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.

Learning curves of microdissection testicular sperm extraction for nonobstructive azoospermia.

OBJECTIVES: Microdissection testicular sperm extraction (micro-TESE) has been performed at some experienced male infertility centers. Micro-TESE in patients with nonobstructive azoospermia (NOA) has been shown to be not only feasible and safe but also a technically demanding operation. To evaluate whether the improvement of surgical outcomes of micro-TESE, we hereby report the learning curves of micro-TESE for NOA patients. DESIGN: Retrospective clinical analysis. SETTING: Male infertility center. PATIENT(S): Since 2006, micro-TESE was performed in 150 patients with NOA by a single surgeon. INTERVENTION(S): After the tunica albuginea was opened, direct examination of the testicular parenchyma was performed at x25 magnification. MAIN OUTCOME MEASURE(S): Sperm retrieval rate and clinical factors in the first 50 patients (group A), the middle 50 patients (group B), and the last 50 patients (group C) were examined. RESULT(S): There were no differences in clinical factors among the three groups. Total operation times were shorter in group B and C than in group A (P<0.05). The sperm retrieval rate in group B (44%) and C (48%) was significantly higher than in group A (32%; P<0.05). CONCLUSION(S): As cases increase, surgical outcomes and sperm retrieval rate have improved. We think that this report showed substantial learning curves for a microscopic surgery such as micro-TESE.

Gene expression analysis of neural cells and tissues using DNA microarrays.

DNA microarrays pose specific challenges to those studying the central and peripheral nervous systems. Probably the most important involve difficulty in obtaining appropriate tissue for study, as well as the problems posed by cellular heterogeneity. This unit describes advances in the available technologies and provides protocols for cDNA microarray hybridization, including the use of PCR amplicons. Protocols are also provided for the two major methods for limiting cellular heterogeneity by study of RNA from single cell populations in high-throughput microarray studies, laser capture microdissection (LCM), and automated fluorescent cell sorting (FACS-array).

Laser-capture microdissection in prostate cancer research: establishment and validation of a powerful tool for the assessment of tumour-stroma interactions.

OBJECTIVES: To describe our experience with the optimization and validation of laser-capture microdissection (LCM) for biomarker analysis in prostate tissues. As LCM allows the separation of benign and malignant epithelial structures and stromal elements, it not only allows identification of the source of the biomarker, but might also accentuate gene or protein expression changes by reducing contamination by other cellular elements. MATERIALS AND METHODS: In all, 19 fresh-frozen prostate tissue samples were subjected to LCM, with the cDNA being analysed using quantitative polymerase chain reaction for several genes, to identify the optimum number of cells for capture, as well as gene markers assessing for the purity of the captured cells. The localization was further confirmed by in situ hybridization. RESULTS: Prostate-specific antigen (PSA) and cytokeratin 8, were expressed solely by epithelial cells, whereas hepatocyte growth factor (HGF) and tissue inhibitor of metalloproteinases-3 (TIMP3) were expressed only by stromal cells, and the levels of transcripts of these genes were unaltered between benign and malignant tissues. CONCLUSIONS: These data suggest that PSA, cytokeratin 8, HGF and TIMP3 are reliable gene markers of purity of epithelial and stromal compartments for LCM of prostate tumours. Although this technique is not new and is increasingly used in laboratories, it needs optimization and stringent validation criteria before data analysis. This applies to all tissue types subjected to LCM.

Effect of mechanical stretch on HIF-1{alpha} and MMP-2 expression in capillaries isolated from overloaded skeletal muscles: laser capture microdissection study.

Under physiological nonhypoxic conditions, angiogenesis can be driven by mechanical forces. However, because of the limitations of the specific gene expression analysis of microvessels from in vivo experiments, the mechanisms regulating the coordinated expression of angiogenic factors implicated in the process remain intangible. In this study, the technique of laser capture microdissection (LCM) was adapted for the study of angiogenesis in skeletal muscles. With a combination of LCM and real-time quantitative PCR it was demonstrated that capillary endothelial cells produce matrix metalloproteinase (MMP)-2 and that mechanical stretch of capillaries within muscle tissue markedly increases MMP-2 mRNA (2.5-fold increase vs. control; P < 0.05). In addition, we showed that transcription factor hypoxia-inducible factor (HIF)-1alpha expression was 13.5-fold higher in capillaries subjected to stretch compared with controls (P < 0.05). These findings demonstrate the feasibility of this approach to study angiogenic gene regulation and provide novel evidence of HIF-1alpha induction in stretched capillary endothelial cells.