Distributed under Creative Commons CC-BY 4.0 Tools4MSP: an open source software package to support Maritime Spatial Planning
Published 1 October 2018

Stefano Menegon firstname.lastname@example.com
Institute of Marine Sciences
National Research Council
VeniceItaly

Stefano Menegon email@example.com
Institute of Marine Sciences
National Research Council
VeniceItaly

Alessandro Sarretta
Institute of Marine Sciences
National Research Council
VeniceItaly

Daniel Depellegrin
Institute of Marine Sciences
National Research Council
VeniceItaly

Giulio Farella
Institute of Marine Sciences
National Research Council
VeniceItaly

Chiara Venier
Institute of Marine Sciences
National Research Council
VeniceItaly

Andrea Barbanti
Institute of Marine Sciences
National Research Council
VeniceItaly

Distributed under Creative Commons CC-BY 4.0 Tools4MSP: an open source software package to support Maritime Spatial Planning
Published 1 October 201810.7717/peerj-cs.165Submitted 13 March 2018 Accepted 4 September 2018Corresponding author Academic editor James Procter Additional Information and Declarations can be found on page 12 OPEN ACCESSSubjects Data ScienceScientific Computing and SimulationSpatial and Geographic Information Systems Keywords PythonSDITools4MSP softwareOpen SourceCumulative Effects AssessmentMaritime spatial planningGeoNode
This paper presents the Tools4MSP software package, a Python-based Free and Open Source Software (FOSS) for geospatial analysis in support of Maritime Spatial Planning (MSP) and marine environmental management. The suite was initially developed within the ADRIPLAN data portal, that has been recently upgraded into the Tools4MSP Geoplatform (data.tools4msp.eu), an integrated web platform that supports MSP through the application of different tools, e.g., collaborative geospatial modelling of cumulative effects assessment (CEA) and marine use conflict (MUC) analysis. The package can be used as stand-alone library or as collaborative webtool, providing userfriendly interfaces appropriate to decision-makers, regional authorities, academics and MSP stakeholders. An effective MSP-oriented integrated system of web-based software, users and services is proposed. It includes four components: the Tools4MSP Geoplatform for interoperable and collaborative sharing of geospatial datasets and for MSP-oriented analysis, the Tools4MSP package as stand-alone library for advanced geospatial and statistical analysis, the desktop applications to simplify data curation and the third party data repositories for multidisciplinary and multilevel geospatial datasets integration. The paper presents an application example of the Tools4MSP GeoNode plugin and an example of Tools4MSP stand-alone library for CEA in the Adriatic Sea. The Tools4MSP and the developed software have been released as FOSS under the GPL 3 license and are currently under further development.

INTRODUCTION

Management and planning of the marine environment require a coordinated development of socio-economic activities, while ensuring a sustainable use of marine resources using an ecosystem-based approach (European Union, 2014;Center for Ocean Solutions, 2011;Douvere, 2008). In the last decade, practical tools to support the implementation of the various steps of Maritime Spatial Planning (MSP) have been developed in various contexts and also analysed to evaluate their usability for different planning purposes (Stelzenmüller et al., 2013;Pınarbaşı et al., 2017).

An extended analysis performed by the ''EBM tools network'' (https://ebmtoolsdatabase. org/) in support of Ecosystem Based Management has recollected and classified various tools, with respect to types, costs, skills, data and technological requirements. Two categories of tools have emerged in the recent years as analytical support to decision-makers: the sea use conflict analysis and the cumulative impacts assessment.

Sea use conflict analysis has been extensively applied in different geographical contexts (Hadjimitsis et al., 2015;White, Halpern & Kappel, 2012) to investigate and spatially identify conflicts between coastal and marine activities for current conditions and for the comparison of possible future scenarios. In particular, the COEXIST project developed the GRID tool (Georeferenced Interactions Database;Gramolini et al., 2010) to analyse the level of coexistence among uses, depicting areas where different sectors more likely overlap in space and time.

In parallel, various authors proposed methodologies to create cumulative impact maps to reconnect effects of human uses of the sea on environmental components, starting from the methodology firstly introduced by Halpern et al. (2008) at global scale, then implemented in several marine regions, such as Baltic Sea (Korpinen, Meidinger & Laamanen, 2013), North Sea (Andersen et al., 2013), Adriatic Sea (Depellegrin et al., 2017) and at regional scale (Barbanti et al., 2017a;Barbanti et al., 2017b). In particular, Stock (2016) developed an open source software for mapping human impacts on marine ecosystems.

The MSP process involves several user categories, from data producers (e.g., domain experts like ecologists and modellers) to stakeholders and planners. It requires a solid command of geographical information to create a more comprehensive understanding of coastal and marine areas and to support management and planning policies. The development and implementation of Spatial Data Infrastructures (SDI) at multiple levels (i.e., local, regional, national and global) matches the need to make geographical data more accessible and interoperable (Georis-Creuseveau, Claramunt & Gourmelon, 2016). However, various authors (Maguire & Longley, 2005) have highlighted the importance of the integration of Geoportals in the context of SDIs and the role of a user-driven and community-based development as fundamental aspects for an effective and efficient use of the resources (De Longueville, 2010;Georis-Creuseveau, Claramunt & Gourmelon, 2016).

This research presents components and functionalities of the Tools4MSP software package, a Python-based Free and Open Source Software (FOSS) which implements a marine use conflict (MUC) analysis module based on the COEXIST methodology (Gramolini et al., 2010) and a cumulative effects assessment (CEA) module based on the methodology developed in Menegon et al. (2018a). Its implementation in the context of a collaborative Geoplatform supporting MSP and environmental management and its utilization as stand-alone library for cumulative effects assessment (CEA) is tested for the Adriatic Sea.

BACKGROUND MUC and CEA analysis

The Maritime Use Conflict (MUC) tool implements the COEXIST methodology to estimate the spatial distribution of the conflicts between sea uses. The inputs of the tool are: (i) the area of analysis; (ii) the grid cell resolution; (iii) layers of presence/absence for each human use present in the area (e.g., location of aquacultures, location of oil and gas platforms); (iv) an expert based characterization for each human use through four attributes (vertical scale, spatial domain, temporal domain and mobility). According to the attributes of each use three pre-defined rules, included in the COEXIST methodology are dynamically applied to estimate the potential conflict score for each pair of uses. The potential score varies from 0 (no conflict) to 6 (very high conflict). Afterwards, the area of analysis is subdivided into regular grid cells according to the specified resolution and, on each cell, information about spatial overlapping human uses are extracted. Finally, on each cell the total MUC score is calculated summarizing the potential conflict score for each pair of overlapping uses. The main output is a geospatial distribution of MUC score over the entire area of analysis. For a detailed explanation of the rules and algorithm we refer to Gramolini et al. (2010) and Barbanti et al. (2015).

The Cumulative Effects Assessment (CEA) tool implements the methodology described in Menegon et al. (2018a). Formally, we consider ''CEA as a systematic procedure for identifying and evaluating the significance of effects from multiple pressures and/or activities on single or multiple receptors. CEA provides management options, by quantifying the overall expected effect caused by multiple pressures and by identifying critical pressures or pressure combinations and vulnerable receptors. The analysis of the causes (source of pressures), pathways, interactions and consequences of these effects on receptors is an essential and integral part of the process'' (Judd, Backhaus & Goodsir, 2015).

The inputs of the Tools4MSP CEA tool are: (i) the area of analysis; (ii) the grid cell resolution; (iii) layers representing intensity or presence/absence of human uses (e.g., intensity of fishery and maritime transport, presence of aquacultures and oil & gas platforms); (iv) layers representing intensity or presence/absence of environmental components (e.g., seabed habitats, probability of presence of nursery habitats, probability of presence of marine mammals); (v) use-specific relative pressure weights and distances of pressure propagation; (vi) environmental component sensitivities related to specific pressures or more general ecological models that describe the response of the environmental components to a specific pressure. Similarly to the MUC tool, the area of analysis is subdivided into regular grid cells. Then, on each cell, information about the presence of human uses and environmental components are extracted. Afterwards, the geospatial layers on human uses are propagated and combined to estimate the geospatial distribution of different pressures (e.g., marine litter, underwater noise, abrasion) for the entire analysis area. Finally, the geospatial distribution of single and cumulative effects and impacts are estimated combining together the pressure layers and the environmental components layers through a sensitivity score. The MSP-oriented integrated system

In Fig. 1 the interactions between the Tools4MSP Geoplatform, external application and potential end-users are presented. As a whole, they represent an integrated system of software, users and web services capable to effectively support MSP activities. Overall, the system can be described by four components:

Component 1: Tools4MSP Geoplatform

The geoplatform is a community-based integrated web application. Data are managed in a SDI over the entire workflow, from the collaborative upload in a web portal, to the creation of metadata, the choice of appropriate visual encodings, the composition of maps, the set up of use cases and the elaboration through specific modules producing final maps and descriptive reports. Internally, the Tools4MSP Geoplatform is divided into two sub-components: the Geospatial Content Management System (CMS; 1.a) and the Tools4MSP package (1.b). A more detailed description of the Geoplatform is given in the next section.

Component 2: Tools4MSP package as stand-alone library

The package can also be downloaded and used as stand-alone library independently from the GeoNode software. The library can be efficiently used through Jupyter Notebook (Kluyver et al., 2016; https://jupyter.org/), a web-based computational environment, which provides one of the most convenient user interfaces for interactive analysis (McGibbon et al., 2015;Shen, 2014). The software allows the authoring of shareable and reproducible notebook documents which allow a combination of input code, rich media representations of the output results, explanatory text, mathematics, images forming a rich computational narrative (Kluyver et al., 2016). Regarding the Tools4MSP development, the Jupyter Notebook supports rapid prototyping of new features, libraries testing and advanced analysis of case studies (Fig. 1, components 2).

Component 3: Desktop applications

Spatial layers and maps managed in the Geoplatform can be downloaded in different formats directly from the web interface or can be reused in desktop GIS applications for further investigation by connecting to standard web services. Data curation can be also improved using the desktop GIS QGIS (QGIS Development Team, 2018) together with the GeoServer explorer plugin (Boundless, 2018). This plugin eases the publication, upload and visual encoding of data and layers, allowing collaborators and domain experts to better contribute to the update and maintenance of the Geoplatform content.

Component 4: Third party data repositories

MSP-related workflows need relevant and updated data to be analysed and processed, the interaction between this component and the Tools4MSP Geoplatform highlights the ability of the system to integrate data from other data portals and SDIs, such as SHAPE Adriatic Atlas (http://atlas.shape-ipaproject.eu/), EMODnet portals (http: //www.emodnet.eu/), EEA services (https://www.eea.europa.eu/data-and-maps), CoCoNet web GIS (http://coconetgis.ismar.cnr.it/) or the European Atlas of the Seas (https: //ec.europa.eu/maritimeaffairs/atlas/maritime_atlas). All these portals use interoperable OGC-compliant web services to exchange spatial information; based on GeoNode features, the Tools4MSP Geoplatform allows users to display external layers (e.g., served from remote Web Map Services; OGC, 2006) and enriches its catalogue with relevant data through the harvesting of standard web services (GeoNode remote services). The creation and maintenance of this network of collaborations allow to harmonize existing multiple efforts and improve the availability of spatial datasets for users interested in MSP-related information.

The Tools4MSP Geoplatform

In Fig. 2 the detailed implementation architecture of the Tools4MSP Geoplatform is presented. The Geospatial CMS is the core of the system and is based on the GeoNode (GeoNode, Developers and Contributors Team, 2018) software, a Django-based web platform for developing community-based SDI. GeoNode facilitates the upload and management of geospatial datasets, making them discoverable and available via standard Open Geospatial Consortium (OGC) protocols and web mapping applications. GeoNode also allows users to automatically upload, describe and share the outputs produced by the Tools4MSP package. The Tools4MSP package is Python-based open source software available on github (Menegon, 2018a). The Tools4MSP core library implements the algorithms for CEA and MUC geospatial modelling and the base functionalities to read the input geospatial datasets and configurations, to apply data transformation operations (such as normalization, Different users can have different modes of interaction with the Geoplatform: administrators perform the case study setup, by specifying the connectors to the geospatial repositories and defining the pre-processing chain for environmental and human use layers harmonization and utilization in the case study (Fig. 2

, Case Study setup GUI).

A broad user community composed by decision-makers, planners, academics, research institutions, MSP stakeholders and the general public can use the case study configuration to run the build-in version of the Tools4MSP library implemented in the Geoplatform (Fig. 2, Webtool GUI). More in detail, the Tools4MSP Webtool GUI implements a four step workflow: (Step 0) Webtool selection (MUC or CEA); (Step 1) case study selection, choosing from a pre-set of case studies; (Step 2) case study configuration, optionally outlining a custom subregion of analysis or a custom combination of human uses, pressures, and environmental components; (Step 3) generation of geospatial and statistical outputs. The outputs are published in GeoNode and accessible through standard interoperable services. The produced reports, graphics and statistical outputs are archived in a dedicated data store catalogue and can be further visualized and re-used also by non technical stakeholders. In the results section an application of the Tools4MSP GeoNode plugin for case study setup and CEA analysis in the Adriatic Sea is provided.

RESULTS

At the current stage, the Tools4MSP modelling framework has been applied in various areas of interest in the Adriatic Sea, such as entire Adriatic Sea (Depellegrin et al., 2017), Italian Adriatic Sea (Menegon et al., 2018a) and regional scale analysis for the Northern Adriatic Sea (Menegon et al., 2018b) and Emilia-Romagna Region (Barbanti et al., 2017a;Barbanti et al., 2017b). In the following section results for Tools4MSP GeoNode plugin and stand-alone library for the Adriatic Sea will be presented.

Application of Tools4MSP GeoNode plugin

The Tools4MSP GeoNode plugin implements two sets of interfaces: the case study setup GUI and the Webtool GUI.

In Fig. 3 an example of GUI illustrating a case study setup is presented. The interface is intended for administrators and was designed to facilitate the configuration of case study input parameters. The GUI is organized in 4 sections: (i) basic parameters (Fig. 3A); (ii) human uses (Fig. 3B); (iii) environmental components (Fig. 3C); (iv) pressures (Fig. 3D).

In the example the case study named ''Adriatic Sea 2017'' has a grid resolution of 500 m and the area of analysis is the Adriatic Sea (Fig. 3E). Figure 3F shows the expanded view of geospatial dataset set up for ''Maritime Transport''. Specific data transformation operations have been configured through the ''Pre-processing expression'' field. The expression is written with a Python-based syntax that allows the user to select and combine one or more layers, apply filters, apply masking conditions, perform grid-cell-based arithmetic and other data transformations (e.g., normalization, logarithmic scaling, gaussian convolution). The resulting geospatial dataset is shown as thumbnail directly into the case study setup GUI (Fig. 3F).

The example reported in Fig. 3F refers to a human use (Maritime Transport) but is equally valid to environmental components or pressures.

The Webtool GUI is the standard interface allowing non-technical users (e.g., decisionmakers, MSP stakeholders, planners) to perform CEA and MUC analyses starting from the pre-set case studies.

The Webtool GUI implements a four-steps workflow: (Step 0) Webtool selection; (Step 1) Case study area selection; (Step 2) Study area selection & Dataset configuration; (Step 3) Geospatial and statistical outputs. An example of the outputs (Step 3: Results) for the case study ''Adriatic Sea 2017'' is shown in Fig. 4. Through the GUI, the geospatial

Application of Tools4MSP package as stand-alone library

In this section we present a cumulative effects assessment based on the stand-alone Tools4MSP package applied for the Adriatic Sea. The case study set up has been downloaded from the Tools4MSP Geoplatform. It consists of a directory named ''adriatic_sea'' containing the input geospatial layers related to human uses, environmental components and pressures and environmental component sensitivities. An example of the case study set up is released within the Tools4MSP software package (https://github.com/CNR-ISMAR/tools4msp/tree/master/data/demo_case_study) and is available for test and demo purposes.

The case study is presented using a workflow implemented through the Jupyter computational environment including the following steps (Fig. 5):

• In [1] Import libraries: Tools4MSP CaseStudy class, rectifiedgrid using the rg alias, matplotlib and numpy. CaseStudy is a comprehensive class that provides the methods to setup a case study (e.g., specify datasets and input parameters) or alternatively to load a predefined case study, perform the analysis and manage the results.

• In [2] Load predefined case study for Adriatic Sea (1 km ×1 km resolution). After instantiating the CaseStudy class specifying the ''adriatic_sea'' case study, the methods load_layers and load_inputs are called in order to import the environmental, pressure and human use layers (including the layer metadata) and the input parameters respectively (e.g., sensitivity scores).

• In [3] Print example case study setup providing general information on spatial extent (in number of cells) and number of available layers (geospatial datasets). The input geospatial layers and the area of analysis layer (.grid parameter) are instances of the RectifiedGrid class. RectifiedGrid is the main class provided by the rectifiedgrid library and it is designed to represent and manipulate 2D georeferenced data arrays. RectifiedGrid provides the ''.plotmap'' method to plot the layer and other information (e.g., coastline, rivers) on a map.

Software details

In Table 1 the summary of the main characteristics and requirements of RectifiedGrid and Tools4MSP is presented.

DISCUSSION AND CONCLUSIONS

This paper presents architecture, implementation and practical application of an MSPoriented software package named Tools4MSP. The tool is presented as GeoNode plugin and as a stand-alone library determining different levels of usability by different user groups. As a plugin, Tools4MSP supports a wide user community that facilitates the implementation of collaborative analyses improving the reusability and sharing of the result outputs. The integration within a Geospatial CMS allows to manage the entire processing data workflow, from the collaborative upload in a web portal, to the creation of metadata, the choice of appropriate visual encodings, the composition of maps, the set up of use cases and the elaboration through specific modules producing final maps and descriptive reports. The usage of the plugin is particularly suitable as it provides a user-friendly interface appropriate to decision-makers, regional authorities, academics and MSP stakeholders (e.g., fishers, eNGOs, industry).

The plugin eases data transformation operations reducing the need of manual data preparation and standardization procedures. Furthermore, archiving the pre-processing expressions in combination with the case study makes the transformation of the input data more explicit and the entire process more transparent and replicable.

As stand-alone library, Tools4MSP requires advanced programming skills for its usage, but provides more flexible integration with other libraries and Python packages also outside Tools4MSP modelling framework. It is particularly suitable for planning authorities seeking advanced modelling procedure for MSP/ICZM and management purposes. The scientific community, consultancies and programmers can use and further develop the library for different research objectives and integration into Decision-Support-Systems.

Compared to other existing decision support tools in MSP, the Tools4MSP ''approach'' is more flexible as it (1) incorporates a multi-objective toolset (CEA and MUC) which can be extended for other analysis purposes (e.g., scenario analysis, comparative MSP or ecosystem services assessment); (2) it enables management and treatment of different datasets and formats and (3) it provides different levels of usability ranging from experienced modellers to more user-friendly modelling through GUIs.

The tools CEA and MUC models implemented can support the development of maritime spatial plans within the implementation process of the MSP Directive (2014/89/CE) in various case study areas and marine waters in the Mediterranean Sea and beyond. The package is regularly upgraded within the Tools4MSP Geoplatform (data.tools4MSP.eu) including ongoing implementations of pressure specific analysis of CEA, CEA backsourcing (CEA-B;Menegon et al., 2018a) and the integration of marine ecosystem services oriented analysis of anthropogenic threats (MES-Threat) in support of environmental management and resource restoration (Depellegrin & Blažauskas, 2013;Menegon et al., 2018b). The Tools4MSP software package was used in ADRIPLAN (ADRiatic and Ionian maritime spatial PLAnning) and RITMARE (Ricerca ITaliana per il MARe) projects and is currently implemented by various research communities within ongoing European Projects around the Mediterranean, such as SUPREME (Supporting maritime spatial Planning in the Eastern Mediterranean) or PORTODIMARE (geoPortal of Tools & Data for sustainable Management of coAstal and maRine Environment).

• Alessandro Sarretta and Daniel Depellegrin contributed reagents/materials/analysis tools, authored or reviewed drafts of the paper, approved the final draft.

Data Availability

The following information was supplied regarding data availability:

Figure 1
1Interaction diagram between the Tools4MSP Geoplatform, external applications and potential end-users. Component 1.b and 2 (blue background) are developed and released with the Tool4MSP package. Full-size DOI: 10.7717/peerjcs.165/fig-1

Figure 2
2Implementation architecture of the Tools4MSP collaborative Geoplatform. Full-size DOI: 10.7717/peerjcs.165/fig-2aggregation, reclassification, gaussian convolution, geospatial filtering) and to manage and write model output results. Tools4MSP adopts a grid-based approach for efficient numerical computation of the geospatial models. The grid-based functionalities are provided through the general purpose RectifiedGrid library (Menegon, 2018b), that ensures direct integration of a multitude of different datasets and facilitates data preparation procedures. It simplifies the access and rasterization of multi-format geospatial data (environmental and anthropogenic datasets) and performs arithmetic and transformation operations on raster map layers. RectifiedGrid combines several FOSS Python projects:(1) NumPy and SciPy for efficient numerical computation (Van der Walt, Colbert &  Varoquaux, 2011)  and (2) rasterio and fiona(Mapbox, Rasterio Development Team, 2018;  Fiona, Developers and Contributors team, 2018)  for multi-format raster and vector data access through the Geospatial Data Abstraction Library (GDAL; Warmerdam, 2008). Interactive graphics for visualization of output results are based on the Bokeh visualization library(Bokeh Development Team, 2018).

Figure 3
3Example of Case Study setup Graphical User Interface. Full-size DOI: 10.7717/peerjcs.165/fig-3distribution of CEA for the analysis area as well as the statistical outputs can be explored and downloaded.A complete example of MUC and CEA analysis through the Tools4MSP webtool GUI including a more in-depth investigation on its strengths and limitations in support of Maritime Spatial Planning is available inMenegon et al. (2018b).

Figure 4
4Example of Graphical User Interface presenting geospatial and statistical results. Full-size DOI: 10.7717/peerjcs.165/fig-4

•[ 4 ]
4In CEA analysis function. The CaseStudy class exposes the ''.cumulative_impact'' method to perform a Cumulative Effects Assessment(Menegon et al., 2018a).• In [5] CEA geospatial results and graphical outputs for the Adriatic Sea. The map of the CEA result is visualized using the ''.plotmap'' method in combination with the distribution of CEA values of the grid cells.

Figure 5
5Workflow for Tools4MSP stand-alone library application using Jupyter computational environment. Full-size DOI: 10.7717/peerjcs.165/fig-5

Stefano Menegon. (2018, February 28). Tools4MSP: Geospatial tools to support Maritime Spatial Planning (Version 1.0.0-beta.3). Zenodo. DOI 10.5281/zenodo.1186160. Stefano Menegon. (2018, February 27). RectifiedGrid: Geospatial python library for grid-based analyses (Version 1.0.0-beta.4). Zenodo. DOI 10.5281/zenodo.1185428.

Table 1
1Software characteristics, requirements and availability for RectifiedGrid library and Tools4MSP package.RectifiedGrid
Tools4MSP

Language
Python
Python

Operating system
Platform-independent; requires Python distribution
Platform-independent; requires Python distribution

Dependencies
Numpy, GeoPandas, Scipy, Rasterio, Fiona, Shapely, Rtree,
Affine, Matplotlib, GDAL

RectifiedGrid, Bokeh, GeoNode (for plugin usage)

Software location
DOI: 10.5281/zenodo.1185428
DOI: 10.5281/zenodo.1186160

Code repository
https://github.com/CNR-ISMAR/rectifiedgrid
https://github.com/CNR-ISMAR/tools4msp

License
GPL 3
GPL 3

Menegon et al. (2018), PeerJ Comput. Sci., DOI 10.7717/peerj-cs.165 3/16
Menegon et al. (2018), PeerJ Comput. Sci., DOI 10.7717/peerj-cs.165 4/16
Menegon et al. (2018), PeerJ Comput. Sci., DOI 10.7717/peerj-cs.165 6/16
Menegon et al. (2018), PeerJ Comput. Sci., DOI 10.7717/peerj-cs.165 8/16
Menegon et al. (2018), PeerJ Comput. Sci., DOI 10.7717/peerj-cs.165 9/16
Menegon et al. (2018), PeerJ Comput. Sci., DOI 10.7717/peerj-cs.165 11/16
Menegon et al. (2018), PeerJ Comput. Sci., DOI 10.7717/peerj-cs.165 12/16
Menegon et al. (2018), PeerJ Comput. Sci., DOI 10.7717/peerj-cs.165 16/16
ACKNOWLEDGEMENTSWe would like to thank the editor and the three reviewers for valuable comments contributing to the overall improvement of the manuscript. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.ADDITIONAL INFORMATION AND DECLARATIONSCompeting InterestsThe authors declare there are no competing interests.Author Contributions• Stefano Menegon analyzed the data, contributed reagents/materials/analysis tools, prepared figures and/or tables, performed the computation work, authored or reviewed drafts of the paper, approved the final draft.
Human uses, pressures and impacts in the eastern North Sea. J H Andersen, A Stock, S Heinänen, M Mannerla, M Vinther, from DCEAarhus: Aarhus University. Available at. 18Technical ReportAndersen JH, Stock A, Heinänen S, Mannerla M, Vinther M. 2013. Human uses, pressures and impacts in the eastern North Sea. Technical Report from DCE- Danish Centre for Environment and Energy No. 18. Aarhus: Aarhus University. Available at http:// www.dmu.dk/ pub/ tr18.pdf .

Developing a maritime spatial plan for the adriatic ionian region. A Barbanti, P Campostrini, F Musco, A Sarretta, E Gissi, 10.5281/zenodo.48231Barbanti A, Campostrini P, Musco F, Sarretta A, Gissi E. 2015. Developing a mar- itime spatial plan for the adriatic ionian region. Zenodo. Venice: CNR-ISMAR. DOI 10.5281/zenodo.48231.

Quadro conoscitivo di riferimento e sua analisi ai fini della pianificazione dello spazio marittimo ICM-MSP nella Regione Adriatico Ionica. A Barbanti, A Sarretta, C Venier, S Bellaciccio, D Depellegrin, G Farella, S Menegon, S Lorito, F Grati, L Bolognini, L Perini, 10.5281/zenodo.11607261Sviluppo ed analisi di proposte di ICZM-MSP in aree specifiche: costa emiliano-romagnolaBarbanti A, Sarretta A, Venier C, Bellaciccio S, Depellegrin D, Farella G, Mene- gon S, Lorito S, Grati F, Bolognini L, Perini L. 2017a. Sviluppo ed analisi di proposte di ICZM-MSP in aree specifiche: costa emiliano-romagnola. Volume 1: Quadro conoscitivo di riferimento e sua analisi ai fini della pianificazione dello spazio marittimo ICM-MSP nella Regione Adriatico Ionica. Zenodo. DOI 10.5281/zenodo.1160726.

Sviluppo ed analisi di proposte di ICZM-MSP in aree specifiche: costa emiliano-romagnola. A Barbanti, A Sarretta, C Venier, S Bellacicco, G Farella, S Menegon, D Depellegrin, S Lorito, F Grati, L Bolognini, L Perini, R Pastres, E Porporato, 10.5281/zenodo.1160720Individuazione ed analisi dei possibili obiettivi gestionali e delle misure per attuarli. 2Barbanti A, Sarretta A, Venier C, Bellacicco S, Farella G, Menegon S, Depellegrin D, Lorito S, Grati F, Bolognini L, Perini L, Pastres R, Porporato E. 2017b. Sviluppo ed analisi di proposte di ICZM-MSP in aree specifiche: costa emiliano-romagnola. Volume 2: Individuazione ed analisi dei possibili obiettivi gestionali e delle misure per attuarli. Zenodo. DOI 10.5281/zenodo.1160720.

Bokeh: Python library for interactive visualization. Bokeh Development Team. 20Bokeh Development Team. 2018. Bokeh: Python library for interactive visualization. Available at http:// www.bokeh.pydata.org/ (accessed on 20 February 2018).

GeoServer explorer plugin for QGIS. Boundless, 20Boundless. 2018. GeoServer explorer plugin for QGIS. Available at http:// boundlessgeo. github.io/ qgis-plugins-documentation/ geoserver/ (accessed on 20 February 2018).

Decision guide for selecting decision support tools for marine spatial planning. Center for Ocean Solutions ; Stanford: The Woods Institute for the Environment, Stanford UniversityTechnical reportCenter for Ocean Solutions. 2011. Decision guide for selecting decision support tools for marine spatial planning. Technical report. Stanford: The Woods Institute for the Environment, Stanford University.

Community-based geoportals: the next generation? Concepts and methods for the geospatial Web 2.0. Computers. B De Longueville, 10.1016/j.compenvurbsys.2010.04.004Environment and Urban Systems. 344De Longueville B. 2010. Community-based geoportals: the next generation? Concepts and methods for the geospatial Web 2.0. Computers, Environment and Urban Systems 34(4):299-308 DOI 10.1016/j.compenvurbsys.2010.04.004.


Indian scorpions collected in Karnataka: maintenance in captivity, venom extraction and toxicity studies

Kambaiah Santhosh
Pavana Nagaraj
Thippeswamy Nayaka Dattatreya
Boramuthi
Indian scorpions collected in Karnataka: maintenance in captivity, venom extraction and toxicity studies
10.1186/s40409-015-0053-4R E S E A R C H Open Access
Background: Maintenance of scorpions under laboratory conditions is ideal for long-term venom collection to explore the therapeutic applications of scorpion venom. Collection of venom by electrical stimulation requires a reliable stimulator and effective restrainer. Thus, the present study was conducted to develop a convenient method to maintain scorpions and to extract their venom for toxicity studies via a modified restrainer and stimulator.Methods: Four different scorpion species were collected, among which three species were maintained in the laboratory in containers that mimic their natural habitat. Venom was extracted from Hottentotta rugiscutis by electrical stimulation at 8 V for 18 months and LD 50 was estimated by the graphic method of Miller and Tainter.Results: A total of 373 scorpions including Hottentotta rugiscutis, Hottentotta tamulus, Lychas tricarinatus and Heterometrus swammerdami were collected, identified and maintained successfully, achieving a 97 % survival rate. Hottentotta rugiscutis yielded 6.0 mL of venom by electrical stimulation. The LD 50 of H. rugiscutis venom was estimated to be 3.02 mg/kg of body weight in female Swiss albino mice. Conclusions: Scorpions were successfully maintained for 18 months. Herein we have also documented a simple, cost-effective method of venom extraction by electrical stimulation using a modified restrainer. Furthermore, Hottentotta rugiscutis was reported for the first time in Karnataka.

Background

Scorpions are arthropods belonging to the class Arachnida and order Scorpiones. Globally, 1988 species are known, among which 113 species from 25 genera belonging to six families have been recorded in India [1]. Scorpions of the family Buthidae are of prime importance due to the lethality of their venom to humans, especially to children in many tropical regions [2]. A Buthidae family scorpion, Hottentotta tamulus (Fabricius, 1798), is widely distributed throughout the Deccan plateau of southern India [3,4].

As shown by an internal survey, Hottentotta rugiscutis (Pocock, 1897) scorpions are prevalent, live alongside the human habitat in the Chirathagundu village (Karnataka, India) and are known to cause considerable public health problems due to envenomations compared to three other regionsnamely Hiriyuru, Nandihalli and Hindaskattewhere human activity is limited. As there is hardly any data on the lethality of H. rugiscutis venom, it is important to research these scorpions and to find out their effects on the local population.

Scorpion venom is a complex mixture of mucopolysaccharides, hyaluronidase, phospholipase, serotonin, histamine, enzyme inhibitors, and toxins, mainly neurotoxins that affect the function of Na + , K + and other ion channelsand is used as investigatory tool in physiological and pharmacological research. The therapeutic properties of scorpion venom includes anticancer, antimicrobial, antiepileptic, analgesic, antimalarial, pesticidal and insecticidal activities and also may be used in modulating cardiovascular effects and autoimmune diseases [5].

To study the biological importance of venom and for the preparation of antivenom to treat human envenomations, a substantial amount of venom is needed and can be obtained by in-field venom collection or from scorpions maintained under laboratory conditions. In comparison with the former, the latter ensures that copious venom can be extracted at frequent intervals for a longer time. Longterm extraction or milking of venom from a pool of scorpions requires an efficient maintenance of scorpions, which otherwise would exhibit a difference in both the quantity and quality of venom obtained. Furthermore, to date, not much information is available on the effective maintenance of scorpions under laboratory conditions.

There are three main methods for the extraction of venom, namely maceration of the telson, manual stimulation and electrical stimulation of scorpions. Maceration involves the snipping and crushing of the telson to access the venom. It is a reliable method but has the obvious drawback of allowing only one extraction per individual animal. In the manual stimulation method, the animal is provoked manually to secrete the venom on a piece of parafilm [6]. But disadvantages include difficulty in provoking the scorpions [7], low venom yield and less toxicity as compared to the venom obtained by the electrical stimulation method [7][8][9]. In the third method, stimulation via mild electric shock results in contraction of muscles around the telson causing the venom to squeeze out of the vesicle. However, extracting the venom by this method requires not only a cost-effective and reliable stimulator but also a restrainer.

Given this context, the present study was conducted to develop a convenient method for scorpion maintenance and venom extraction for toxicity studies with the aid of a modified restrainer and stimulator.

Methods

Animals

All the experiments were performed according to the guidelines approved by the Institutional Animal Ethics Committee (National College of Pharmacy, Shivamogga, Karnataka; no: NCP/IAEC/CL/206/01). Five groups of female Swiss albino mice aged two months (weighing 20-25 g), with each group containing five mice, were used for testing each dose of venom for toxicity and a sixth group was used as control. The mice were kept under room temperature where they had ad libitum access to rodent chow and tap water throughout the experiment.

Collection of scorpions

The scorpions were collected from four different locations namely, Hiriyuru, Hindaskatte, Nandihalli (Chitradurga district) and Chirathagundu (Bellary district) of Karnataka, India (Fig. 1). Brown colored scorpions (Hottentotta rugiscutis, Hottentotta tamulus and Lychas tricarinatus) were found underneath the stone and wood materials over damp soil. Black scorpions (Heterometrus swammerdami) were found burrowed in soil at depths of 5-10 inches. These were captured carefully using long forceps by holding the tip of their tail without harming the animal. Black and brown scorpions were then transferred into separate containers with soil of about two inches in depth upon which small stones were placed. A maximum of 15 scorpions were kept in each container, which contained tiny holes for adequate ventilation.

Identification of scorpions

Based on the morphological characteristics such as body color, the shape and size of pedipalps, shape of sternum, color of telson and number of pectine teeth, the collected scorpions were identified to the species level [10,11].

Maintenance of scorpions

In the laboratory, scorpions were placed in large plastic tubs (diameter: 54 cm; height: 22 cm) and cages [43 (l) × 27 (w) × 15 (h) cm] on top of a soil bed of about 3 inches in depth, above which proper size stones (~15 × 8 × 15 cm) were kept, mimicking their natural habitat. As a source of drinking water, a Petri dish lid containing tap water was kept at the center of the plastic tub/cage (Fig. 2). About 8-10 brown scorpions such as Hottentotta rugiscutis (Pocock, 1897), Hottentotta tamulus (Fabricius, 1798), and the black scorpion, Heterometrus swammerdami (Simon, 1872), were housed in cages and plastic tubs respectively, and then covered with a mosquito net of proper dimensions.

Scorpions were fed once weekly such insects as grasshoppers, crickets and occasionally cockroaches [12]. The size of the insects fed to the scorpions was directly proportional to that of the scorpions themselves. At threeday intervals, the water content in the Petri dish was checked and, if necessary, refilled. Whenever necessary, soil was moistened with adequate water and unused dead insects and debris were removed. Scorpions found to be weak and mother scorpions with their young brood were housed in separate plastic containers to avoid any cannibalistic activity on the part of other scorpions. After 2 to 3 weeks, mother scorpions were placed in the common cage and scorplings were fed tiny insects. Molted scorpions were not fed insects for 3 to 4 days to avoid any physical damage to themselves, nor were subjected to venom extraction for about 20 days. The scorpions were manipulated only on the day of venom extraction and during the replacement of soil, which was carried out once every two months. The venom was not extracted on the day immediately after feeding and also no food was added until three days after the venom extraction [13,14].

Extraction of scorpion venom

Extraction of venom was performed at monthly intervals by the electrical stimulation method using a restrainer developed by Deoras and Vad [15] with slight modifications. Scorpions were restrained over a rectangular shaped thermocol board (20 × 16 cm) affixed at the center with a thick layer of cotton. Above this, a Petri dish lid with cotton inside and an opening on its side sufficiently large for protrusion of the scorpion tail, was placed in an inverted position. The tail was held firmly using forceps and electrically stimulated by pointing two electrodes connected to a step-down transformer (Chandra Scientific Industries, India) at junctions between the tail segments, one next to the telson and the other at the junction between IV and V metasomal segment (Fig. 3). Specimens of Hottentotta tamulus, Hottentotta rugiscutis and Heterometrus swammerdami were stimulated twice at 8 V and 10 V respectively, for 2 to 3 seconds, with a short interval between two stimulations. The venom was allowed to secrete over a piece of parafilm placed at the base of its tail and then transferred to a vial. The venom obtained in a single episode from many scorpions of a species were pooled, mixed with excess double-distilled water and centrifuged at 15,000 rpm for 20 minutes to remove the mucus; the supernatant was lyophilized and stored at -20°C until use. For protein estimation and toxicity studies, venom was resuspended in phosphate buffered saline, pH 7.0 [16,17].
A B C D

Lethality of scorpion venom

Results and discussion

A total of 351 brown scorpions and 22 black scorpions were collected at different time intervals from different regions of eastern Karnataka (Table 1). Transporting the scorpions in containers mimicking their natural habitat was apparently helpful in minimizing any sort of stress on them. Brown scorpions collected from the Hindaskatte and Chirathagundu regions were identified as Hottentotta rugiscutis, whereas the scorpions from Nandihalli and Hiriyuru were identified as Hottentotta tamulus. The black scorpion from Chirathagundu was identified as Heterometrus swammerdami. Heretofore, the scorpion species, H. rugiscutis has not been reported from any part of Karnataka [10]. Although this study was focused on the collection of scorpions from the regions previously stated, we also collected another scorpion species haphazardly from the premises of Kuvempu University, Shivamogga district, with the aim of displaying the diversity of scorpions in Karnataka; it was identified as Lychas tricarinatus (Simon, 1884), but maintenance and venom collection were not carried out for this species (Fig. 1). The scorpions maintained in cages and plastic tubs were active and healthy with a survival rate of 97 %. Employment of a Petri dish as a water reservoir makes water easily available to all scorpions (Fig. 2). This has proved to be a more efficient and easier method than the use of wet cotton balls placed in a container [13,14]. Adequate spraying of water restores the moisture content of the soil bed and is also helpful in the molting of scorpions [19].

Feeding of scorpions once a week was noteworthy as it was observed that most of them did not consume insects when supplied at an interval of 3-4 days. It was crucial that insects be smaller than the scorpion to ensure that the latter can grab the prey easily with its pincers. All the insects were added to tubs/cages with their wings and forelimbs chopped so that they were easily accessible to the scorpions.

During the period of captivity, molting was observed at least twice in Heterometrus swammerdami, and only once in few Hottentotta rugiscutis and Hottentotta tamulus. Few scorpions gave birth to offspring, which may have been conceived after captivity, as we observed spermatophores (sperm containing tube-like structures) attached to mud particles or stones (Fig. 4) in many plastic tubs/cages, which is an indication of mating activity in laboratory conditions. Although it was not our motive to rear the scorplings, they were maintained on moral grounds, but ended up with only a 2 % survival rate. Rearing of scorplings in captivity is a difficult task with a much lower success rate as compared to the maintenance of adult scorpions [20], as also reported by Candido and Lucas and Gopalakrishnakone [14,21]. As it was observed, such factors as the size and type of feed, moisture content of soil and temperature of the environment may affect the survival of these scorplings.

Extraction of venom by electrical stimulation method

In total, 6.0 mL of venom was obtained from Hottentotta rugiscutis species at monthly intervals for the duration of 18 months (data not shown). The venom was also collected from other species for a short period in order to Fig. 3 Extraction of venom by electrical stimulation method. Setup for milking of venom from Hottentotta rugiscutis by the electrical stimulation method using step-down transformer and restrainer validate the stimulator and modified restrainer used. The H. rugiscutis and H. tamulus scorpions secreted a transparent watery pre-venom on initial stimulation, whereas on second stimulation, the venom obtained was viscous and whitish in color. But this secretion pattern was not consistently observed in all the scorpions of the particular species stimulated. However, Heterometrus swammerdami secreted relatively more viscous and whitish venom in all the stimulations. The quantity of venom procured from an individual scorpion varied with its size. The average amount of venom secreted from each individual scorpion in a single extraction procedure was~8 μL during the period of first 12 months, but reduced to~5 μL per scorpion for the next six months. Thus, maintaining the scorpions for more than 18 months may not be sufficiently productive in the context of venom yield, which was also noted by Candido and Lucas [14]. H. swammerdami secretes venom which is less toxic as they are relatively large in size and possess strong pedipalps, thus enabling them to attack the prey without the need of highly toxic venom. In contrast, H. rugiscutis and H. tamulus scorpions, which are small in size and possess relatively weak pedipalps, produce a more highly toxic venom [22]. H. rugiscutis is responsible for many envenomations in the region of Chirathagundu village. In an extensive survey of the literature, we found hardly any reports on the lethality of this species. In this context, further studies were carried out only on H. rugiscutis venom.

The stimulator used in the present study for the extraction of venom was more affordable than the one mentioned by Lowe and Farrell [8] and can be employed in the field if connected to a small battery. The voltage required to stimulate the scorpions varies according to species and size. The venom from the species Androctonus mauretanicus and Buthus occitanus tunetanus were extracted by stimulating at 12 V, whereas Parabuthus sp., Tityus serrulatus and Heterometrus gravimanus were stimulated at 40 V, 12.5 V and 8 V, respectively [9,13,14,16]. The present study also revealed similar variations. H. rugiscutis and H. tamulus secreted the venom when stimulated at 8 V, while the H. swammerdami secreted the venom only at 10 or 12 V based on species size. The expression time reported in this study (2-3 s) was shorter than the finding of 15 s by Gopalakrishnakone et al. [21]. These variations can also be due to different stimulators and conditions used in those studies.

The restrainer used herein was simple, cheap and effective, which eliminated any possibility of injury to the animal or the handler. The spontaneous expulsion of venom is a concern that was avoided by anesthetizing the scorpion with 5 % CO 2 or by placing it in a refrigerator (0°C) for 3 to 5 minutes prior to venom collection [13,16]. However, during this study, proper handling and use of the modified restrainer drastically reduced the problem of spontaneous expulsion of venom. Although the use of thermocol in the restrainer does not cause any problem, in the future it should be replaced with a wooden base to which a petri dish can be fixed with a spring holder to enable an easy restraining process.

Toxicity of scorpion venom

The protein content of venom from Hottentotta rugiscutis and H. tamulus were 0.79 mg and 0.70 mg/mg of lyophilized venom. The only report found until the present on the lethality of H. rugiscutis scorpion venom records an LD 50 value of 4.2 mg/kg of body weight via intraperitoneal route [23], but no report was found on the amount of protein present in that venom. In this study, the LD 50 of this venom was estimated to be 3.02 mg/kg of body weight via subcutaneous route. Lethality of H. rugiscutis scorpion venom was determined using different doses after which the death or survival ratio was recorded over a 24-hour period ( Table 2). The symptoms observed in the experimental animals after venom injection included agitation, hyper-excitability, sweating, paralysis, salivation, squeaking, hunched back movements, convulsions, weakness and death.

Conclusions

The method followed in this study for scorpion maintenance in the laboratory is valuable. The modified restrainer and stimulator are significantly efficient in venom extraction for both smaller and larger scorpions. This was the first report for the presence of H. rugiscutis in Karnataka. Furthermore, the elevated population of H. rugiscutis in the Chirathagundu region presents a threat to human life that requires the development of a therapeutic anti-venom.

Ethics committee approval

All the experiments were performed according to the guidelines approved by the Institutional Animal Ethics Committee (National College of Pharmacy, Shivamogga, Karnataka; no: NCP/IAEC/CL/206/01).

Fig. 1
1Map of Karnataka showing the locations of different scorpion species. At the bottom: a Hottentotta rugiscutis (Pocock, 1897); b Hottentotta tamulus (Fabricius, 1798); c Lychas tricarinatus (Simon, 1884), d Heterometrus swammerdami (Simon, 1872) Fig. 2 A scorpion habitat in the laboratory (left) and a black scorpion drinking the water (right). A plastic tub having soil substrate and stones mimicking scorpion habitat along with water-filled petri dish at the center as a water supplement. A black scorpion, Heterometrus swammerdami, drinking the water placed in the petri dish

PBS). Control group was injected with equivalent volume of PBS only. The animals were monitored for 24 hours and the LD 50 was calculated by the graphical method of Miller and Tainter[18].Toxicity of venom from Hottentotta rugiscutis of Chir-
athagundu region was studied by estimating the LD 50
value under in vivo conditions. Five different doses cov-
ering the range of mortality from 0 to 100 % were tested.
The test group mice were subcutaneously injected with
one of five concentrations (2.60, 2.80, 3.02, 3.26, 3.52 mg/
kg b.w.) of venom dissolved in 0.2 mL of phosphate buff-
ered saline (

Table 1
1Numbers of six scorpion species collected from five different regions of KarnatakaFig. 4 Spermatophore and mother scorpion with young brood. A spermatophore attached on the surface of a soil clod found in the container (left) and mother scorpion with its young brood which were delivered during captivity (right)Region
Chirathagundu
Hiriyuru
Nandihalli
Hindaskatte
Shankaraghatta

Species
Hottentotta rugiscutis
(Pocock)

Heterometrus
swammerdami (Simon)

Hottentotta tamulus
(Fabricius)

Hottentotta tamulus
(Fabricius)

Hottentotta rugiscutis
(Pocock)

Lychas tricarinatus
(Simon)

No. of
scorpions

181
22
10
45
95
20

Nagaraj et al. Journal of Venomous Animals and Toxins including Tropical Diseases (2015) 21:51
Page 5 of 7

Table 2
2LD 50 of H. rugiscutis venom injected to mice subcutaneously (n = 5) and calculated by graphical method of Miller and Tainter Group Dose (mg/kg bw) Log of dose Volume of injection (mL) Mice (death/total) Percentage of death (%) Corrected percentage Probit value1.
2.60
0.414
0.2
0/5
0
5 a
3.36

2.
2.80
0.447
0.2
1/5
20
20
4.16

3.
3.02
0.480
0.2
3/5
60
60
5.25

4.
3.26
0.513
0.2
4/5
80
80
5.84

5.
3.52
0.544
0.2
5/5
100
100 a
6.64

a

Correction formula: 0% ¼ 100 0:25

n

À Á
100% ¼ 100 n−0:25=5
ð
Þ ; bw: body weight

AcknowledgmentsThe authors would like to thank to Dr. Deshabhushan Bastawade, Retired Scientist, Zoological Survey of India, Western Regional Station, Pune, for helping us in the identification of collected scorpions. We are also thankful to UGC, New Delhi, for their funding of this research [grant n.Competing interestsThe authors declare that there are no competing interests.Authors' contributions NBT, SKN and PD collected and maintained the scorpions in the laboratory during the study period. SKN and PD worked in the extraction of venom by the electrical stimulation method and drafted the manuscript. SKN designed the restrainer used in this study. PD did LD 50 estimation. NBT provided the idea, gave supervision for experimental designs and finalized the manuscript. All authors approved the manuscript.  Submit your next manuscript to BioMed Central and we will help you at every step:
. D B Bastawade, S S Jadhav, R M Sharma, Scorpionida, Zool Surv India. 46Bastawade DB, Jadhav SS, Sharma RM. Scorpionida. Zool Surv India. 2012; 4(6):1-16.

The action of red scorpion (Mesobuthus tamulus coconsis, Pocock) venom and its isolated protein fractions on blood sodium levels. R V Badhe, A B Thomas, A D Deshpande, N Salvi, A Waghmare, J Venom Anim Toxins incl Trop Dis. 131Badhe RV, Thomas AB, Deshpande AD, Salvi N, Waghmare A. The action of red scorpion (Mesobuthus tamulus coconsis, Pocock) venom and its isolated protein fractions on blood sodium levels. J Venom Anim Toxins incl Trop Dis. 2007;13(1):82-93.

Preparation of a potent antiscorpion-venom serum against the venom of red scorpion. R C Kankonkar, D G Kulkurni, C B Hulikavi, Buthus tamulusKankonkar RC, Kulkurni DG, Hulikavi CB. Preparation of a potent anti- scorpion-venom serum against the venom of red scorpion (Buthus tamulus).

. J Postgrad Med. 444J Postgrad Med. 1998;44(4):85-92.

Scorpion sting: Update. H S Bawaskar, P H Bawaskar, J Assoc Physicians India. 60Bawaskar HS, Bawaskar PH. Scorpion sting: Update. J Assoc Physicians India. 2012;60:46-55.

Scorpion toxins and its applications. B Joseph, J George, Int J Toxicol Pharmacol Res. 43Joseph B, George J. Scorpion toxins and its applications. Int J Toxicol Pharmacol Res. 2012;4(3):57-61.

A simple device for collecting scorpion venom. E Zlotkin, A S Sholov, Toxicon. 74Zlotkin E, Sholov AS. A simple device for collecting scorpion venom. Toxicon. 1969;7(4):331-2.

Cost of venom regeneration in Parabuthus transvaalicus (Arachnida: Buthidae). Z Nisani, S G Dunbar, W K Hayes, Comp Biochem Physiol A Mol Integr Physiol. 1472Nisani Z, Dunbar SG, Hayes WK. Cost of venom regeneration in Parabuthus transvaalicus (Arachnida: Buthidae). Comp Biochem Physiol A Mol Integr Physiol. 2007;147(2):509-13.

A portable device for the electrical extraction of scorpion venom. R M Lowe, P M Farrell, Toxicon. 572Lowe RM, Farrell PM. A portable device for the electrical extraction of scorpion venom. Toxicon. 2011;57(2):244-7.

Comparison between two methods of scorpion venom milking in Morocco. N Oukkache, F Chgoury, M Lalaoui, A A Cano, N Ghalim, 10.1186/1678-9199-19-5J Venom Anim Toxins incl Trop Dis. 195Oukkache N, Chgoury F, Lalaoui M, Cano AA, Ghalim N. Comparison between two methods of scorpion venom milking in Morocco. J Venom Anim Toxins incl Trop Dis. 2013;19(5). doi:10.1186/1678-9199-19-5.

Taxonomic studies on a collection of scorpions (Scorpiones: Arachnida) from Western Ghats in Kerala, India with two new distribution records. P M Sureshan, D B Bastawade, C Radhakrishnan, Zoos Print J. 2212Sureshan PM, Bastawade DB, Radhakrishnan C. Taxonomic studies on a collection of scorpions (Scorpiones: Arachnida) from Western Ghats in Kerala, India with two new distribution records. Zoos Print J. 2007;22(12): 2903-8.

Diversity and identification key to the species of scorpions (Scorpiones: Arachnida) from Jaffna Peninsula, Sri Lanka. K Veronika, K Akilan, A Murugananthan, T Eswaramohan, J Entomol Zool Stud. 15Veronika K, Akilan K, Murugananthan A, Eswaramohan T. Diversity and identification key to the species of scorpions (Scorpiones: Arachnida) from Jaffna Peninsula, Sri Lanka. J Entomol Zool Stud. 2013;1(5):70-7.

General biochemical and immunological characterization of the venom from the scorpion Tityus trivittatus of Argentina. A R De Roodt, F I Coronas, N Lago, M E Gonzalez, R D Laskowicz, J C Beltramino, Toxicon. 552-3de Roodt AR, Coronas FI, Lago N, Gonzalez ME, Laskowicz RD, Beltramino JC, et al. General biochemical and immunological characterization of the venom from the scorpion Tityus trivittatus of Argentina. Toxicon. 2010;55(2-3):307-19.

Studies of scorpion antivenins. 2. Venom collection and scorpion colony maintenance. F W WhittemoreJr, H L Keegan, C M Fitzgerald, H A Bryant, J F Flanigan, Bull World Health Organ. 284Whittemore Jr FW, Keegan HL, Fitzgerald CM, Bryant HA, Flanigan JF. Studies of scorpion antivenins. 2. Venom collection and scorpion colony maintenance. Bull World Health Organ. 1963;28(4):505-11.

Maintenance of scorpions of the genus Tityus Koch (scorpiones, buthidae) for venom obtention at Instituto Butantan, São Paulo, Brazil. D M Candido, S Lucas, J Venom Anim Toxins incl Trop Dis. 101Candido DM, Lucas S. Maintenance of scorpions of the genus Tityus Koch (scorpiones, buthidae) for venom obtention at Instituto Butantan, São Paulo, Brazil. J Venom Anim Toxins incl Trop Dis. 2004;10(1):86-97.

The milking of scorpions. P J Deoras, N E Vad, Toxicon. 141Deoras PJ, Vad NE. The milking of scorpions. Toxicon. 1962;1:41.

Collection of venom from southern African scorpions. J L Du Plessis, Toxicon. 455du Plessis JL. Collection of venom from southern African scorpions. Toxicon. 2005;45(5):681-2.

Purification of the immunogenic fractions and determination of toxicity in Mesobuthus eupeus (Scorpionida: Buthidae) venom. M Khoobdel, T Zahraei-Salehi, B Nayeri-Fasaei, M Khosravi, Z Omidian, M H Motedayen, J Arthropod Borne Dis. 72Khoobdel M, Zahraei-Salehi T, Nayeri-Fasaei B, Khosravi M, Omidian Z, Motedayen MH, et al. Purification of the immunogenic fractions and determination of toxicity in Mesobuthus eupeus (Scorpionida: Buthidae) venom. J Arthropod Borne Dis. 2013;7(2):139-46.

Calculation of LD 50 values from the method of Miller and Tainter. M A Randhawa, J Ayub Med Coll Abbottabad. 213Randhawa MA. Calculation of LD 50 values from the method of Miller and Tainter, 1944. J Ayub Med Coll Abbottabad. 2009;21(3):184-5.

Scorpions: Everything about purchase, care, feeding, and housing. M Rubio, Barrons Educational Series. Rubio M. Scorpions: Everything about purchase, care, feeding, and housing. Hauppauge: Barrons Educational Series; 2008.

Field and laboratory methods. W D Sissom, G A Polis, D D Watt, The Biology of Scorpions. Polis GAStanfordStanford University pressSissom WD, Polis GA, Watt DD. Field and laboratory methods. In: Polis GA, editor. The Biology of Scorpions. Stanford: Stanford University press; 1990. p. 449-450.

Black scorpion (Heterometrus longimanus) as a laboratory animal: maintenance of a colony of scorpion for milking of venom for research, using a restraining device. P Gopalakrishnakone, J Cheah, M C Gwee, Lab Anim. 294Gopalakrishnakone P, Cheah J, Gwee MC. Black scorpion (Heterometrus longimanus) as a laboratory animal: maintenance of a colony of scorpion for milking of venom for research, using a restraining device. Lab Anim. 1995; 29(4):456-8.

Medicine update: Scorpion envenomation: What is new? Assoc Physicians India. A Muruganathan, 23Muruganathan A. Medicine update: Scorpion envenomation: What is new? Assoc Physicians India. 2013;23:421-3.

Procoagulant property of venoms of some medically important scorpion species from Sindh Region, Pakistan. R Amir, J M Alam, Jma Khan, Pak J Zool. 263Amir R, Alam JM, Khan JMA. Procoagulant property of venoms of some medically important scorpion species from Sindh Region, Pakistan. Pak J Zool. 1994;26(3):261-3.


Flat epithelial atypia -classification, pathologic features and clinical significance
2003

Stuart J Schnitt email@example.com
Department of Pathology
Beth Israel Deaconess Medical Center
Harvard Medical School
BostonMassachusettsUSA

Flat epithelial atypia -classification, pathologic features and clinical significance

Breast Cancer Res
5200310.1186/bcr625)263 ADH = atypical ductal hyperplasia; DCIS = ductal carcinoma in situ. Available online Review The diagnosis and management of pre-invasive breast disease Correspondence: Stuart J Schnittatypiabreastcolumnar cellductal carcinoma in situflat
Flat epithelial atypia is a descriptive term that encompasses lesions of the breast terminal duct lobular units in which variably dilated acini are lined by one to several layers of epithelial cells, which are usually columnar in shape and which display low-grade cytologic atypia. Observational studies have suggested that at least some of these lesions may represent either a precursor of ductal carcinoma in situ (DCIS) or the earliest morphological manifestation of DCIS. In contrast, the limited available clinical follow-up data suggest that the risk of both local recurrence and progression of these lesions to invasive cancer is extremely low, supporting the notion that categorizing such lesions as 'clinging carcinoma' and managing them as if they were fully developed DCIS will result in overtreatment of many patients. Additional studies are needed to better understand the biological nature and clinical significance of these lesions.

Introduction

Mammographic screening has resulted in the increased detection of invasive breast cancers as well as the detection of certain lesions regarded to be precursors of invasive breast cancer (such as ductal carcinoma in situ [DCIS]) and risk factors for the subsequent development of breast cancer (such as atypical ductal hyperplasia [ADH]). In addition, several breast lesions that were only uncommonly seen in the premammographic era are currently being encountered with increasing frequency in breast biopsies performed because of a mammographic abnormality, particularly microcalcifications. Included within this group are lesions that have recently been designated 'flat epithelial atypia' by the World Health Organization Working Group on the Pathology and Genetics of Tumours of the Breast [1]. 'Flat epithelial atypia' is better considered a descriptive term that encompasses several different lesions, rather than a specific pathologic diagnosis per se. Despite the diversity of published descriptions and the wide assortment of names applied to breast lesions that would currently be included in the category of flat epithelial atypia [2][3][4][5][6][7][8][9][10][11] (Table 1), for practical purposes these lesions can be grouped into one of two diagnostic categories: columnar cell change with atypia or columnar cell hyperplasia with atypia [12]. Although 'flat epithelial atypia' will be used for purposes of discussion in the present review, we use the more specific diagnostic terms 'columnar cell change with atypia' and 'columnar cell hyperplasia with atypia' in clinical practice and in surgical pathology reporting.

Classification and pathologic features

Columnar cell change with atypia

Columnar cell change with atypia is characterized by terminal duct lobular units that display variably dilated acini lined by one or two layers of epithelial cells that are columnar in shape, although the height of these columnar cells varies. Apical cytoplasmic blebs or snouts are often present at the luminal surface of the epithelial cells and, in some cases, may be prominent or exaggerated, which may impart an irregular contour to the luminal aspect of the acini.

Flocculent secretions are often present in the acinar lumina. In addition, the acini often contain luminal calcifications that may be irregular or may have the appearance of psammoma bodies (Fig. 1a). The cells lining the involved acini show cytologic atypia, which is usually of low grade. This atypia is characterized by the presence of columnar epithelial cells with round to ovoid nuclei that are not regularly oriented perpendicular to the basement membrane, with a slight increase in the nuclear/cytoplasmic ratio. The nuclear chromatin may be evenly dispersed or slightly mar-ginated, and the nucleoli are variably prominent. Mitotic figures may be seen but are uncommon (Fig. 1b). In other cases, the columnar cells may resemble the cells comprising the tubules of tubular carcinoma. The cytologic atypia in these cases may be quite subtle.

In some cases of columnar cell change with atypia, the lesion may be overlooked entirely on low-power microscopic examination due to the lack of significant cellular proliferation and the subtle nature of the cytologic atypia [8]. In fact, terminal duct lobular units exhibiting this alteration are often misinterpreted on low-power microscopic examination as either normal or as showing only microcysts. It is only after examination of such foci under high magnification that the subtle cytologic atypia becomes evident.

Columnar cell hyperplasia with atypia

The term columnar cell hyperplasia with atypia is used to describe lesions composed of terminal duct lobular units with variably dilated acini lined by columnar cells that have cytologic features similar to those seen in columnar cell change with atypia but that also show cellular stratification of more than two cell layers. The proliferating epithelial cells may form small mounds, tufts or short micropapillations. However, complex architectural patterns, such as well-developed micropapillations, rigid cellular bridges, bars and arcades, or sieve-like fenestrations, with evidence of cellular polarization within the micropapillations and bars or around the fenestrations, are absent (Fig. 2). Thus, while these lesions are included under the heading 'flat epithelial atypia', it should be apparent that 'flat' is a relative term, simply denoting the absence of complex architectural patterns as described earlier. Exaggerated apical cytoplasmic snouts and abundant flocculent intraluminal secretions are often present, and some of the cells comprising such lesions may have a hobnail appearance. These lesions frequently show intraluminal calcifications, Table 1 Other names used to describe lesions within the category of flat epithelial atypia

Description Reference

Atypical cystic duct [2] Atypical cystic lobules [3] Atypical lobules type A (some examples) [4] Clinging carcinoma (monomorphic type) [5,6] Columnar alteration with prominent apical snouts and [7] secretions with atypia Columnar cell change with atypia a [12] Columnar cell hyperplasia with atypia a [12] Ductal intraepithelial neoplasia of the flat monomorphic type [8] Hypersecretory hyperplasia with atypia (some examples) [9] Pretubular hyperplasia [10] Small ectatic ducts lined by atypical ductal cells with [11] apocrine snouts a Preferred diagnostic terms.  High-grade cytologic atypia with nuclear pleomorphism of the type seen in high-grade DCIS is not a feature of lesions we include in the categories of columnar cell change with atypia or of columnar cell hyperplasia with atypia [12]. The presence of such high-grade nuclear features merits the designation of high-grade DCIS, even if the cells comprise only a single cell layer [6]. However, such lesions are rarely seen in the absence of high-grade DCIS exhibiting other architectural patterns.

It is important to note that while the flat lesions we have designated columnar cell change with atypia and columnar hyperplasia with atypia may be observed in isolation, they often coexist with more complex proliferative lesions that are composed of cytologically similar cells and that fulfill the diagnostic criteria for ADH or DCIS. In fact, their presence should prompt a diligent search for such areas.

Biological markers

Studies of the expression of various biological markers in flat epithelial atypia are fairly limited. The available data have indicated that the majority of the cells comprising these lesions exhibit expression of cytokeratin 19 [13], and they consistently lack expression of high molecular weight cytokeratins as defined by antibody 34 beta E-12 and antibodies to cytokeratin 5 and cytokeratin 6 [14] (Carlo V, Fraser J, Pliss N, Connolly JL, Schnitt S, unpublished data, 2003). In addition, the cells typically show intense nuclear expression of the estrogen receptor [3,15,16] and the progesterone receptor [13] in the majority of the cells. These cells also show strong cytoplasmic expression of the bcl-2 protein [15] and variable expression of cyclin D 1 [13]. In most examples of flat epithelial atypia, very few of the cells show staining for the Ki-67 antigen, indicating that the cells comprising these lesions have a low proliferative rate [15].

Clinical significance

Assessment of the clinical significance of the flat epithelial atypias has been hampered by variations in the terminology used to describe these lesions and by the limited number of cases that have been studied in a systematic fashion. There have been two types of studies that have addressed the potential clinical importance of these lesions: observational studies and clinical outcome studies.

Observational studies

A number of authors have noted that flat epithelial atypias are often seen in association with DCIS and with some types of invasive breast carcinoma, particularly tubular carcinoma. Oyama and colleagues studied 21 cases of the flat lesion they termed "atypical cystic lobules" [3], and found these lesions to be more common in specimens containing DCIS than in specimens without DCIS (36% versus 3%). They also noted geographic proximity between the flat lesion and the DCIS. Weidner described a lesion composed of small ectatic ducts lined by one or two layers of columnar cells with apical snouts similar to those seen in tubular carcinoma [17]. He considered this flat lesion to represent an example of low-grade DCIS. In a more detailed analysis, Goldstein and O'Malley found an association between a similar lesion they called "small ectatic ducts lined by atypical cells with apocrine snouts" with both low-grade DCIS and tubular carcinoma [11]. Other authors have also noted an association between various flat atypical lesions and DCIS and/or invasive carcinoma [4,9,10].

In addition, cytologic, immunophenotypic, and genetic similarities between flat epithelial atypias and DCIS and/or tubular carcinoma have been described by various authors. Among 16 cases with both atypical cystic lobules and low-grade DCIS, Oyama and colleagues noted that the cells comprising the atypical cystic lobules were cytologically similar to the cells of the fully developed DCIS present in the same specimen [3]. Other investigators have also noted that the cells comprising some flat atypical lesions are cytologically similar to or identical to the cells comprising some forms of DCIS or to the cells comprising the glands of tubular carcinoma [6,8,10,11,18]. Oyama and colleagues further noted an immunophenotypic identity between atypical cystic lobules and coexistent low-grade DCIS (the cells of both lesions being Available online http://breast-cancer-research.com/content/5/5/263  positive for the estrogen receptor, the progesterone receptor, keratin 19 and cyclin D 1 ) in their 16 cases [3]. In a recent genetic study of 13 cases of the flat atypical lesion designated 'DIN-flat monomorphic type', Moinfar and colleagues found that 70% showed loss of heterozygosity at one or more of the eight loci evaluated [8]. Furthermore, they found that the genetic alterations in these columnar cell lesions were the same as those in the associated DCIS or invasive cancer [8].

Based on the foregoing observations, it is reasonable to conclude that at least some flat epithelial atypias are probably neoplastic proliferations that may well represent either a precursor of or the earliest morphologic manifestation of low-grade DCIS, as well as being a precursor to invasive carcinoma, particularly tubular carcinoma. While this is of great interest from a biological point of view, the clinical implications of these observations can only be determined from follow-up studies. Unfortunately there is a paucity of data on this subject, and the few available studies are characterized by very small numbers of patients.

Clinical outcome studies

Only two follow-up studies have directly addressed the clinical significance of flat epithelial atypia to date. In a review of more than 9000 breast biopsies that were initially considered benign, Eusebi and colleagues retrospectively identified 25 patients with so-called "clinging carcinoma" of the flat, monomorphic (low nuclear grade) type [6]. Only one of these patients (4%) is reported to have developed a 'local recurrence' after an average follow-up period of 19.2 years. However, the local recurrence in this patient consisted of a clinging carcinoma lesion histologically identical to the original lesion, and it is not possible to determine whether this simply reflected persistence of the original lesion due to inadequate exci-sion or whether this represented a true local recurrence. Of note, none of these 25 patients developed an invasive breast cancer within the follow-up period.

In another study, 59 patients with clinging carcinoma of the low nuclear grade type were identified among the patients entered into European Organization for Research and Treatment of Cancer trial 10853, a randomized clinical trial comparing excision and radiation therapy with excision alone for the treatment of women with DCIS [19]. None of these 59 patients developed a local recurrence or an invasive breast cancer with a median follow-up period of 5.4 years. The very limited available data thus suggest that, among patients with so-called clinging carcinoma of the low nuclear grade/monomorphic type (lesions that we would categorize as columnar cell change with atypia or columnar cell hyperplasia with atypia), the likelihood of local recurrence or progression to invasive breast cancer is exceedingly low, at least with the available follow-up (Table 2). Additional clinical follow-up studies are clearly needed, however, to better understand the relationship between these flat epithelial atypias and the risk of subsequent breast cancer.

Practical considerations

The appropriate pathology work-up and clinical management of patients whose biopsy specimens show flat epithelial atypias are evolving as information regarding these lesions begins to accumulate. The limited available data suggest that when flat epithelial atypia is encountered in a core needle biopsy specimen, subsequent excision shows a more advanced lesion in about one-third of cases. This is sufficiently frequent to recommend excision in such cases as a matter of routine [20,21].

The presence of flat epithelial atypia in an excisional biopsy specimen should prompt a careful search for areas Breast Cancer Research Vol 5 No 5 Schnitt Table 2 Outcome of patients with 'clinging carcinoma' (flat type, with low nuclear grade/monomorphic nuclei) European Organization for Research and Eusebi and colleagues [6] Treatment of Cancer Trial 10853 [19] Number Number with subsequent invasive 0 0 breast cancer a The 'local recurrence' in this case consisted of a 'clinging carcinoma' lesion histologically identical to the original lesion. It is therefore not possible to determine whether this simply reflects persistence of the original lesion due to inadequate excision or whether this represents a true local recurrence. b Number of patients in each treatment arm, with pure 'clinging carcinoma' arm not provided.

with diagnostic features of ADH or DCIS, by examining additional sections from the block or blocks containing the lesion and by the submission of the remainder of the tissue for histological examination. There are several other considerations regarding the identification of flat epithelial atypia in excisional biopsy specimens that merit discussion. When a proliferation that fulfills the diagnostic criteria for ADH or DCIS is found to arise in a background of flat epithelial atypia, it seems most prudent to manage the patient as one would manage ADH or DCIS in any other setting. However, there are two issues that remain to be resolved when the flat atypia is found to coexist with diagnostic areas of DCIS, particularly in cases in which the cytologic features of the cells comprising the flat atypia are similar to those of the cells comprising the diagnostic areas of DCIS. The first issue is whether the flat atypia should be taken into consideration in determining the size or the extent of the DCIS lesion. The second is whether the presence of flat atypia at the excision margins is sufficient to render the margins 'positive', requiring further surgical resection. As noted earlier, the limited available clinical data suggest that flat atypias are associated with a very low risk of recurrence or progression to invasive carcinoma. Therefore, we believe that these lesions should not be taken into consideration when determining the size of a coexistent DCIS lesion or in the evaluation of the status of the margins of excision, even when they are composed of cells that are cytologically similar to those in the diagnostic areas of DCIS [12].

Another problem is the management of patients whose breast biopsies, after thorough examination, show flat epithelial atypia (columnar cell change with atypia and/or columnar cell hyperplasia with atypia) without diagnostic areas of ADH or DCIS. Again, pathologists that consider these flat lesions to represent 'cancerization of lobules' by the cells of low-grade DCIS or 'clinging carcinoma' would argue that they should be considered variants of DCIS, and managed as such [11]. Others would argue that, despite the fact that these flat lesions may well be neoplastic and may even be composed of cells that are identical to those seen in some forms of DCIS or even tubular carcinoma, the few available clinical follow-up studies suggest that they are associated with a risk of subsequent breast cancer that is considerably lower than that seen with fully developed forms of low-grade DCIS. The concern, therefore, is that managing patients with such lesions as if they had DCIS would result in overtreatment of many patients.

Conclusions

Lesions included within the category of flat epithelial atypia (i.e. lesions that we currently designate columnar cell change with atypia and columnar cell hyperplasia with atypia) are being encountered with increasing frequency due to the widespread use of screening mammography.

Recent studies have begun to provide insights into the biological and clinical significance of these lesions. However, additional morphologic, immunophenotypic and genetic studies are needed to better define the relationships between these flat atypical lesions to DCIS and invasive breast cancer. Emerging data suggest that these flat epithelial atypias may be neoplastic lesions that represent the earliest form of low-grade DCIS. Despite this, the limited available clinical follow-up data suggest that the risk of local recurrence or progression of these lesions to invasive cancer is quite low, supporting the notion that categorizing such lesions as 'clinging carcinoma' and managing them as if they were fully developed DCIS will result in overtreatment of many patients. Thus, at this time, the appropriate management of patients whose breast biopsies show flat epithelial atypia in the absence of diagnostic areas of ADH or DCIS is unknown and requires evaluation in further clinical outcome studies.

Figure 1
1Figure 1

Flat epithelial atypia: columnar cell change with atypia. (a) This terminal duct lobular unit shows variably dilated acini, many of which contain intraluminal secretions and calcifications. Most acini are lined by only one or two layers of columnar epithelial cells, many of which show prominent apical cytoplasmic snouts (hematoxylin & eosin, original magnification 10×). (b) The columnar epithelial cells lining the acini show cytologic atypia, characterized by enlarged, monotonous nuclei; the nuclear/cytoplasmic ratio is increased. Nucleoli are evident in some of the nuclei (hematoxylin & eosin, original magnification 40×).which in some instances may have the configuration of psammoma bodies.

Figure 2
2Figure 2

Flat epithelial atypia: columnar cell hyperplasia with atypia. The acini in this terminal duct lobular unit are lined by a few layers of columnar epithelial cells that show low-grade cytologic atypia, characterized by relatively round, monotonous nuclei (hematoxylin & eosin, original magnification 20×).
AcknowledgementThe author thanks Dr Anne Vincent-Salomon, Institut Curie, Paris, France for many helpful discussions concerning this manuscript.Competing interestsNone declared.This article is the fourth in a review series on The diagnosis and management of pre-invasive breast disease -current challenges, future hopes, edited by Sunil R Lakhani.Other articles in the series can be found at http://breast-cancer-research.com/articles/reviewseries.asp?series=bcr_Thediagnosis
Intraductal proliferative lesions. In: Pathology and Genetics of Tumours of the Breast and Female Genital Organs. F A Tavassoli, H Hoefler, J Rosai, R Holland, I Ellis, S Schnitt, Lakhani, Sr, W Boecker, S H Heywang-Kobrunner, F Moinfar, J Peterse, F A Tavassoli, Devilee P Lyon, IARC Pressin pressTavassoli FA, Hoefler H, Rosai J, Holland R, Ellis I, Schnitt S, Lakhani, SR, Boecker W, Heywang-Kobrunner SH, Moinfar F, Peterse J: Intraductal proliferative lesions. In: Pathology and Genetics of Tumours of the Breast and Female Genital Organs. Edited by Tavassoli FA, Devilee P. Lyon: IARC Press; in press.

Atypical ductal hyperplasia, atypical lobular hyperplasia, and interpretation of a new borderline lesion. S Tsuchiya, Jpn J Cancer Clin. 44Tsuchiya S: Atypical ductal hyperplasia, atypical lobular hyper- plasia, and interpretation of a new borderline lesion. Jpn J Cancer Clin 1998, 44:548-555.

Atypical cystic lobule of the breast: an early stage of low-grade ductal carcinoma in-situ. T Oyama, K Iijima, H Takei, J Horiguchi, Y Iino, T Nakajima, F Koerner, Breast Cancer. 7Oyama T, Iijima K, Takei H, Horiguchi J, Iino Y, Nakajima T, Koerner F: Atypical cystic lobule of the breast: an early stage of low-grade ductal carcinoma in-situ. Breast Cancer 2000, 7: 326-331.

An atlas of subgross pathology of the human breast with special reference to possible precancerous lesions. S R Wellings, H M Jensen, R G Marcum, J Natl Cancer Inst. 55Wellings SR, Jensen HM, Marcum RG: An atlas of subgross pathology of the human breast with special reference to possi- ble precancerous lesions. J Natl Cancer Inst 1975, 55:231-273.

J G Azzopardi, Problems in Breast Pathology. Philadelphia, PA: WB SaundersAzzopardi JG: Problems in Breast Pathology. Philadelphia, PA: WB Saunders; 1979.

Long-term follow-up of in situ carcinoma of the breast. V Eusebi, E Feudale, M P Foschini, A Micheli, A Conti, C Riva, S Dipalma, F Rilke, Semin Diagn Pathol. 11Eusebi V, Feudale E, Foschini MP, Micheli A, Conti A, Riva C, DiPalma S, Rilke F: Long-term follow-up of in situ carcinoma of the breast. Semin Diagn Pathol 1994, 11:223-235.

Columnar alteration with prominent apical snouts and secretions: a spectrum of changes frequently present in breast biopsies performed for microcalcifications. J L Fraser, S Raza, K Chorny, J L Connolly, S J Schnitt, Am J Surg Pathol. 22Fraser JL, Raza S, Chorny K, Connolly JL, Schnitt SJ: Columnar alteration with prominent apical snouts and secretions: a spectrum of changes frequently present in breast biopsies performed for microcalcifications. Am J Surg Pathol 1998, 22: 1521-1527.

F Moinfar, Y G Man, G L Bratthauer, M Ratschek, F A Tavassoli, Genetic abnormalities in mammary ductal intraepithelial. Moinfar F, Man YG, Bratthauer GL, Ratschek M, Tavassoli FA: Genetic abnormalities in mammary ductal intraepithelial neo- Available online http://breast-cancer-research.com/content/5/5/263

clinging ductal carcinoma in situ'): a simulator of normal mammary epithelium. plasia-flat type. 88plasia-flat type ('clinging ductal carcinoma in situ'): a simula- tor of normal mammary epithelium. Cancer 2000; 88:2072- 2081.

Hypersecretory hyperplasia with atypia in breast biopsies. What is the proper level of clinical concern? Pathol Case Rev. D L Page, M Kasami, R A Jensen, 1Page DL, Kasami M, Jensen RA: Hypersecretory hyperplasia with atypia in breast biopsies. What is the proper level of clini- cal concern? Pathol Case Rev 1996, 1:36-40.

Columnar cell hyperplasia is associated with lobular carcinoma in situ and tubular carcinoma. P P Rosen, Rosen PP: Columnar cell hyperplasia is associated with lobular carcinoma in situ and tubular carcinoma [letter].

. Am J Surg Pathol. 231561Am J Surg Pathol 1999, 23:1561.

Cancerization of small ectatic ducts of the breast by ductal carcinoma in situ cells with apocrine snouts: a lesion associated with tubular carcinoma. N S Goldstein, O Malley, B A , Am J Clin Pathol. 107Goldstein NS, O'Malley BA: Cancerization of small ectatic ducts of the breast by ductal carcinoma in situ cells with apocrine snouts: a lesion associated with tubular carcinoma. Am J Clin Pathol 1997, 107:561-566.

Columnar cell lesions of the breast. S J Schnitt, A Vincent-Salomon, Adv Anat Pathol. 10Schnitt SJ, Vincent-Salomon A: Columnar cell lesions of the breast. Adv Anat Pathol 2003, 10:113-124.

Atypical cystic lobules: an early stage in the formation of low-grade ductal carcinoma in situ. T Oyama, H Maluf, F Koerner, Virchows Arch. 435Oyama T, Maluf H, Koerner F: Atypical cystic lobules: an early stage in the formation of low-grade ductal carcinoma in situ. Virchows Arch 1999, 435:413-421.

Cytokeratin 5/6 immunohistochemistry assists the differential diagnosis of atypical proliferations of the breast. F Otterbach, A Bankfalvi, S Bergner, T Decker, R Krech, W Boecker, Histopathology. 37Otterbach F, Bankfalvi A, Bergner S, Decker T, Krech R, Boecker W: Cytokeratin 5/6 immunohistochemistry assists the differ- ential diagnosis of atypical proliferations of the breast. Histopathology 2000, 37:232-240.

Immunophenotype of columnar alteration with prominent apical snouts and secretions (CAPSS) [abstract. J L Fraser, S Raza, K Chorny, J L Connolly, S J Schnitt, Lab Invest. 8021Fraser JL, Raza S, Chorny K, Connolly JL, Schnitt SJ: Immunophenotype of columnar alteration with prominent apical snouts and secretions (CAPSS) [abstract]. Lab Invest 2000, 80:21A.

Histological and biological evolution of human premalignant breast disease. D C Allred, S K Mohsin, S A Fuqua, Endocr Relat Cancer. 8Allred DC, Mohsin SK, Fuqua SA: Histological and biological evolution of human premalignant breast disease. Endocr Relat Cancer 2001, 8:47-61.

Malignant breast lesions that may mimic benign tumors. N Weidner, Semin Diagn Pathol. 12Weidner N: Malignant breast lesions that may mimic benign tumors. Semin Diagn Pathol 1995, 12:2-13.

Rosen's Breast Pathology. P P Rosen, Philadelphia, PA: Lippincott-RavenRosen PP: Rosen's Breast Pathology. Philadelphia, PA: Lippin- cott-Raven; 2001.

Risk factors for recurrence and metastasis after breast-conserving therapy for ductal carcinoma in-situ: analysis of European Organization for Research and Treatment of Cancer Trial 10853. N Bijker, J L Peterse, L Duchateau, J P Julien, I S Fentiman, C Duval, S Dipalma, J Simony-Lafontaine, I De Mascarel, M Van De Vijver, J Clin Oncol. 19Bijker N, Peterse JL, Duchateau L, Julien JP, Fentiman IS, Duval C, DiPalma S, Simony-Lafontaine J, de Mascarel I, van de Vijver M: Risk factors for recurrence and metastasis after breast-con- serving therapy for ductal carcinoma in-situ: analysis of Euro- pean Organization for Research and Treatment of Cancer Trial 10853. J Clin Oncol 2001, 19:2263-2271.

Findings at excisional biopsy (EBX) performed after identification of columnar cell change (CCC) of ductal epithelium in breast core biopsy (CBX) [abstract. E Brogi, L K Tan, Mod Pathol. 15Brogi E, Tan LK: Findings at excisional biopsy (EBX) per- formed after identification of columnar cell change (CCC) of ductal epithelium in breast core biopsy (CBX) [abstract]. Mod Pathol 2002, 15:29A-30A.

Columnar cell alteration diagnosed on mammotome core biopsy for indeterminate microcalcifications: results of subsequent mammograms and surgical excision. M Harigopal, D X Yao, S A Hoda, R A Delellis, M F Vazquez, Mod Pathol. 1536Harigopal M, Yao DX, Hoda SA, DeLellis RA, Vazquez MF: Columnar cell alteration diagnosed on mammotome core biopsy for indeterminate microcalcifications: results of subse- quent mammograms and surgical excision [abstract]. Mod Pathol 2002, 15:36A.

. J Stuart, M D Schnitt, Department Of Pathology, Beth Israel Deaconess Medical Center. 330fax: +1 617 975 5620; e-mail: firstname.lastname@example.com J Schnitt, MD, Department of Pathology, Beth Israel Deaconess Medical Center, 330 Brookline Avenue, Boston, MA 02215, USA. Tel: +1 617 667 4344; fax: +1 617 975 5620; e-mail: email@example.com