Application Of Immunomagnetic Cell Separation In Cancer Cell Detection
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| Author | : Liying Yang |
| Publisher | : |
| Total Pages | : |
| Release | : 2007 |
| Genre | : Cancer |
| ISBN | : |
Abstract: Detection of rare, circulating tumor cells (CTCs) in peripheral blood is a potential prognostic/diagnostic tool in oncology. The use of immunomagnetic cell separation has been shown to improve the target cell purity and thus detection sensitivity. In this dissertation, a repeatable enrichment process including a flow through immunomagnetic cell separation system, the quadrupole magnetic cell sorter (QMS), was continuously developed and optimized. Molecular analysis technologies such as immunocytochemical assay and the Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) were combined with the enrichment process to reliably and accurately detect the presence of CTCs in peripheral blood. The novel technique was applied involving samples from head and neck patients undergoing surgery. Immunochemical staining and RT-PCR analysis of the same, enriched sample result in congruent outcome in all but one cases. Furthermore, the data with respect to the quantitative detection of CTCs is generally consistent with the pathological report on these patients. Data suggested that if a sample had 10 or more CTCs per ml of blood, a metastatic disease was present in the corresponding patient. iii In order to further improve the final purity of cancer cells, to be eligible for studies including cDNA microarray, continuous development and optimization of the novel system was desired. A kinetics model was used to describe the process of ligand binding to cell surface receptors, which demonstrated that the ratio between the initial free antibody concentration and its dissociation constant (L0/KD) is the limiting factor for a given magnetic labeling system. Based on the theory, an optimal labeling scheme is identified, including the use of a tetrameric antibody complex, resulting in significantly better separation performance with order of magnitude higher log depletion. In the second part of the study, a reaction-diffusion model was constructed to describe the in vitro tissue dissociation process. It is not only useful in recognizing the rate-limiting factor in the tissue dissociation process, but also provides quantitative guidelines to establish an optimal tissue dissociation technology. A rapid tissue dissociation process is established and characterization followed by a positive selection of EGFR targeted cancer cells.
| Author | : |
| Publisher | : |
| Total Pages | : |
| Release | : 2003 |
| Genre | : Cell separation |
| ISBN | : |
Abstract: Immunomagnetic-based cell separation techniques, developed over the last decade, have shown to be extremely efficient at obtaining highly purified cell populations from a mixture of heterogeneous cells. Commercially available immunomagnetic cell separation devices are valuable tools that enrich cell suspensions by targeting the desired cells with a suitable monoclonal antibody conjugated to magnetic particles. However, one of the main drawbacks of these devices is their limited capacity to remove undesired cells that express low numbers of surface receptors. The development of flow-through devices such as the Quadrupole Magnetic Cell Sorter (QMS) has led to further application of immunomagnetic cell separation techniques in the clinical field. QMS has already proved efficient in selecting hematopoietic stem cells, T-helper, T-cytotoxic cells and cancer cells. The use of QMS offers the following advantage: the possibility of predicting outcomes of the sorted fractions, the capability of continuous operation, and the flexibility of changing operational conditions according to the magnetophoretic mobility of the labeled cell population to obtain the desired results. QMS can operate in positive selection (when cells of interest are labeled) or negative selection (when cells of interest are not targeted). Our investigation focuses on the challenges presented in negative selection. Presently, studies involving rare cell detection (cancer cells circulating in blood), T-cell depletion, and allogeneic T-cell depletion studies are being performed to evaluate the operational suitability of the device in the medical arena. Studies conducted on T-cell depletion and allogeneic T-cell depletion indicate that the sorting efficacy using QMS outperforms past methods. These studies are vital in order to develop a device that can expand the donor pool for cells needed for therapies, such as bone marrow transplants or stem cells transplant. Our results lead us to believe that the goal to obtain cell grafts free or with very low number of both T- or allogeneic T-cells is possible. The impact of those results is vast, as our research has demonstrated cell depletion levels below those detected by flow cytometry. We are confident that the Quadrupole Magnetic Cell Sorter has many future applications including use in cell selection for bone marrow or cell therapy treatment. Additionally, QMS may become one of the first FDA-approved devices for cell selection before cell therapy.
| Author | : Diether Recktenwald |
| Publisher | : CRC Press |
| Total Pages | : 352 |
| Release | : 1997-11-04 |
| Genre | : Medical |
| ISBN | : 1482273640 |
"Offers complete coverage and assessment of cell separation technologies for analytical and preparative isolations of biological cell populations-demonstrating how to select and devise optimal sorting strategies for applications in biochemistry, immunology, cell and molecular biology, and clinical research. "
| Author | : Peng Chen |
| Publisher | : |
| Total Pages | : 290 |
| Release | : 2014 |
| Genre | : |
| ISBN | : |
Circulating tumor cells (CTCs) are the cells that are shed from a primary tumor into the vasculature and circulate in the bloodstream. CTCs may trigger cancer metastasis, which leads to most cancer-related deaths. CTCs are widely studied due to their value in cancer diagnosis, prognosis, and oncology studies. The major challenges with CTCs lie in their extremely low concentration in blood, thus requiring an effective enriching system to enable downstream analyses. The immunomagnetic assay has proved to be a promising CTC detection tool with high sensitivity and throughput. Key factors related to the immunomagnetic assay include the capture rate, which indicates the sensitivity, and distributions of target cells after capture, which impact the cell integrity and other biological properties. In this dissertation, we build a sedimentation model, a partial viscosity model, and a cell-tracking model to address the principle of the immunomagnetic cell separation. We examine the channel orientations and determine the favorable inverted condition. In addition, we develop a micromagnet approach to modulate the in-channel magnetic field toward enhanced cell detection and distribution. Through numerical studies, we calculate the magnetic field generated by the thin-film micromagnets, determine its effective ranges, and demonstrate its value in optimizing cell distribution. In the experimental demonstration, we present two types of micromagnets based on e-beam Ni deposition and inkjet printing technology, respectively. In the screening experiments, the Ni micromagnet integrated system achieves over 97% capture rate. It shows a 14% increase in capture rate, and a 14% improvement in distribution uniformity compared with plain slides. We also successfully isolate CTCs from metastatic cancer patients with the micromagnet assay. The inkjet-printed patterns yield a similarly high capture rate of 103%. With the pixel permanent magnet array, the inkjet patterns further increase the distribution uniformity for 20%. The proposed models lay the theoretical foundations for future modification of the immunomagnetic assay, and the micromagnet-integrated system provides a promising tool for translational applications in cancer diagnose and clinical cancer management.
| Author | : Thomas G. Pretlow |
| Publisher | : Academic Press |
| Total Pages | : 399 |
| Release | : 2014-05-10 |
| Genre | : Science |
| ISBN | : 1483219410 |
Cell Separation: Methods and Selected Applications, Volume 4 provides information pertinent to the design and application of methods for the separation of cells. This book covers a variety of topics, including liver cells, epidermal Langerhans cells, isolation of oval cells, clonal analysis, and the purification of polymorphonuclear leukocytes. Organized into 17 chapters, this volume begins with an overview of the central role of the liver in the metabolism of the body. This text then provides the analysis of Langerhans cells that allow modulation of their function and provide approaches to the treatment of skin disease. Other chapters consider the biological significance of oval cells. This book discusses as well the elucidation of the mechanisms of cellular proliferation, function, and differentiation in living tissues. The final chapter deals with the important applications of cell culture that involve continuous cell lines. This book is a valuable resource for cell biologists, experimental oncologists, hematologists, immunologists, and endocrinologists.
| Author | : Yu-Yen Huang |
| Publisher | : |
| Total Pages | : 216 |
| Release | : 2013 |
| Genre | : |
| ISBN | : |
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 | : Wouter De Brouwer |
| Publisher | : |
| Total Pages | : |
| Release | : 2017 |
| Genre | : |
| ISBN | : |
Abstract text (max. 300 words)Please use the following headings for the structure of your abstract: ObjectivesMethodsResultsConclusionObjective: Minimal residual disease (MRD) after treatment is associated with reduced progression-free survival in myeloma. Detection of circulating tumor cells (CTCu2019s) could represent a non-invasive method to evaluate MRD. The aim of this study is to develop a sensitive and reliable technique to detect CTCu2019s.Methods: In a series of spike-in experiments, we contaminated a fixed amount of peripheral blood mononuclear cells (PBMCu2019s) with LP-1 cells (human myeloma cell line) in decreasing concentrations. After a CD138-based double immunomagnetic enrichment-step (Auto MACS) we tried to detect the myeloma cells with allele-specific oligonucleotide polymerase chain reaction (qASO-PCR). We determined the efficiency of enrichment, the best method of DNA extraction and the optimal qASO-PCR conditions.Results: Immunomagnetic cell separation allowed to enrich the tumor cells more than 150-fold. We used the QiAmp DNA extraction kit for all our samples. When paired samples (with equal starting numbers) were analyzed, there was no real significant difference between detection rates in unseparated versus enriched fractions (limit of detection 10-5). However, the tumor cell enrichment step depleted PBMCu2019s sufficiently to remain within the capacity limits of the Qiagen DNA extraction method and qASO-PCR. This allowed to start with higher cell numbers (108) and to increase the qASO-PCR detection level at least ten-fold (10-6).Conclusion: Our data indicate that CTC enrichment prior to molecular tumor detection allows to analyze larger blood sample volumes while preserving the lower detection limit at the currently standard 10-6 level. Compared with the standard qASO-PCR technique with non-enriched cell suspensions, our method allows for more tumor cells to be analyzed. Future experiments have to reveal whether the enrichment efficiency of CTC (in terms of purity and yield) can be increased and whether the use of Ig targeted next-generation sequencing can further improve the tumor detection sensitivity in this already promising dual-platform strategy.
| Author | : Mark Jesus M. Magbanua |
| Publisher | : Humana |
| Total Pages | : 0 |
| Release | : 2018-08-03 |
| Genre | : Medical |
| ISBN | : 9781493983971 |
This volume explores various approaches for enrichment, detection, isolation, and molecular profiling of circulating tumor cells (CTCs). Each chapter provides comprehensive descriptions and guidelines on how to perform innovative experiments in CTC research. Included are protocols for capture of CTCs via filtration and density gradient centrifugation; microfluidic and immunomagnetic separation of CTCs; detection of CTCs by immunocytochemistry, fluorescence in situ hybridization, and flow cytometry; assays designed for genomic characterization and functional analyses of CTCs, and many more. Written in the highly successful Methods in Molecular Biology series format, the chapters in this book 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. Cutting-edge and authoritative, Circula ting Tumor Cells: Methods and Protocols is a valuable resource for laboratory researchers and clinicians who are interested in furthering their studies on CTCs.
| Author | : Malisha Pattanaik |
| Publisher | : |
| Total Pages | : 59 |
| Release | : 2009 |
| Genre | : |
| ISBN | : |
The ability to identify, characterize and isolate cancer cell subpopulations from a global cell population is important and fundamental for effective cancer diagnostic treatments. Cells that have shed from a primary tumor site at early stages of malignant progression and then enter the blood circulation are called circulating tumor cells (CTC). Although only a small percentage of CTC survive (roughly 0.05%), the detection of these cells provides an excellent indicator of metastatic primary tumor sites. The necessity for effective measurement of CTC requires engineering a standardized detection method capable of processing whole blood samples. An alternative method to cell selection is the alternating current (AC) electrokinetic technique, dielectrophoresis (DEP), which employs the use of alternating current (AC) fields to separate cells. Previous studies have confirmed the diagnostic ability of identification and separation of live cells from dead ones using DEP. A novel approach utilized to kill explicit cell lines in order to perform DEP separations of live and dead cells, employs a developed theory that tumor microenvironments are more acidic than normal microenvironments. By combining the selection of cancer cells from normal cells through pH control and subsequently separating these cells using dielectrophoresis an effective circulating tumor cell detection system can be produced.
| Author | : A. M. Cheret |
| Publisher | : |
| Total Pages | : 358 |
| Release | : 1982 |
| Genre | : Cell separation |
| ISBN | : 9780125645010 |
