Sensitive and High-throughput Detection, Separation and Analysis of Circulating Tumor Cells PDF Download
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Author: Mengxia Zhao
Publisher:
ISBN:
Category :
Languages : en
Pages : 113
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Book Description
This dissertation describes novel methods to detect, separate and analyze circulating tumor cells (CTCs) from peripheral blood, with a high sensitivity and throughput. The ensemble-decision aliquot ranking (eDAR) platform is a new method for isolating CTCs from cancer patients with a recovery ratio of 93% and a zero false positive rate. We have validated this method by analyzing the samples from breast, pancreatic and lung cancer patients. The capability to perform downstream analyses on the isolated CTCs were also tested and integrated into the eDAR platform. We developed a method that can monitor eight protein markers, using a sequential staining and photobleaching procedure. Single-cell isolation and culture of the trapped CTCs were also tested. Based on these, we re-designed the eDAR platform by applying a new active cell sorting scheme and a new further purification method. Because many applications in the CTC area are focused on the enumeration, we also developed an automated method for counting tumor cells in whole blood samples. This enumeration method was validated by a side-by-side comparison with CellSearch, the only FDA approved method at present, in 90 metastatic breast cancer patient samples. These tools facilitate fundamental investigation of rare cells, as well as developing non-invasive biopsy to improve the clinical treatment.
Author: Mengxia Zhao
Publisher:
ISBN:
Category :
Languages : en
Pages : 113
Get Book Here
Book Description
This dissertation describes novel methods to detect, separate and analyze circulating tumor cells (CTCs) from peripheral blood, with a high sensitivity and throughput. The ensemble-decision aliquot ranking (eDAR) platform is a new method for isolating CTCs from cancer patients with a recovery ratio of 93% and a zero false positive rate. We have validated this method by analyzing the samples from breast, pancreatic and lung cancer patients. The capability to perform downstream analyses on the isolated CTCs were also tested and integrated into the eDAR platform. We developed a method that can monitor eight protein markers, using a sequential staining and photobleaching procedure. Single-cell isolation and culture of the trapped CTCs were also tested. Based on these, we re-designed the eDAR platform by applying a new active cell sorting scheme and a new further purification method. Because many applications in the CTC area are focused on the enumeration, we also developed an automated method for counting tumor cells in whole blood samples. This enumeration method was validated by a side-by-side comparison with CellSearch, the only FDA approved method at present, in 90 metastatic breast cancer patient samples. These tools facilitate fundamental investigation of rare cells, as well as developing non-invasive biopsy to improve the clinical treatment.
Author: Michail Ignatiadis
Publisher: Springer Science & Business Media
ISBN: 3642281605
Category : Medical
Languages : en
Pages : 245
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Book Description
This important book provides up-to-date information on a series of topical issues relating to the approach to minimal residual disease in breast cancer patients. It first explains how the study of minimal residual disease and circulating and disseminated tumor cells (CTCs/DTCs) can assist in the understanding of breast cancer metastasis. A series of chapters then discuss the various technologies available for the detection and characterization of CTCs and DTCs, pinpointing their merits and limitations. Detailed consideration is given to the relevance of CTCs and DTCs, and their detection, to clinical research and practice. The role of other blood-based biomarkers is also addressed, and the closing chapters debate the challenges facing drug and biomarker co-development and the use of CTCs for companion diagnostic development. This book will be of interest and assistance to all who are engaged in the modern management of breast cancer.
Author: Yu-Yen Huang
Publisher:
ISBN:
Category :
Languages : en
Pages : 216
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Book Description
Circulating tumor cells (CTCs) are known to escape from the primary tumor site and may settle down at the distant organ to grow a second tumor. CTCs are one of causes initiating carcinoma metastasis. Detection of CTCs has been considered to be valuable for cancer management, including diagnosis, prognosis, and clinical treatment management. However, efficient isolation, enumeration, characterization, and genetic analysis of CTCs in whole-blood samples from cancer patients are very challenging due to their extremely low concentration and rare nature (per CTC in blood cells is 1:106-109). With the increasing worldwide death rate associated with cancer, there is a desperate demand for a high-sensitivity, high-throughput, and low-cost detection and separation system. My doctoral research focused on the design and fabrications of the screening system for the detection of CTCs with further analysis of captured CTCs, such as immunofluoresce staining and fluorescence in-situ hybridization (FISH). The distinct significance of this research is that the development of the computer-controlled rotational holder with a series of six inverted microfluidic chips reduced the cost by significantly reducing the consumption of magnetic carriers (25% of the consumed amount used in the commercial CellSearch® system), increasing the capture efficiency by manipulating the blood sedimentation in the microchannel, enhancing the system stability by integrating the micromagnets on the plain glass slide substrate, and achieving high throughput because of the high flow rate (2.5 mL/hr) and large screening volume (screening up to six chips in parallel with each containing 2.5 mL of blood). Immunofluorescence staining and the FISH method have been performed to prove the capability of the system. In addition, the system has been successfully applied for patient samples screening. The incorporation of micromagnets has demonstrated that micromagnets provide localized magnetic forces to scatter the target cancer cells and free nanoparticles throughout the whole channel substrate to increase the channel space usage by 13%. Four cancer cell lines, including COLO 205 (colorectal cancer), SK-BR-3 (breast cancer), MCF-7 (breast cancer), and PC3 (prostate cancer), were spiked in blood samples from healthy donors to verify high capture efficiency of the developed system. On average, over a 97% capture rate was demonstrated for all cell lines. Moreover, the developed screening system has been successfully screened over 40 patient samples, including metastatic lung cancer, breast cancer, prostate cancer, and colorectal cancer. After capture of CTCs, immunofluorescence staining was used to identified the captured cancer cells and the FISH method was performed to characterize the isolated cancer cells by studying the gene expression of CTCs from breast cancer. The proposed automated immunomagnetic microchip-based screening system shows high capture efficiency (average 97% for three spiked cell lines), high throughput (15 mL of blood sample per screening), high sensitivity, high specificity, and low nanoparticle consumption (75% less than CellSearch® system). The screening system provides great promise as a clinical tool for early cancer diagnosis, diagnosis, personalized therapy, and treatment monitoring.
Author: Dawson J. Wong
Publisher:
ISBN:
Category :
Languages : en
Pages :
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Book Description
The presence of circulating tumor cells (CTCs) in the blood of metastatic cancer patients can be an indicator of poor prognosis and survival. However, CTC detection assays that enable non-invasive "liquid biopsy" are generally limited by the lack of high-throughput, high-efficiency enrichment of CTCs from whole blood and rapid, accurate enumeration of the collected CTCs. Moreover, molecular analysis of single CTCs, which may recapitulate primary and metastatic tumor biology, remains challenging because current platforms have limited throughput, are expensive, and are not easily translatable to the clinic. This doctoral dissertation describes research work that aims to address these challenges by enabling a comprehensive image and transcriptomic analysis of CTCs from cancer patients. Image analysis of CTCs by immunocytochemistry comprises the immunofluorescent staining of CTCs followed by image analysis to identify CTCs. After CTCs were immunomagnetically enriched from whole blood samples by MagSifter and immunofluorescently stained, fluorescence microscope images of CTCs were then acquired on prepared cytospin slides. To achieve automated image cytometry of CTCs, CTC candidate images were first identified using an automated cell image segmentation method. Subsequently, a Random Forest machine learning algorithm was developed to classify each CTC candidate image as positive or negative, based on parameters such as size, shape, and fluorescence intensities in different channels. This approach was applied to identify and enumerate CTCs enriched from non-small cell lung cancer (NSCLC) patients' blood samples with high correlation to pathological assessment. This approach for automated image cytometry has demonstrated high-throughput CTC identification with excellent accuracy and reproducibility. For downstream molecular analysis of CTCs at the single-cell level, a massively parallel, multiplex gene expression profiling platform has been developed to enable compartmentalization and analysis of hundreds of single CTCs. After magnetic collection of CTC from blood, a single-cell Nanowell assay performs CTC mutation profiling using modular gene panels. Using this approach, multigene expression profiling of individual CTCs from NSCLC patients has been demonstrated with unprecedented sensitivity. These results represent the first demonstration of a high-throughput, multiplexed strategy for comprehensive single-cell mutation profiling of individual lung cancer CTCs toward non-invasive cancer therapy prediction and disease monitoring.
Author: Shay Soker
Publisher: Humana Press
ISBN: 3319605119
Category : Medical
Languages : en
Pages : 225
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Book Description
Cancer cell biology research in general, and anti-cancer drug development specifically, still relies on standard cell culture techniques that place the cells in an unnatural environment. As a consequence, growing tumor cells in plastic dishes places a selective pressure that substantially alters their original molecular and phenotypic properties.The emerging field of regenerative medicine has developed bioengineered tissue platforms that can better mimic the structure and cellular heterogeneity of in vivo tissue, and are suitable for tumor bioengineering research. Microengineering technologies have resulted in advanced methods for creating and culturing 3-D human tissue. By encapsulating the respective cell type or combining several cell types to form tissues, these model organs can be viable for longer periods of time and are cultured to develop functional properties similar to native tissues. This approach recapitulates the dynamic role of cell–cell, cell–ECM, and mechanical interactions inside the tumor. Further incorporation of cells representative of the tumor stroma, such as endothelial cells (EC) and tumor fibroblasts, can mimic the in vivo tumor microenvironment. Collectively, bioengineered tumors create an important resource for the in vitro study of tumor growth in 3D including tumor biomechanics and the effects of anti-cancer drugs on 3D tumor tissue. These technologies have the potential to overcome current limitations to genetic and histological tumor classification and development of personalized therapies.
Author: Natasha S. Barteneva
Publisher: Humana
ISBN: 9781493933006
Category : Medical
Languages : en
Pages : 0
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Book Description
This detailed volume for the first time explores techniques and protocols involving quantitative imaging flow cytometry (IFC), which has revolutionized our ability to analyze cells, cellular clusters, and populations in a remarkable fashion. Beginning with an introduction to technology, the book continues with sections addressing protocols for studies on the cell nucleus, nucleic acids, and FISH techniques using an IFC instrument, immune response analysis and drug screening, IFC protocols for apoptosis and cell death analysis, as well as morphological analysis and the identification of rare cells. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, Imaging Flow Cytometry: Methods and Protocols will be a critical source for all laboratories seeking to implement IFC in their research studies.
Author: Robert C. Bast, Jr.
Publisher: John Wiley & Sons
ISBN: 111900084X
Category : Medical
Languages : en
Pages : 2004
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Book Description
Holland-Frei Cancer Medicine, Ninth Edition, offers a balanced view of the most current knowledge of cancer science and clinical oncology practice. This all-new edition is the consummate reference source for medical oncologists, radiation oncologists, internists, surgical oncologists, and others who treat cancer patients. A translational perspective throughout, integrating cancer biology with cancer management providing an in depth understanding of the disease An emphasis on multidisciplinary, research-driven patient care to improve outcomes and optimal use of all appropriate therapies Cutting-edge coverage of personalized cancer care, including molecular diagnostics and therapeutics Concise, readable, clinically relevant text with algorithms, guidelines and insight into the use of both conventional and novel drugs Includes free access to the Wiley Digital Edition providing search across the book, the full reference list with web links, illustrations and photographs, and post-publication updates
Author: Rosalyn D. Blumenthal
Publisher: Humana Press
ISBN: 9781588295866
Category : Medical
Languages : en
Pages : 442
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Book Description
A state-of-the art collection of readily reproducible laboratory methods for assessing chemosensitivity in vitro and in vivo, and for assessing the parameters that modulate chemosensitivity in individual tumors. Chemosensitivity,Volume 2: In Vivo Models, Imaging, and Molecular Regulators contains cutting-edge protocols for classifying tumors into response categories and for customizing therapy to individuals. These readily reproducible techniques allow measurements of DNA damage, apoptotic cell death, and the molecular and cellular regulators of cytotoxicity, as well as in vivo animal modeling of chemosensitivity. A companion volume, Volume 1: In Vitro Assays contains in vitro and in vivo techniques to identify which new agents or combination of agents are effective for each type of tumor.
Author: Fan Xia
Publisher: Springer
ISBN: 9811078351
Category : Science
Languages : en
Pages : 220
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Book Description
This book shows the various sandwich assays that are constructed from recognition molecules, such as antibodies, oligonucleotide sequences and aptamers, developed as a result of nano- and biotechnology advances. It consists of ten chapters presenting interesting examples of these assays, organized according to the type of analytic methods (colorimetric, fluorescence, electrochemical, etc.) and detected objects (protein, nucleic acid, small-molecule, ion, etc.). It also includes a chapter discussing the introduction of sandwich assays as biosensors for the detection of a range of targets. It is an interesting and useful resource for a wide readership in various fields of chemical science and nanotechnology.
Author: Alice Longobardi Givan
Publisher: John Wiley & Sons
ISBN: 1118688392
Category : Science
Languages : en
Pages : 309
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Book Description
Flow cytometry continually amazes scientists with its ever-expanding utility. Advances in flow cytometry have opened new directions in theoretical science, clinical diagnosis, and medical practice. The new edition of Flow Cytometry: First Principles provides a thorough update of this now classic text, reflecting innovations in the field while outlining the fundamental elements of instrumentation, sample preparation, and data analysis. Flow Cytometry: First Principles, Second Edition explains the basic principles of flow cytometry, surveying its primary scientific and clinical applications and highlighting state-of-the-art techniques at the frontiers of research. This edition contains extensive revisions of all chapters, including new discussions on fluorochrome and laser options for multicolor analysis, an additionalsection on apoptosis in the chapter on DNA, and new chapters onintracellular protein staining and cell sorting, including high-speed sorting and alternative sorting methods, as well as traditional technology. This essential resource: Assumes no prior knowledge of flow cytometry Progresses with an informal, engaging lecture style from simpleto more complex concepts Offers a clear introduction to new vocabulary, principles of instrumentation, and strategies for data analysis Emphasizes the theory relevant to all flow cytometry, with examples from a variety of clinical and scientific fields Flow Cytometry: First Principles, Second Edition provides scientists, clinicians, technologists, and students with the knowledge necessary for beginning the practice of flow cytometry and for understanding related literature.
