White matter bundle segmentation using diffusion MRI fiber tractography has become the method of choice to identify white matter fiber pathways in vivo in human brains. However, like other analyses of complex data, there is considerable variability in segmentation protocols and techniques. This can result in different reconstructions of the same intended white matter pathways, which directly affects tractography results, quantification, and interpretation. In this study, we aim to evaluate and quantify the variability that arises from different protocols for bundle segmentation. Through an open call to users of fiber tractography, including anatomists, clinicians, and algorithm developers, 42 independent teams were given processed sets of human whole-brain streamlines and asked to segment 14 white matter fascicles on six subjects. In total, we received 57 different bundle segmentation protocols, which enabled detailed volume-based and streamline-based analyses of agreement and disagreement among protocols for each fiber pathway. Results show that even when given the exact same sets of underlying streamlines, the variability across protocols for bundle segmentation is greater than all other sources of variability in the virtual dissection process, including variability within protocols and variability across subjects. In order to foster the use of tractography bundle dissection in routine clinical settings, and as a fundamental analytical tool, future endeavors must aim to resolve and reduce this heterogeneity. Although external validation is needed to verify the anatomical accuracy of bundle dissections, reducing heterogeneity is a step towards reproducible research and may be achieved through the use of standard nomenclature and definitions of white matter bundles and well-chosen constraints and decisions in the dissection process.

AINOSTICS Limited London United Kingdom

Athinoula A Martinos Center for Biomedical Imaging Massachusetts General Hospital and Harvard Medical School Boston Massachusetts USA

Brain Cognition and Brain Disease Institute Shenzhen Institutes of Advanced Technology Chinese Academy of Sciences Shenzhen 518055 China

Brain MRI 3T Research Center IRCCS Mondino Foundation Pavia Italy

Brigham and Women's Hospital and Harvard Medical School Boston Massachusetts USA

Cardiff University Brain Research Imaging Centre Cardiff University Cardiff United Kingdom

Center for Integrative Connectomics Stevens Neuroimaging and Informatics Institute Keck School of Medicine of USC University of Southern California Los Angeles CA United States

Center for Magnetic Resonance Research Department of Radiology University of Minnesota Minneapolis MN USA

Center for Research and Interdisciplinarity INSERM U1284 Université de Paris Paris France

Centre for Medical Image Computing University College London London United Kingdom

Centro de Investigación Científica y de Educación Superior de Ensenada Cátedras CONACyT Ensenada Mexico

Centro de Investigación en Matemáticas A C Guanajuato Mexico

CIBM Center for BioMedical Imaging Lausanne Switzerland

Departamento de Anatomía Facultad de Medicina Universidad Complutense de Madrid Madrid Spain

Departamento de Psicobiología y Metodología en Ciencias del Comportamiento Universidad Complutense de Madrid Spain Laboratorio de Análisis de Imagen Médica y Biometría Universidad Rey Juan Carlos Madrid Spain

Department of Advanced Biomedical Sciences University Federico 2 Naples Italy

Department of Applied Mathematics and Computer Science Technical University of Denmark Kongens Lyngby Denmark

Department of Biomedicine Unit of Anatomy Faculty of Medicine of the University of Porto Al Professor Hernâni Monteiro Porto Portugal

Department of Brain and Behavioral Sciences University of Pavia Italy

Department of Clinical and Experimental Epilepsy University College London London United Kingdom

Department of Computer Science Indiana University Bloomington IN USA

Department of Computer Science University of Verona Italy

Department of Electrical Engineering and Computer Science Vanderbilt University Nashville TN United States

Department of Intelligent Systems Engineering Indiana University Bloomington IN USA

Department of Neurological Surgery University of Pittsburgh Pittsburgh PA United States

Department of Neuroscience and Biomedical Engineering Aalto University School of Science Espoo Finland

Department of Neuroscience Brighton and Sussex Medical School University of Sussex Brighton United Kingdom

Department of Neuroscience Karolinska Institutet Stockholm Sweden

Department of Neuroscience University of Minnesota Minneapolis MN USA

Department of Neurosciences and Reproductive and Odontostomatological Sciences University Federico 2 Naples Italy

Department of Neurosurgery Charité Universitätsmedizin Berlin Berlin Germany

Department of Neurosurgery Neuroscience Advanced Clinical Imaging Suite Royal Children's Hospital Parkville Melbourne Australia

Department of Neurosurgery School for Mental Health and Neuroscience Maastricht University

Department of Paediatric Neurology University Hospital and Medicine Faculty Masaryk University Brno Czech Republic

Department of Physiology and Biochemistry Faculty of Medicine and Surgery University of Malta Malta

Department of Psychology Stanford University Stanford California USA

Department of Psychology The University of Texas at Austin TX 78731 USA

Department of Radiology and Radiological Sciences Vanderbilt University Medical Center Nashville TN United States

Department of Radiology and Radiological Sciences Vanderbilt University Medical Center Nashville TN USA

Department of Radiology Juntendo University Graduate School of Medicine Tokyo Japan

Department of Radiology University of Calgary 2500 University Drive NW Calgary AB Canada T2N 1N4

Department of Radiology University of Pennsylvania Philadelphia PA United States

Developmental Behavioral Pediatrics Division Department of Pediatrics Stanford School of Medicine Stanford CA United States

Developmental Imaging and Biophysics Section UCL GOS Institute of Child Health London

Developmental Imaging Murdoch Children's Research Institute Melbourne Australia

Division of Clinical Behavioral Neuroscience Department of Pediatrics University of Minnesota Minneapolis MN USA

Division of Neuroscience and Experimental Psychology Faculty of Biology Medicine and Health The University of Manchester Manchester Academic Health Science Centre Manchester United Kingdom

Epilepsy Society MRI Unit Chalfont St Peter United Kingdom

Facultad de Ciencias de la Salud Universidad Rey Juan Carlos Madrid Spain

Groupe dImagerie Neurofonctionnelle Institut Des Maladies Neurodegeneratives CNRS CEA University of Bordeaux Bordeaux France

Institute for Radiological Research Chang Gung University and Chang Gung Memorial Hospital Taoyuan Taiwan

Kimel Family Translational Imaging Genetics Laboratory Research Imaging Centre Centre for Addiction and Mental Health Toronto Ontario

KU Leuven Department of Imaging and Pathology Translational MRI B 3000 Leuven Belgium

Laboratorio de Análisis de Imagen Médica y Biometría Universidad Rey Juan Carlos Madrid Spain

Laboratory of Neuro Imaging Stevens Neuroimaging and Informatics Institute Keck School of Medicine of USC University of Southern California Los Angeles CA United States

Lysholm Department of Neuroradiology National Hospital for Neurology and Neurosurgery UCL Hospitals NHS Foundation Trust London United Kingdom

Memory and Aging Center UCSF Weill Institute for Neurosciences University of California San Francisco USA

MRI Clinical Science Specialist General Electric Healthcare Australia

National Intrepid Center of Excellence Walter Reed National Military Medical Center Bethesda MD USA

National Neuroscience Institute Singapore

Neurology Department UCSF Weill Institute for Neurosciences University of California San Francisco

Neurosurgery department Hôpital Pasteur University Hospital of Nice Côte d'Azur University France

NMR Research Unit Queen Square MS Centre Department of Neuroinflammation UCL Queen Square Institute of Neurology Faculty of Brain Sciences University College London London United Kingdom

Poitiers University Hospital France

PROVIDI Lab UMC Utrecht The Netherlands

School of Biomedical Engineering The University of Sydney Sydney Australia

SCIL Université de Sherbrooke Québec Canada

Signal Processing Lab École Polytechnique Fédérale de Lausanne Lausanne Switzerland

Sir Peter Mansfield Imaging Centre School of Medicine University of Nottingham UK

Sydney Imaging and School of Biomedical Engineering The University of Sydney Sydney Australia

Translational Imaging in Neurology Department of Medicine and Biomedical Engineering University Hospital and University of Basel Basel Switzerland

Tri institutional Center for Translational Research in Neuroimaging and Data Science Georgia State University Georgia Institute of Technology Emory University Atlanta GA 30303 United States

UMC Utrecht Brain Center Department of Neurology and Neurosurgery Utrecht the Netherlands

Universidad de Concepción Faculty of Engineering Concepción Chile

Universidad Nacional Autonoma de Mexico Institute of Neurobiology Mexico City Mexico

Université Paris Saclay CEA CNRS Neurospin Gif sur Yvette France

University of Arkansas for Medical Sciences Little Rock AR USA

University of Southern California Keck School of Medicine Neuroimaging and Informatics Institute Los Angeles California United States

University of Wisconsin Madison Madison WI USA

Victorian Infant Brain Studies Murdoch Children's Research Institute Melbourne Australia

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