA glycosylase, methyl transferase, and thiotransferase genes were identified. However, the majority of amplified gene cassettes contained open reading frames with no identifiable homologues in databases. Accumulation analysis of the gene cassettes amplified from soil samples showed no signs of saturation, and soil samples taken at 1-m intervals along transects demonstrated different amplification profiles. Taken together, the genetic novelty, steep accumulation curves, and spatial heterogeneity of genes recovered show that this method taps into a vast pool of unexploited genetic diversity. The success of this approach indicates that mobile gene cassettes and, by inference, integrons are widespread in natural environments and are likely to contribute significantly to bacterial diversity.

Departmental audit in histopathology.

Audit is now part of any laboratory service. Histopathology is no exception, and we have set up a system which allows us to review 4% of our specimens. These specimens are identified using a random number generator and reviewed by a consultant pathologist. Both slides and wet specimens are reviewed and graded according to a set scheme. The results from the first year of operation (1990) show a high rate of accuracy with no serious diagnostic disagreements between the auditor and the reporting pathologist. However, some errors which we would wish to prevent were detected and the audit has allowed us to take corrective measures. In our opinion, this form of audit is useful and necessary to maintain good clinical practice. The cost is considerable--histopathology is by its nature labour intensive. Recognition of this fact by health boards is essential if such systems are to continue.

Plexiform fibrohistiocytic tumour: clinicopathological, immunohistochemical and ultrastructural analysis in favour of a myofibroblastic lesion.

Plexiform fibrohistiocytic tumour is a recently described, seemingly benign neoplasm of superficial soft tissue which is poorly recognized and the differentiation pattern of which remains obscure. Fourteen new cases are presented here. These presented predominantly in the upper limb of infants and children, although the age-range was wide. A morphological spectrum depending on the relative proportions of the spindle cellular and nodular histiocyte-like components was evident. Immunohistochemical analysis revealed positivity of tumour cells in both components for smooth muscle actin, suggestive of myofibroblastic differentiation, as was borne out ultrastructurally in two cases. In addition, a minority of the histiocyte-like cells were also CD68 positive but negative for leucocyte common antigen, HLA-DR, Mac387 and lysozyme. In view of the ultrastructural and other immunohistochemical results, this is regarded as further evidence that the CD68 epitope recognized by KP-1 is not confined to cells of monocyte/macrophage or myeloid lineage. Plexiform fibrohistiocytic tumour appears to be a clinicopathologically distinctive myofibroblastic neoplasm which may warrant reclassification in due course.

Successful chemical cholecystectomy: a laparoscopic guided technique.

A laparoscopic guided technique of percutaneous chemical destruction of the gall bladder is described. The procedure entails the creation of a percutaneous access cholecystostomy for stone removal/fragmentation. After endoscopic occlusion of the cystic duct, the alkali corrosive, sodium carbonate was instilled into the gall bladder for 10-15 minutes. The treatment was applied in 11 animals with three deaths because of technical mishaps and eight survivors, in five of which a histologically confirmed complete destruction of the gall bladder (chemical cholecystectomy) was achieved without damage to the rest of the biliary tract or liver parenchyma.

Epithelioid haemangioendothelioma of the liver: objective response to hepatic intra-arterial 5-FU.

A case of an epithelioid haemangioendothelioma of the liver is presented. The tumour was unresectable at laparotomy because of extensive involvement of both lobes of the liver. The histological appearances of the biopsy taken at operation suggested that the lesion was at the more malignant end of the spectrum for these tumours. The patient was treated by cycles of hepatic intra-arterial 5-fluorouracil with relief of symptoms and prolonged survival. It is important to recognize this type of neoplasm which bears resemblance to other liver pathologies, in particular, to sclerosing cholangiocarcinoma.

Percutaneous stone clearance of the gallbladder through an access cholecystostomy. Laparoscopic-guided technique.

A laparoscopic-guided technique of percutaneous gallstone fragmentation/removal has been developed in the pig. The procedure entails the creation of a percutaneous access cholecystostomy. The access tract can be safely dilated after 7 days to F16, thereby allowing the introduction of both the Olympus flexible and the Berci-Shore rigid choledochoscopes. Following endoscopic occlusion of the cystic duct by a biliary balloon catheter, stone fragmentation can be conducted under direct visual control. In this particular study, electrohydraulic lithotripsy was performed of human cholesterol and bile-pigment stones inserted into the gallbladder of 16 pigs. The gallstone debris resulting from lithotripsy was then washed out with saline. Larger residual fragments could easily be extracted with the Dormia basket under visual guidance. There was a significant positive correlation between stone size (r = 0.98) and weight (r = 0.96) and the number of pulses needed to achieve satisfactory stone fragmentation. The gross composition of the stones (predominantly cholesterol or pigment) did not influence the number of pulses required. Electrohydraulic lithotripsy caused an explosion effect (the fragments hit the gallbladder wall), causing submucosal haematoma formation. This, however, was not followed by any untoward effect until sacrifice of the animals 10-16 weeks later. Electrohydraulic shocks delivered to the gallbladder wall itself resulted in larger haematoma formation and breach of the gallbladder mucosa with active bleeding into the gallbladder lumen, but again no instance of gallbladder perforation was encountered.(ABSTRACT TRUNCATED AT 250 WORDS)

Parietal cell carcinoma of the stomach: association with long-term survival after curative resection.

Following the recent identification of gastric parietal cell carcinoma (Capella et al., 1984), a histological and clinical review of 125 consecutive cases of gastric cancers treated surgically during a 9-year period was undertaken. The pathology was reviewed blind and in addition to H & E sections, staining with Luxol Fast Blue, phosphotungstic acid haematoxylin and E-M studies were performed to identify parietal cell differentiation. The surgical procedures performed were curative R2 gastrectomy (n = 56), palliative resection (n = 30), gastro-enterostomy (n = 25) and intubation (n = 14). The 30-day operative mortality was 12/125 (10%) overall and 4/56 (7%) in the curative resection group. Two parietal cell cancers were identified and a further 4 tumours showed areas of parietal cell differentiation. All occurred in male patients (mean age 55 years, range 43-62). Sixteen patients out of the 56 patients (29%) who underwent curative R2 resection have survived long-term (mean 5.5 years, range 2.5-11): 4/5 mucosal/submucosal cancers (T1N0), 5/29 intestinal cancers (T2N0-2) 2/16 diffuse cancers (T2N1) and 5/6 with parietal cell cancer/differentiation (T2-3N0-2). There were no survivors beyond 14 months in the patients who were treated by palliative resection, bypass or intubation irrespective of histology. This study suggests that gastric parietal cell carcinoma carries a good prognosis after curative resection despite the advanced stage at presentation.

Evaluation of the Ca 1 antibody in the diagnosis of invasive breast cancer.

An evaluation of Ca 1 antibody staining was performed on paraffin sections from 136 breast lesions (64 benign and 72 malignant). Although cytoplasmic staining was encountered significantly more often in malignant lesions, the false negative rate was 6.9% and the false positive rate 56.2%. Benign lesions which showed positive staining included gynaecomastia, cystic mastopathy and fibroadenomata. Various other monoclonal antibodies showed staining similar to Ca 1 antibody. Ca 1 antibody was observed to bind to epithelial membrane antigen-coated sepharose beads.

Evaluation of the Cal antibody in the detection of malignant cells in fine needle aspirates from breast lumps.

In a study involving 86 fine needle cell aspirates from patients with breast lumps attending a primary Breast Referral Clinic, we have been unable to confirm that cellular binding of Cal antibody reliably differentiates between benign and malignant disease of the breast. The test carried unacceptable predictive values (46% when positive and 70% when negative). The false positive and negative rates were 47.2% and 36.4% respectively. A skilled cytologist using conventional Papanicolaou staining gave a much more accurate interpretation of the benign or malignant nature of the aspirates than did Cal antibody binding.

Total and subtotal replacement of the common bile duct: effect of transhepatic silicone tube stenting.

The effect of silicone tube stenting on the outcome of total and subtotal replacement of the bile duct with autologous vein was investigated in experiments performed in pigs. All but one of the unstented animals developed dense strictures of the extrahepatic ductal system. This complication was abolished by the use of silicone stents passed transhepatically. The vein autografts in the stented animals underwent progressive shortening with elongation of the distal common bile duct so that beyond four months only one suture line was encountered, and no recognisable venous element was detectable on histological examination. Apart from minor dilatation of the intrahepatic biliary tree encountered in some animals, all the surviving stented animals progressed well and had normal serial liver function tests.

Tuberculin testing in hospital personnel.

Tuberculin testing of 176 personnel in a Liverpool hospital showed a total incidence of 68% negative reactors. This remarkably high percentage was similar for each of the various groups of workers tested. We did not find the Tine test to be a satisfactory method of screening for tuberculin sensitivity because 30% were doubtful reactors. In these, further tuberculin testing by the Mantoux reagent was always positive.

Bacteria in flower vase water: incidence and significance in general ward practice.

Culture of flower vase water from wards in the David Lewis Northern Hospital, Liverpool, revealed large numbers of potentially pathogenic bacteria. The types of organisms isolated may reflect the particular ecology of this hospital as they differed in some ways from those reported from other centres. The incidence of wound infections during the period of study was low (5-2 per cent) despite the regular overgrowth of bacteria in flower vases, and there did not appear to be any definite correlation between the types of bacteria isolated from flower vase water and those responsible for wound infections. The addition of hydrogen peroxide to flower vases proved a very effective antiseptic and is recommended for general use as a precautionary measure.