Semi-automated standardisation of melanin bleaching procedures of heavily pigmented melanocytic lesions for immunohistochemical analysis on an automated platform.

Background: The diagnosis of heavily pigmented melanocytic lesions is problematic. This is often compounded by lack of visibility of nuclear detail of tumour cells due to physical masking by melanin pigment. Similarly, there can be colour merging of chromogenic final reaction products with melanin, making an evidence of antigenic localisation problematic. There are a number of melanin bleaching techniques available for immunohistochemical assessments.Material and methods: All methods to date have involved the bleaching of melanin as a manually performed primary step before loading subsequently bleached slides onto automated immunohistochemical platforms. Here we define a semi-automated bleaching procedure that allows full integration on one of the most widely employed automated IHC staining platforms (Roche Ventana BenchMark Ultra). The bleaching protocol was defined on the BenchMark Ultra and involved the assessment of 24 histological cases of heavily pigmented malignant melanoma lesions (13 cutaneous and 11 metastatic) routinely fixed processed and paraffin wax embedded.Results: Completion of the bleaching was assessed on H&E preparations performed following the semi-automated bleaching step and employing the Roche Ventana BenchMark Ultra machine for 60 min at 42°C. Complete immunohistochemical staining was achieved on the automated platform within 5-6 h including the bleaching step. Results were consistent across all tissue evaluated.Discussion: This data provides evidence that the hydrogen peroxide bleaching procedure can be adapted for integration on one of the most widely employed automated IHC staining platforms and as a result, improve the efficiency and reproducibility of the technique.

A multicentre study of the precision and accuracy of the TruSlice and TruSlice Digital histological dissection devices.

BACKGROUND: Five key factors enabling a good surgical grossing technique include a flat uniformly perpendicular specimen cutting face, appropriate immobilisation of the tissue specimen during grossing, good visualisation of the cutting tissue face, sharp cutting knives and the grossing knife action. TruSlice and TruSlice Digital are new innovative tools based on a guillotine configuration. The TruSlice has plastic inserts whilst the TruSlice Digital has an electronic micrometre attached: both features enable these dissection factors to be controlled. The devices were assessed in five hospitals in the UK. MATERIAL AND METHODS: A total of 267 fixed tissue samples from 23 tissue types were analysed, principally the breast (n = 32) skin (30), rectum (28), colon (27) and cervix (17). Precision and accuracy were evaluated by measuring the defined thickness, and the consistency of achieving the defined thickness of tissue samples taken respectively. Both parameters were expressed as a total percentage of compliance for the cohort of samples accessed. RESULTS: Overall, the mean (standard deviation) score for precision was 81 (11) % whilst the accuracy score was 82 (11) % (both p < 0.05, chi-squared test), although this varied with type of tissue. Accuracy and precision were strongly correlated (rp = 0.83, p < 0.001). CONCLUSION: The TruSlice Digital devices offer an assured precision and accuracy performance which is reproducible across an assortment of tissue types. The use of a micrometre to set tissue slice thickness is innovative and should comply with laboratory accreditation requirements, alleviating concerns of how to tackle issues such as the 'measurement of uncertainty' at the grossing bench.

Development of new and accurate measurement devices (TruSlice and TruSlice Digital) for use in histological dissection: an attempt to improve specimen dissection precision.

Histological dissection of human tissue has relied on conventional procedures, which have largely remained unchanged for decades. Practices to determine measurement parameters employed in these procedures have largely relied on the use of rulers and weighing scales. It is well documented in the scientific literature that both fixation and processing of tissue can significantly affect the viability of the of tissue sections both for tinctorial and immunocytochemical investigations. Both of these factors can be compounded in their negative effects by inappropriate sampling of tissue at histological cut up. There are five key factors to ensure good surgical grossing technique, flat uniformly perpendicular specimen cutting face, appropriate immobilisation of the tissue specimen during grossing, good visualisation of the cutting tissue face, sharp cutting knives and the grossing knife action. Meeting these factors implies the devices are fit for purpose. Here we describe an innovative approach to designing cut up devices to improve accuracy and precision, which take these five key requirements into consideration. The devices showed accuracy and precision, enabling tissue slices to be produced in a uniformly perpendicular fashion to within 2 mm in thickness and to enable consistency and reproducibility of performance across a series of tissue types. The application of a digital rule on one of these devices ensures accuracy and also enables quality control issues to be clearly assessed. As cellular pathology laboratories conform to ever increasing standards of compliance and performance in practice, the advent of assured precision and accuracy at cut up is awaited. Recommendations from accreditation bodies such as the United Kingdom Accreditation Service (UKAS) continue to push for improvements in this area of histological investigation. These newly designed devices may give the answers to these requirements and provide the impetus for a new generation of innovative equipment for histological dissection.

Investigation into a new softening agent for use on formalin-fixed, paraffin wax-embedded tissue.

The use of tissue softening agents to improve microtomy of keratotic tissues is employed widely. Many of these softeners contain hazardous constituents such as phenol. In this study, the use of non-ionic surfactants or non-toxic ingredients are investigated with the aim of creating a new softening agent. The new agent should be more effective in facilitating the sectioning of hardened tissue while reducing toxicity and complications associated with sectioning hard tissue compared to a commercially available phenol-based formulation. Four formulations are compared against the commercial product for their capability to section routinely processed paraffin-embedded tissue under standard operating procedure parameters. The trial formulations were shown to be fast acting and enabled improved serial sectioning of hard keratotic tissue in nearly all the cases tested. There was no evidence of adverse staining using either tinctorial or immunohistochemical methods. The new formulations had advantages over the commercially available solutions, improving on the number and quality of sections attainable from the tissue blocks, as well as offering a composition less toxic than phenol-based products.