The Executive Management is responsible for the overall strategies of the consortium and for managing the overall program:
Prof Mathias Uhlen, Program Director
Prof Fredrik Ponten, Vice Program Director
Prof Sophia Hober, Director of Protein Science
Assoc prof Caroline Kampf, Site Director Uppsala
Dr Cecilia Lindskog Bergström, Deputy Site Director, Uppsala
Dr Jenny Ottosson Takanen, Site Director AlbaNova, Stockholm
Prof Peter Nilsson, Site Director SciLifeLab, Stockholm
Kalle von Feilitzen, Director of Bioinformatics
Assoc prof Emma Lundberg, Director for Subcellular Atlas
Dr Sanjay Navani, Site Director Mumbai, India
The Scientific Advisory Board (SAB) consists of representatives mainly from the academic field in areas such as bioinformatics, protein science and cell biology. The role of the SAB is to evaluate the scientific out-put and to advise in new developments and external collaborations.
Dr Cristina Al-Khalili Szigyarto is responsible for external relations.
AlbaNova site, Stockholm (site director: Jenny Ottosson Takanen)
SciLifeLab site, Stockholm (site director: Peter Nilsson)
Uppsala site (site director: Caroline Kampf)
Lab SurgPath, Mumbai site (site director: Sanjay Navani)
AlbaNova site, Stockholm
||Dr Jenny Ottosson Takanen
Group leader:Dr Hanna Tegel
Personnel: Roxana Astefanei (technician), Anna Berling (research engineer), Jenny Blomqvist (research engineer),
Malin Cammenberg (research engineer), Jenny Fall (technician), Lili Gong (research engineer), Emma Grundell (technician),
Anneli Halldin (research engineer), Madeleine Kronqvist (technician), Katarina Malm (research engineer), Sofia Moberg (technician),
Mehri Salahi (technician), Petter Stanley (technician), Anne-Sophie Svensson (laboratory engineer) and Lan Lan Xu (laboratory engineer).
Responsibility: Production of recombinant PrEST expression clones including cDNA synthesis, cloning, and plasmid purification. PrEST-proteins are then express and purify and used for preparation of antigens and affinity columns.
Description: PrEST regions are first amplified with RT-PCR from a total RNA template pool with the specific oligonucleotide primers designed in the Bioinformatics module. Three different RNA pools are used, two consisting of total RNA from six individual human tissues, and one comprising total RNA from 10 different cell lines. For PrESTs not found in any RNA pool, cDNA clones are used as template (Ultimate ORF LITE Clones (Human collection), Life Technologies, Carlsbad, CA and Mammalian Gene Collection (The MGC Project team, Genome Res. 2009)). Amplicons are automatically processed with solid phase restriction, and ligated into the plasmid vector pAff8c (Larsson, M. et al, 2000) where the human gene fragment is fused to a histidine tag and albumin binding protein (His6ABP). After transformation into E. coli Rosetta(DE3), inserts are verified by DNA sequencing to omit clones with mutations and approved clones are single cell streaked. Plasmids are collected from all purified clones for deposition in the clone library and glycerol stocks are prepared and used as starting material for protein production.
All proteins are expressed as His6ABP fusions in E. coli shake flask cultures upon induction with IPTG. A fully automated protein purification system has been developed to allow for purifications of up to 60 cell lysates at a time. One-step purification is enabled by the hexahistidine affinity tag and metal affinity chromatography (IMAC) and performed under denaturing conditions. After evaluation of protein concentration and purity, the molecular weight of the PrEST proteins is determined by mass spectrometry as a final quality control. The purified proteins are then used to prepare antigens and affinity columns with PrEST-ligands. In addition, affinity resin with His6ABP-ligand is also produced.
ABP - Albumin Binding Protein
IPTG - Isopropyl-B-D-Thiogalactopyranoside
IMAC - Immobilized Metal Affinity Chromatography
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Group leader: Marica Hamsten
Personnel: Delaram Afshari (research engineer), Annelie Cajander (research engineer), Anna Johansson (technician), Klas Linderbäck (research engineer), Atefeh Mohsenchian (laboratory engineer), Mona Moradi (laboratory engineer), Camilla Ohlsson (technician) and Martin Östberg (technichian).
Responsibility: (i) Management of immunization outsourcing, (ii) Generation of purified antibodies through affinity purification of polyclonal antisera, (iii) Western blot (WB) analysis of antibodies approved in protein array analysis, (iv) WB antibody validation using over-expression lysates, (v) storage and distribution of antibodies within the program.
Description: The Immunotechnology group is responsible for the generation, purification and Western Blot analysis of all antibodies produced in the Human Protein Atlas (HPA) program. Polyclonal antisera generated together with collaborative partners are carefully purified in a three-step fashion consisting of: depletion of unwanted specificity, capture of wanted specificity and a final buffer exchange step. A manual process using gravity-flow columns carries out depletion of antibodies with unwanted specificity. The following steps are performed on the ÄKTAxpress chromatography system enabling a high-throughput semi-automated process where captured antibodies are eluted by a low pH glycine buffer and automatically loaded onto a desalting column for buffer exchange. Antibodies are supplemented with 50% glycerol and 0.02% sodium azide for long-term storage at -20°C. The binding specificity of all antibodies is determined on protein microarrays to certify that only antibodies with high specificity and low background binding are approved for immunohistochemistry analysis. All approved antibodies are further analyzed in a high-throughput WB platform using protein lysates from human cell lines (RT-4 and U-251 MG), human plasma depleted of IgG and HSA and whole tissue lysates from human liver and tonsil. Published antibodies scored as non-supportive, in the standard WB panel, are subsequently revalidated in a WB set-up comprising an over-expression lysate (VERIFY Tagged Antigen, OriGene Technologies, Rockville, MD) as a positive control.
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Epitope Mapping and Therapeutic Antibodies
Group leader: Dr Johan Rockberg
Personnel: Björn Forsström (PhD-student), Francis Jingxin Hu (PhD-student), Magnus Lundqvist (PhD-student), Dr Helena Persson (senior scientist) and Anna-Luisa Volk (PhD-student).
Responsibility: (i) To determine antibody epitopes for HPA antibodies (ii) To generate monoclonal reagents towards selected targets
and screen them in functional assays for therapeutic effect.
Description: Epitope mapping is performed on a selection of the HPA's monospecific and monoclonal reagents using a combination of platforms including: (i) Cellsurface display of target directed peptide libraries using staphylococcal display (ii) Suspension bead arrays for mapping using overlapped or alanine mutated synthetic peptides (iii) Large scale synthetic peptide arrays.
Monoclonal antibodies to clinically relevant target proteins are selected using a combination of phage display and cell surface display of antibody fragments. These are subsequently screened for desired effect in cell-based assays and cloned for production in mammalian cells as full-length antibodies.
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Group leader: Prof Sophia Hober
Personnel: Dr Anja Persson (project leader), Dr Johan Nilvebrant (researcher), Mikael Åstrand (PhD-student), Tove Boström (PhD-student) and Sara Kanje (PhD-student).
Responsibility: Protein science research.
Description: The module coordinates and conducts research projects aiming to extend the scientific outcome of data generated within the
Human Protein Atlas project as well as improving current methodology. Both biological and technical research projects based on the vast amount
of data generated are performed to further explore the function, localization and interactions of human proteins.
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SciLifeLab site, Stockholm
||Prof Peter Nilsson
Protein Array Technologies
Group leader: Prof Peter Nilsson
Personnel: Eni Andersson (research engineer), Burcu Ayoglu (postdoc), Cecilia Hellström (research engineer), Anna Häggmark (PhD-student), David Just (PhD-student), Cecilia Mattsson (research engineer), Maria Mikus (PhD-student), Maja Neiman (researcher), Ulrika Qundos (researcher), Ronald Sjöberg (research engineer / PhD-student) and Arash Zandian (PhD-student)
Responsibility: To validate the specificity and selectivity of all purified HPA antibodies. To develop and utilize peptide, antigen and antibody based microarray methodologies for large scale analysis of body fluids in the context of biomarker discovery and autoantibody profiling.
Description: Methodology for microarray based analysis of antibody specificity has been developed, where all purified
antibodies are analyzed on protein arrays with immobilized PrESTs. Each microarray is divided into 21 replicated subarrays with
384 PrESTs, enabling the analysis of 21 antibodies simultaneously. The antibodies are detected through a fluorescently labeled
secondary antibody. A specificity plot is generated for each antibody, where the signal from the binding to its antigen is
compared to the unspecific binding to all the other PrESTs. A dual color system is used in order to verify the presence of
the spotted PrESTs. Several complementary microarray formats for systematic analysis of bodyfluids are being utilized and under
constant development. The PrEST-arrays have been implemented for systematic antigen-based plasma profiling for the screening of
new autoimmunity components. The antibody microarrays with the possibility for simultaneous analysis of large amounts of
analytes with high sensitivity and the reverse phase serum microarrays which enable serum from very large patient cohorts to
be analyzed simultaneously are both utilizing in-house produced planar microarrays. The main platform for systematic
antibody-based plasma profiling, is although the suspension bead array format with capacity for multiplexing in two dimensions,
enabling the simultaneous profiling of 384 antibodies on 384 samples, see Plasma profiling.
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Group leader: Assoc prof Emma Lundberg
Personnel: Martin Hjelmare (lab manager), Hammou Ait Blal (research engineer), Ellinor Bäcklin-Bergh (lab engineer), Anna Bäckström (research engineer), Frida Danielsson (PhD-student), Magnus Lennartsson (technician), Diana Mahdessian (PhD-student), Natalia Orlowicz (technician), Linnea Pettersson (technician), Rutger Schutten (research engineer), Marie Skogs (PhD-student), Dr Charlotte Stadler (post-doc), Dr Peter Thul (post-doc), Mikaela Wiking (PhD-student), Viktoria Wiking (technician), Ulla Wrethagen (technician) and Annica Åbergh (technician).
Responsibility: (i) Analyze the subcellular distribution of proteins using high-resolution confocal microscopy, (ii) validation of antibody specificity using siRNA (iii) annotation and knowledge-based curation of subcellular distribution profiles.
Description: The Cell Profiling module is responsible for the subcellular localization of proteins using antibodies generated in the Human Protein Atlas (HPA) program.
For each protein the subcellular localization is studied in three different human cell lines selected from a panel of eighteen cell lines. In order to localize the whole human proteome in one cell line, U-2 OS cells are always used. The cell lines are cultured in vitro and immunofluorescently stained using automated protocols before high-resolution confocal images are acquired. All images are manually annotated and for each protein the subcellular localization, characteristics and intensity of the staining is described. To validate the results, the corresponding target protein may be downregulated by siRNA prior to immunostaining. Subsequently the loss of antibody staining is quantified and antibody specificity assessed.
In the end, a knowledge-based curation of the subcellular distribution is performed in a gene-centric manner, taking into account the staining of one or multiple antibodies. Here, the subcellular distribution is classified as main and additional locations.
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Group leader: Assoc prof Jochen M. Schwenk
Personnel: Elin Birgersson (research engineer), Sanna Byström (PhD-student), Kimi Drobin (research engineer / PhD-student), Dr Claudia Fredolini (post-doc), Dr Mun-Gwan Hong (biostatistician), Dr MariaJesus Iglesias (researcher), Dr Elisa Pin (post-doc)
Responsibility: Utilizing HPA antibodies in multiplexed affinity arrays for protein profiling of human body fluids (serum, plasma, CSF) for the discovery, identification and verification potential biomarker candidates.
Description: Antibodies are immobilized on fluorescent color-coded beads to create antibody arrays in suspension. Each bead array is composed of 384 antibodies that are selected either from defined lists of targets or by antibody availability. Fluid samples are then biotinylated and heat treated for the analysis without involving purification, fractionation or depletion steps. In each analysis, bead arrays generate profiles in up to 384 samples.
We also are working on expanding the profiling procedure into other body fluids such as urine and are conducting studies within cancer, cardiovascular, neurodegenerative as well as other diseases. The data from such multiplexed these single-binder assays is processed and analyzed with biostatistical models to identify antibodies that are indicative for diseases. Such antibodies then enter further analysis with regard to technical and biological verification, and are used to develop dual-binder sandwich immunoassays. Subsequent studies are then designed to study larger numbers of samples from preferably independent cohorts.
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Group leader: Dr Jan Mulder
Personnel: Dr Tony Jimenez-Beristain (technician) and Dr Nicholas Mitsios (researcher)
Responsibility: (i) Validation of antibodies against human targets on rodent tissues, (ii) profiling the distribution of proteins in the developing, adult and diseased nervous system, (iii) quantification, annotation and presentation of whole brain protein distribution profiles.
Description: Mice are widely used in biomedical research and due to its size the mouse brain is very suitable to study regional and cellular protein distributions in the mammalian nervous system. HPA antibodies against proteins expressed in the mouse nervous system are validated on mouse brain tissue using western blot and immunohistochemistry. Antibodies that pass validation are used to generate detailed protein distribution profiles using 30-35 coronal sections of the mouse brain with a 400 µm section interval covering all major brain nuclei. Whole slide immunofluorescence captured at 20x primary objective is analyzed and regional, cellular and subcellular protein distributions are quantified. Data and images are optimized for online publication.
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Group leader: Kalle von Feilitzen
Personnel: Dr Tove Alm (research engineer), Dr Linn Fagerberg (researcher), Mattias Forsberg (research engineer), Dr Björn Hallström (researcher), Fredric Johansson (research engineer),
Per Oksvold (research engineer), Lukas Persson (research engineer), Dr Åsa Sivertsson (researcher), Anders Sjöland (technician) and Martin Zwahlen (research engineer).
Responsibility: (i) To deliver custom made software solutions for all operations in the Human Protein Atlas project,
(ii) to provide the collected data to the public via the Human Protein Atlas,
(iii) to map and quantify RNA-seq data, and
(iv) to initiate the analysis of human proteins by in silico selection and design of Protein Epitope Signature Tags (PrESTs).
Description: With the LIMS (Laboratory Information Management System) as the backbone, data is collected from each module in the pipeline.
The protein expression profiles, RNA-seq data and raw data from the project is published on the Human Protein Atlas public web site through annual releases.
The Bioinformatics group performs computer-based analysis of protein sequences for selection of protein fragments (PrESTs) to be used as immunogens for generation
of target-specific polyclonal antibodies. A custom made pipeline analyzes RNA-seq data from a large number of tissue, cancer and cell line samples.
The group is also heavily involved in all research performed in the project, such as data collection, data interpretation and visualization as well as statistical analysis.
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|PI and Clinical Director:||Prof Fredrik Ponten|
|Site Director:||Assoc prof Caroline Kampf|
|Deputy Site Director:||Dr Cecilia Lindskog Bergström|
|Project coordinator:||Dr Per-Henrik Edqvist|
Tissue Microarray Production and Scanning
Group leader: Ing-Marie Olsson (technician)
Personnel: Sofie Gustafsson (biomedical analyst), Erik Lindahl (biomedical analyst), Maria Aronsson (technician), Lillemor Källström (biomedical analyst).
Responsibility, TMA production: (i) Production of tissue microarrays (TMAs) and cell microarrays (CMAs), (ii) handling of tissues (biobank material), cells and cell lines for TMA and CMA production and protein extraction, (iii) sectioning of tissue- and TMA-blocks, (iv) quality control of TMA-blocks, (v) maintenance and cultivation of cell lines and (vi) protein extraction for Western blot analysis.
Description, TMA production:The TMA-group is responsible for the production of tissue microarrays to be used in the basic protein profiling for the Human Protein Atlas and for handling the digital images of stained TMAs. Each protein expression profile is based on 8 TMAs and 1 CMA. The TMAs include normal tissues from 144 different individuals (triplicate samples of 48 different tissue types) and cancer tissues from 216 different patients (duplicate samples of tumor tissues representing the 20 most common forms of human cancer). The CMA includes duplicate samples of 47 cell lines and 12 clinical cell samples. In total, 708 tissue cores are immunostained and analyzed for each antibody.
Formalin fixed, paraffin embedded tissue specimens are collected from the Department of Pathology, Uppsala University Hospital. Representative areas to include on the TMAs are defined for each tissue specimen by visual inspection of a corresponding hematoxylin-eosin stained section under a microscope. Tissue cores from these selected areas are then taken and used for TMA production. Cell lines are cultured in vitro, harvested, fixed in formalin and dispersed in agarose prior to embedding in paraffin blocks. These cell-blocks are used for the construction of CMAs.
Responsibility, Scanning: (i) Scanning of immunohistochemically stained TMA slides, (ii) export and processing of scanned digital images from TMAs, and (iii) administration of TMAx for automated image analysis.
Description, Scanning:Immunostained TMA slides are scanned to generate high-resolution digital images, using 20x (tissue) or 40x (cells) magnification. Images from scanned TMAs are separated into spot images, representing immunostained tissue (1 mm diameter) or cell sample (0.6 mm diameter), and exported as individual TIFF files. Manual, pathology-based evaluation of images and annotation of protein expression in tissue is performed using an in-house built web based annotation software. Automated image-analysis algorithms using the TMAx system are used to generate protein expression data from images of immunostained CMAs.
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Group leader: Ing-Marie Olsson (technician)
Personnel: Dennis Kesti (biomedical analyst), Urban Ryberg (technician), Eva Wahlund (biomedical analyst)
Responsibility: (i) Handling and storage of antisera, (ii) test and titration of antibodies for immunohistochemistry, (iii) immunostaining of TMAs and CMAs, and (v) Western blot analysis.
Description: Antibodies are titrated using a specially designed test-TMA, representing a limited selection of tissues and cells. The titration is done according to a defined schedule, according to which different antibody dilutions are tested. The primary working dilution is based on the protein concentration for each antibody. When an optimal antibody dilution is found automated immunostaining is performed on sections from eight TMAs and one CMA for basic protein profiling in the Human Protein Atlas project. Instruments and commercially available detection kits are used to ensure standardization and reproducibility of immunohistochemistry. In addition, protein extractions are prepared from fresh frozen tissues and cell lines for the purpose of running Western blot on commercial antibodies.
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Group leader: Evelina Sjöstedt (PhD-student)
Personnel: Dijana Cerjan (biomedical analyst), Hanna Emanuelsson (technician), Stina Strid (technician), Karin Elmén (technician), Feria Hikmet Noraddin (technician), Eléne Kunze (technichian)
Responsibility: (i) Validation of antibody target specificity (ii), maintenance of immunostaining reproducibility and quality, (iii) coordination of evaluation of antibodies submitted by commercial vendors and academic scientists and (iv) evaluation of protein profiles.
Description: Optimal antibody dilution and target specificity is assessed by microscopical examination. Available information in public databases on gene, RNA and protein level, as well as in-house technical validation, such as protein arrays and Western blots, are considered in the decision process. For each approved antibody, a final immunostaining protocol is defined and subsequently applied to the full-scale TMAs. The images are then scanned by the TMA-group and annotated at the Mumbai-site.
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Protein Profiling and Antibody Destiny
Group leader: Dr Cecilia Lindskog Bergström
Personnel: Marcus Runeson (technician), Borbala Katona (technician), Nusa Pristovsek (technician), Linda Oskarsson (technician) and Groom Alemayehu (technician)
Responsibility: (i) annotation of immunohistochemically stained images (ii) final approval of annotated protein profiles (destiny), (iii) generation of annotated protein expression profiles and (iv) continuous curation and quality assurance of protein profiles.
Description: Protein expression is annotated on immunohistochemically stained images corresponding to 44 different normal tissues and 20 different cancer types. After annotation, protein profiles are evaluated and the data is compared with previously published literature, RNA and gene characterization data. Antibodies that are approved at this stage are assigned a reliability score and released for publication in the next version of the Human Protein Atlas. If a protein has annotated profiles from two or more independent antibodies, an annotated protein expression is performed. The antibody-based data from these multiple antibodies are thus merged manually, while also taking into consideration the performance of the respective antibodies, and reviewing the publically available protein/RNA/gene characterization data, to arrive at a best estimate of true expression pattern.
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Group leader: Dr Anna Asplund
Personnel: Sandra Andersson (PhD-student), Kristina Magnusson (PhD-student), Sofie Gustafsson (technician), Nusa Pristovsek (technician)
Responsibility: (i) To develop and set up of methods for validation and characterization of antibodies and their performance, and (ii) to develop technical resources for external and internal research projects, and (iii) to improve and expand the cell line atlas, including expression profiling in 44 different cell lines on both RNA and protein level, within the Human Protein Atlas.
Description: The HPA continuously uses antibodies as protein probes for both large-scale protein expression mapping in normal and diseased tissues and 44 different cell-lines as well as for a number of external and internal research projects. Methods generating a greater understanding of antibody performance and characteristics constitute a valuable resource in both pursuits. Technical development aims to expand the panel of methods at hand at the Uppsala site, to include for example siRNA knock-down of expression, PLA for bright-field microscopy, LigandTracer analysis of binding kinetics, functional analyses in cell lines and image analysis of protein expression in both tissues and cells.
In addition, the HPA has mapped the gene expression in the 44 cell lines using next generation RNA-sequencing, and both immunohistochemical (IHC) data and RNA data are assembled and visualized in a cell line atlas. The group works to continuously add and curate data in the atlas, and based on the comparative analysis of RNA and protein levels across all cell lines, antibodies not likely to target the correct protein are disapproved.
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Group leader: Prof Fredrik Ponten
Personnel: Sandra Andersson (PhD-student), Dr Julia Bergman (MD, PhD-student), Kristina Magnusson (PhD-student), Dijana Djureinovic (PhD-student), Dr Cecilia Lindskog Bergström (researcher)
Responsibility: (i) Develop strategies to identify potential biomarkers based on the HPA database and other efforts, (ii) validate proteins that can be used as clinical biomarkers for disease, (iii) participate in clinical studies, collect tumor material and clinical data to generate specific cancer TMAs coupled to clinical databases, (iv) perform statistical analysis and validate the clinical usefulness of identified biomarkers.
Description: The HPA database is actively mined for potential biomarkers with the aim to identify protein expression patterns that could be of medical or biological significance. Projects include various forms of human disease with an emphasis on cancer. Most projects are focused on identification and validation of biomarker candidates that can fulfill currently unmet clinical needs related to diagnostics, prognostics and treatment prediction. To address such questions, patient cohorts representing different cancers are defined and tumor material as well as clinical data is collected. These specifically designed cancer TMAs are produced and used for extended analysis of protein expression patterns to test and validate candidate proteins as useful biomarkers. The biomarker discovery and validation efforts include both internal projects and external collaborative projects.
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Lab SurgPath, Mumbai site
|Site Director:||Dr Sanjay Navani|
Group leader: Dr Sanjay Navani
Personnel: Dr Tushar Patil (research associate), Nitin Pardule (technician) and Avadhesh Vishwakarma (IT administrator)
Responsibility: (i) Organization and administration of pathology-based annotation and curation of immunohistochemistry-based protein profiling in normal and cancer tissues, (ii) recruitment and training of personnel for the HPA-Mumbai site, (iii) initiation, supervision and collaboration for India-based research projects. (iv) Comparison of immunohistochemical expression pattern with RNA-seq data.
Description: Manual annotation of immunohistochemistry is performed on high-resolution images generated in the tissue microarray module. A web-based in-house developed software is used to record the intensity and fraction of immunoreactive cells for each given cell population, and to determine the subcellular localization of immunoreactivity. A text comment summarizing the characteristics for each antibody is added to the annotation. The results are visualized in a summary view as color codes corresponding to the protein expression level in each given cell type. In total 83 normal cell types from 144 individuals and 20 different cancer cell types from 216 different tumors are annotated for each antibody. An independent pathologist or specially trained personnel curates all finished annotations to ensure uniform annotations of high quality.
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