Quantification of tumour vascularity in squamous cell carcinoma of the tongue using CARD amplification, a systematic sampling technique, and true colour image analysis
Affiliations: Department of Pathology, University Medical Centre Nijmegen, The Netherlands | Department of Cranio and Maxillofacial Surgery, University Hospital Maastricht, The Netherlands | Department of Otorhinolaryngology, Head and Neck Surgery, University Hospital Maastricht, The Netherlands
Note: [] Corresponding author: E.J.M. Hannen, DMD, MD, Department of Cranio and Maxillofacial Surgery, University Hospital Maastricht, PO Box 5800, 6202 AZ Maastricht, The Netherlands. Tel.: +31 43 387 2010; Fax: +31 43 387 2020, E‐mail: ehan@mkg.azm.nl.
Abstract: The aims of this study of head and neck tissue samples were to develop an immunohistochemical protocol based on the catalysed reporter deposition (CARD) technique to enhance staining results for use in automated true colour image analysis, to assess the reproducibility of systematic tissue sampling in the angiogenic hot spot selection, and quantification of microvessel density (MVD) and other vessel characteristics. The latter data were compared between six metastasised tongue squamous cell carcinomas, vs. four non‐metastasised. In comparison to the standard immunohistochemical protocol with anti‐CD34 antibodies, CARD amplification resulted in both more intensely stained and larger numbers of vessels. Averaging the 10 most vascularised fields of the 40 to 60 systematically sampled fields in a tissue section resulted in an overall acceptable interobserver reproducibility for most assessed vessel parameters (r≥0.76 and p≤0.01). The percentage vessels with diameter <5 μm was significantly higher in the non‐metastasised tongue carcinomas (p=0.02). However, for a number of tumours the effect of tissue sampling was significant. We conclude that CARD amplification is needed for reliable segmentation of vessels by image analysis systems, and that tumour heterogeneity is a limiting factor for all procedures in which tumour vascularity is assessed in a single tissue section. Figures on http://www.esacp.org/acp/2001/22‐4/hannen.htm.
