Analytical Cellular Pathology 25 (2003) 63–75 63 IOS Press An original approach for quantification of blood vessels on the whole tumour section Nga Tran Kim a,d,∗, Nicolas Elie a,d, Benoît Plancoulaine b,d, Paulette Herlin a,d and Michel Coster c,d a GRECAN, Equipe Universitaire d’Accueil 1772, Centre F. Baclesse, Route de Lion sur Mer, F-14076 Caen, France b Département de Mesures Physiques, IUT de Caen, Boulevard du Maréchal Juin, F-14032 Caen, France c LERMAT-ISMRA, UPRES-A CNRS 6004, F-14032 Caen, France d Pôle Traitement et Analyse d’Images de Basse-Normandie, France Received 11 March 2002 Accepted 22 August 2002 Abstract. Relative abundance of tumour angiogenesis has been shown to be of clinical relevance in cancers of various locations such as the ovary. Nevertheless, several problems are encountered when quantifying tumour microvessels: (i) as many other tumour markers, vascularity pattern is often heterogeneous within the tumour mass and even within the same histological section. As a consequence, an adequate acquisition method must be developed for accurate field sampling. (ii) Manual microvessel counting is long, tedious and subject to poor reproducibility. Introduction in routine practice requires a fast, reproducible and reliable automatic image processing. In this study we present an original procedure combining a slide scanner image acquisition and a fully automatic image analysis sequence. The slide scanner offers the advantage of recording an image of the whole histological section for subsequent automatic blood vessel detection and hot spot area location. Microvessel density and surface fraction were measured for the whole section as well as within hot spots. Different immunostaining methods were tested in order to optimise the procedure. Moreover, the method proposed was sub- mitted to a quality control procedure, with reference to interactive identification of microvessels at scanner level. This experiment showed that 93 to 97% of blood vessels were detected, according to the staining protocol used. Colour figures can be viewed on http://www.esacp.org/acp/2003/25-2/kim.htm. Keywords: Angiogenesis, quantification, image analysis, slide scanner 1. Introduction Currently, tumour angiogenesis is assessed by quan- tifying, at the microscopical level, immunostained It is now well established that tumour growth and blood vessel profiles. More often, tumour microves- spread are highly dependent on neo-vascularization sels are visually counted at high magnification [3,10, [14,32]. A more disputed statement is the prognostic 11,13,17,19,27], but this method is tedious and sub- value of tumour angiogenesis. While several studies ject to poor reproducibility. Compared to this proce- have demonstrated its predictive significance for clin- dure, some authors have found the stereological Chalk- ical outcome in cancers of many locations [1,2,10,12, ley point counting method, more reliable [15,23]. An- 17–19,30,39], others failed to find any correlation be- other step towards an objective quantification of tu- tween tumour vascularity and behaviour [3,4,16,25,27, mour angiogenesis was proposed by introducing semi- 28,36]. These discrepancies could be partly explained automatic procedures [6,39]. Nevertheless, these meth- by differences in microvessel counting methodology, ods still require human intervention for interactive cor- applied to heterogeneous tumours [5,16]. rection or thresholding. Few fully automatic proce- dures have been recently introduced [7,22,40,41,47]. *Corresponding author: Tel.: +33 231 45 51 37; Fax: +33 231 45 Whatever the quantification method used, the most 51 72; E-mail: [email protected]. critical step remains the identification of the fields to be 0921-8912/03/$8.00 2003 – IOS Press. All rights reserved 64 N.T. Kim et al. / Quantification of blood vessels measured. Usually, and according to Weidner’s proce- in 0.3% H2O2. A buffered casein solution (Power dure, the quantification is performed in one or several BlockTM, Biogenex Menarrini, France) was then used highly vascularized fields (called “hot spots”), previ- to block unspecific binding sites (for 10 minutes). Af- ously selected at low magnification by a trained pathol- ter incubation with the rabbit anti-Von Willebrand Fac- ogist [43,46]. Other authors have chosen to systemat- tor antibody (primary antibody, Ab1), 13 and 12 slides ically sample fields over the whole histological sec- were respectively treated by a classical biotin–strep- tion [22] and to average blood vessel density within tavidin peroxidase technique (series 1) or submit- the most vascularized fields. However, as these authors ted to signal amplification (series 2) (En VisionTM noted, the reproducibility in systematic tumour sam- kit, Dako). Briefly, for classical procedure, after Ab1 pling is determined by both tumour heterogeneity de- incubation (dilution 1 : 50, 1 hour), a biotinylated gree and sampled tumour area, which is defined by the anti-rabbit antibody (Dako) and then a streptavidin– number of sampled fields. The choice of the micro- peroxidase complex (Dako) were applied (dilution scopical magnification also influences the final tumour 1 : 40, 30 minutes for each reagent). For signal amplifi- area analysed, as well as the reliability of the measured cation, slides were incubated with peroxidase labelled parameters [22,34]. polymer, conjugated to goat anti-rabbit immunoglob- Whether the measures obtained within hot spots or ulins for 30 minutes. Staining was completed by in- all over the section carry the same biological informa- cubation, either with diamino-benzidine (DAB) for tion remains questionable. 10 minutes (series 2a), which stains vessels brown, In this context, we propose in this paper a fully au- or with amino-ethylcarbazole (AEC) for 15 minutes tomatic method for tumour angiogenesis quantification (series 2b), which highlights them in red (6 samples over the whole histological section and inside automat- for each chromogen, on 2 consecutive sections). Light ically detected hot spots. The originality of the study is counterstaining was performed during 10 seconds with to assess the degree of neovascularization by process- Gill’s haematoxylin [20] for all specimens. ing a single numerical image of the whole tumour sec- tion, using a photographic slide scanner, provided with 2.3. Image acquisition a glass slide holder. A quality control was performed in order to assess the robustness of the quantification Images of the whole immunostained sections were method and to find the most suitable immunostaining captured using a 2700 dpi (1 pixel = 9.4 × 9.4 µm) procedure. slide scanner (SprintScan ss35Plus, Polaroid, France) provided with a special medical slide holder (PathScan EnablerTM, Meyer Instruments, Inc., USA) and con- 2. Material & methods nected to a personal computer (Pentium III, 450 MHz, 128 Mo). Large images (up to 30 megabytes, for sec- 2.1. Specimens tions up to 8 cm2)werestoredasbitmap files. These large images of the whole tumour tissue section were Formalin-fixed paraffin-embedded samples of 19 directly processed for automatic vessel detection. human ovarian tumours were cut into 5 µm thick sec- tions and mounted on polylysine coated slides for im- 2.4. Image processing munohistochemical staining. Image processing was performed by chaining oper- 2.2. Immunohistochemistry ators of an image analysis toolbox Aphelion software, v. 3.0 (Adcis, France) implemented on a personal com- Sections were immunolabelled with a polyclonal puter and running under Windows operating system. anti-Von Willebrand Factor antibody (Dako, France), The procedure can be divided into 5 steps: back- using the Optimax automatic machine (Biogenex) to ground correction, tissue detection and necrosis elim- guarantee staining reproducibility. After deparaffiniza- ination, immunostained structure detection, hot spot tion and rehydration, slides were heated 30 minutes at identification and location, and measures (Figs 1, 4, 95◦C in a citrate buffer (ChemMateTM, Dako) for anti- Table 1). gen retrieval. Following incubations were performed Before any analysis, the user can discard normal tis- at room temperature. Endogenous peroxidase activity sue surrounding the tumour, by drawing regions of in- was inhibited by incubating sections for 20 minutes terest. N.T. Kim et al. / Quantification of blood vessels 65 Fig. 1. Strategy for automatic microvessel isolation. The procedure was divided into 5 steps: background correction (1), detection of tumour tissue limits (2), isolation of vessel (3), identification and location of hot spots (4) and measures (5). Table 1 2.4.2. Tissue detection and necrosis elimination Measures obtained by the automatic procedure (case presented in A fixed threshold, set on the green component of the Fig. 3). All microvessel profile density (MVD) were expressed as number per mm2 of tumour tissue. All surface proportions were original image, allowed the detection of the histolog- + given as a percentage ical section limits (TSnec ). Tumour tissue can con- Parameters tain necrotic areas and mucus which might be deeply stained by chromogens and could appear as large red or Mean microvessel profile density, MVD 40 brown fields. They were easily removed by threshold- Mean microvessel surface proportion, MSP 4.5 ing an “excess blue image”, a grey scale image gener- Mean MVD within hot spot 144 ated from the linear combination of the 3 components Max MVD within hot spot 387 RGB [50] and computed as follows: Mean MSP within hot spot 19 Max MSP within hot spot 30 f(ExcB) = 2f(B) − f(R) − f(G). Surface