Flow cytometric quantification of tumour endothelial cells; an objective alternative for microvessel density assessment

Assessment of microvessel density by immunohistochemical staining is subject to a considerable inter-observer variation, and this has led to variability in correlation between microvessel density and clinical outcome in different studies. In order to improve the method of microvessel density measurement in tumour biopsies, we have developed a rapid, objective and quantitative method using flow cytometry on frozen tissues. Frozen tissue sections of archival tumour material were enzymatically digested. The single-cell suspension was stained for CD31 and CD34 for flow cytometry. The number of endothelial cells was quantified using light scatter- and fluorescence-characteristics. Tumour endothelial cells were detectable in a single cell suspension, and the percentage of endothelial cells detected in 32 colon carcinomas correlated highly (r=0.84, P<0.001) with the immunohistochemical assessment of microvessel density. Flow cytometric endothelial cells quantification was found to be more sensitive especially at lower levels of immunohistochemical microvessel density measurement. The current method was found to be applicable for various tumour types and has the major advantage that it provides a retrospective and quantitative approach to the angiogenic potential of tumours

Expression of endoglin (CD105) in cervical cancer

In this study, we have investigated the role of endoglin (CD105), a regulator of transforming growth factor (TGF)-β1 signalling on endothelial cells, basic fibroblast growth factor (bFGF) and vascular endothelial growth factor-A (VEGF-A) in cervical cancer. We have measured the number and determined the location of both newly formed (CD105-positive) and the overall number of (CD31-positive) blood vessels, and bFGF and VEGF-A expression using immunohistochemistry in 30 cervical carcinoma specimens. Vascular endothelial growth factor-A mRNA expression was determined using RNA-in situ hybridisation. CD105- and CD31-positive vessels and bFGF- and VEGF-A-positive cells were predominantly present in the stroma. The presence of CD105- and CD31-positive vessels in the stroma did neither correlate with the number of VEGF-A-positive cells nor the number of bFGF-positive cells. However, the number of CD105- and CD31-positive vessels was associated with the expression of VEGF-A mRNA in the epithelial cell clusters (P=0.013 and P=0.005, respectively). The presence of CD105-positive and CD31-positive vessels was associated with the expression of αvβ6 (a TGF-β1 activator; P=0.013 and P=0.006, respectively). Clinically, the number of CD105-positive vessels associated with the number of lymph node metastasis (P<0.001). Furthermore, the presence of CD105-positive vessels within the epithelial cell clusters associated with poor disease-free survival (P=0.007)

Intra-tumoural microvessel density in human solid tumours

Over the last decade assessment of angiogenesis has emerged as a potentially useful biological prognostic and predictive factor in human solid tumours. With the development of highly specific endothelial markers that can be assessed in histological archival specimens, several quantitative studies have been performed in various solid tumours. The majority of published studies have shown a positive correlation between intra-tumoural microvessel density, a measure of tumour angiogenesis, and prognosis in solid tumours. A minority of studies have not demonstrated an association and this may be attributed to significant differences in the methodologies employed for sample selection, immunostaining techniques, vessel counting and statistical analysis, although a number of biological differences may account for the discrepancy. In this review we evaluate the quantification of angiogenesis by immunohistochemistry, the relationship between tumour vascularity and metastasis, and the clinicopathological studies correlating intra-tumoral microvessel density with prognosis and response to anti-cancer therapy. In view of the extensive nature of this retrospective body of data, comparative studies are needed to identify the optimum technique and endothelial antigens (activated or pan-endothelial antigens) but subsequently prospective studies that allocate treatment on the basis of microvessel density are required

HIGH-RESOLUTION ULTRASONOGRAPHY OF THE CUTANEOUS NERVE BRANCHES IN THE HAND AND WRIST

Ultrasonography can be used in the diagnosis of various neuropathies, including nerve injury. Nerves often involved in traumatic and iatrogenic injury are small cutaneous branches in the hand and wrist, which cannot be seen in detail using current ultrasound probes. This study explored the potential of high-resolution ultrasonography in seeing these nerve branches in the human. The VisualSonics Vevo 770 system with a 15-82.5 MHz probe was compared to a commonly used 5-12 MHz probe and ultrasound machine. The accuracy was validated by ultrasound guided dye injection into cadaver nerves, with subsequent anatomical dissection and verification. Results were confirmed in two healthy volunteers. The Vevo 770 system was able to accurately identify the small cutaneous nerves. It could also depict the median nerve and its fascicles in greater detail. This may be useful for clinical diagnosis, localisation and follow-up of neuropathies and nerve injuries

Minimally Invasive Autopsy: An Alternative to Conventional Autopsy?

Purpose: To determine the diagnostic performance of minimally invasive autopsy (MIA) for detection of causes of death and to investigate the feasibility of MIA as an alternative to conventional autopsy (CA) in the clinical setting. Materials and Methods: The institutional review board approved the MIA procedure and study, and informed consent was obtained for all deceased patients from relatives. Thirty deceased patients (19 men, 11 women; age range, 46-79 years), for whom family permission for CA on medical grounds had already been obtained, underwent additional evaluation with MIA prior to CA. MIA consisted of whole-body 16-section computed tomography (CT) and 1.5-T magnetic resonance (MR) imaging, followed by ultrasonography-guided 12-guage needle biopsy of heart, both lungs, liver, both kidneys, and spleen. Percentage agreement between MIA and CA on cause of death was evaluated. Sensitivity and corresponding 95% confidence intervals (CIs) of MIA for detection of overall (major plus minor) findings, with CA as the reference standard, were calculated. Specificity was calculated for overall findings. Sensitivity analysis was performed to explore the effect of the clustered nature of the data. Results: In 23 patients (77%), MIA and CA were in agreement on the cause of death. Sensitivity of MIA for detection of overall findings and detection of major findings was 93% (95% CI: 90%, 96%) and 94% (95% CI: 87%, 97%), respectively. Specificity was 99% (95% CI: 98%, 99%) for detection of overall findings. MIA failed to demonstrate acute myocardial infarction as the cause of death in four patients. Sensitivity analysis indicated a negligible correlation between observations within each patient. CT was superior to MR for detection of pneumothorax and calcifications. MR was superior to CT for detection of brain abnormalities and pulmonary embolus. With biopsy only, detection of disease in 55 organs was possible, which included 27 major findings. Conclusion: MIA is a feasible procedure with high diagnostic performance for detection of common causes of death such as pneumonia and sepsis; MIA failed to demonstrate cardiac diseases, such as acute myocardial infarction and endocarditis, as underlying cause of death. (c) RSNA, 200