Sample Preparation Technology Research Articles

Analytical validation and initial clinical testing of quantitative microscopic evaluation for PD-L1 and HLA I expression on circulating tumor cells from patients with non-small cell lung cancer

IntroductionPD-L1 expression in non-small cell lung cancer (NSCLC) predicts response to immune checkpoint blockade, however is an imperfect biomarker given tumor heterogeneity, and the antigen presentation pathway requiring other components including HLA I expression. HLA I downregulation may contribute to resistance, warranting its evaluation in attempts to guide patient selection. In addition, earlier detection of acquired resistance could prompt earlier change in treatment and prolong patient survival. Analysis of circulating tumor cells (CTCs) captures heterogeneity across multiple sites of metastases, enables detection of changes in tumor burden that precede radiographic response, and can be obtained in serial fashion.MethodsTo quantify the expression of both PD-L1 and HLA I on CTCs, we developed exclusion-based sample preparation technology, achieving high-yield with gentle magnetic movement of antibody-labeled cells through virtual barriers of surface tension. To achieve clinical-grade quantification of rare cells, we employ high quality fluorescence microscopy image acquisition and automated image analysis together termed quantitative microscopy.ResultsIn preparation for clinical laboratory implementation, we demonstrate high precision and accuracy of these methodologies using a diverse set of control materials. Preliminary testing of CTCs isolated from patients with NSCLC demonstrate heterogeneity in PD-L1 and HLA I expression and promising clinical value in predicting PFS in response to PD-L1 targeted therapies.ConclusionsBy confirming high performance, we ensure compatibility for clinical laboratory implementation and future application to better predict and detect resistance to PD-L1 targeted therapy in patients with NSCLC.

Optimization of the In Situ Biasing FIB Sample Preparation for Hafnia-Based Ferroelectric Capacitor.

Hafnia-based ferroelectric (FE) thin films have received extensive attention in both academia and industry, benefitting from their outstanding scalability and excellent CMOS compatibility. Hafnia-based FE capacitors in particular have the potential to be used in dynamic random-access memory (DRAM) applications. Obtaining fine structure characterization at ultra-high spatial resolution is helpful for device performance optimization. Hence, sample preparation by the focused ion beam (FIB) system is an essential step, especially for in situ biasing experiments in a transmission electron microscope (TEM). In this work, we put forward three tips to improve the success rate of in situ biasing experiments: depositing a carbon protective layer to position the interface, welding the sample on the top of the Cu column of the TEM grid, and cutting the sample into a comb-like shape. By these means, in situ biasing of the FE capacitor was realized in TEM, and electric-field-induced tetragonal (t-) to monoclinic (m-) structure transitions in Hf0.5Zr0.5O2 FE film were observed. The improvement of FIB sample preparation technology can greatly enhance the quality of in situ biasing TEM samples, improve the success rate, and extend from capacitor sample preparation to other types.

Experimental Investigation of sample preparation and grouting technology on microbially reinforced tailings

The nonuniformity of samples in reinforcement treatment is a bottleneck that restricts the development of microbial geotechnical technologies, which severely affects the reinforcement effect of microbially reinforced tailings and other fine-grained geotechnical materials. This study presents a mixing method that represents an innovative sample preparation and grouting technology for reinforcing tailings using microbially induced calcite precipitation (MICP) based on the characteristics of tailings discharged in the form of mud and accumulated layer-by-layer. A large number of mechanical experiments and microanalyses were conducted to compare the effects of various methods on tailings reinforcement, including the methods of adding bacterial solution (i.e., the traditional grouting method and the proposed mixing method) and the treatment methods of cementing solution (i.e., the grouting method and soaking). The results of the shear test showed that the strength of the samples generated by the mixing method increased compared with the original tailings samples and samples generated using the traditional grouting method. Moreover, miniature cone penetration test results showed that the samples made using the mixing method had better uniformity, which could be attributed to the fine particles (silt and clay particles) of tailings that strongly adsorb bacteria and the low permeability of the fine particle tailings, which is not conducive to the spread of bacteria. In the tailings samples, the traditional grouting method only makes the bacteria gather at the region where the bacterial solution initially makes contact. However, the mixing method evenly distributed the bacteria among the tailings particles, which significantly improved the reinforcement effect of bacteria, as the reinforcement reaction occurred throughout each region of the samples. Therefore, the mixing method can be used for reinforcement research and in the application of MICP to tailings or other fine-grained geotechnical materials.

Preparation of a new coating of graphene oxide/nickel complex on a nickelized metal surface for direct immersion solid phase microextraction of some polycyclic aromatic hydrocarbons

BackgroundSolid-phase microextraction (SPME) is a versatile sampling and sample preparation technology that possess a significant application in the extraction and pre-concentration of a broad range of micro-pollutants from different kind of matrices. Selection and preparation of an appropriate fiber substrate and coating materials have always been the main challenges of the SPME procedure. This paper introduces a high-efficiency metal-based SPME fiber with a new chemical coating of nickel/graphene oxide/nickel tetraazamacrocyclic complex (Ni/GO/NiTAM).ResultThe Ni/GO/NiTAM sorbent was electroless deposited onto the surface of an aluchrom (Alu) wire, and then the prepared fiber was employed for the extraction and pre-concentration of some PAHs before their HPLC–UV analysis. The prepared fiber characterization data were assessed using FE-SEM, EDX, XRD, FT-IR, and BET techniques. The method validation parameters, including the linearity range (LRs: 0.10 to 200.0 µg L−1), the limit of detection (LODs: 0.03‒0.30 µg L−1), and the limit of quantification (LOQs: 0.10–1.00 µg L−1), under optimal conditions. The relative standard deviations (RSDs) of intra-day, inter-day, and single fiber repeatability (for the samples spiked at 25 µg L‒1) were in the range of 0.32–2.94, 1.20–4.09, and 1.42‒4.39%, respectively. In addition, the technique recoveries (RR %) and enrichment factors (EF) were in the range of 83.10‒107.80% and 83–164, respectively.ConclusionThe fiber fabrication was simple, and the applied materials were also economical and easily accessible. Alu metal has high physicochemical and mechanical stability and thus can be a good alternative for the substrate of the fragile commercial SPME fibers. High rigidity and durability, long service life, and high extraction capability are some of the other advantages of the offered fiber.

Reversible Click Chemistry Tag for Universal Proteome Sample Preparation for Top-Down and Bottom-Up Analysis.

Successful proteome analysis requires reliable sample preparation beginning with protein solubilization and ending with a sample free of contaminants, ready for downstream analysis. Most proteome sample preparation technologies utilize precipitation or filter-based separation, both of which have significant disadvantages. None of the current technologies are able to prepare both intact proteins or digested peptides. Here, we introduce a reversible protein tag, ProMTag, that enables whole proteome capture, cleanup, and release of intact proteins for top-down analysis. Alternatively, the addition of a novel Trypsin derivative to the workflow generates peptides for bottom-up analysis. We show that the ProMTag workflow yields >90% for intact proteins and >85% for proteome digests. For top-down analysis, ProMTag cleanup improves resolution on 2D gels; for bottom-up exploration, this methodology produced reproducible mass spectrometry results, demonstrating that the ProMTag method is a truly universal approach that produces high-quality proteome samples compatible with multiple downstream analytical techniques. Data are available via ProteomeXchange with identifier PXD027799.

Extractive Freezing-Out with Centrifugation—A New Technology of Sample Preparation in Chemical Analysis on an Example of Organic Bases

Extractive Freezing-Out with Centrifugation—A New Technology of Sample Preparation in Chemical Analysis on an Example of Organic Bases

Sample preparation technologies for serial synchrotron crystallography and single-particle cryo-EM

Sample preparation technologies for serial synchrotron crystallography and single-particle cryo-EM

Green bioanalytical sample preparation: fabric phase sorptive extraction.

Fabric phase sorptive extraction (FPSE) is a recently introduced sample preparation technique that has attracted substantial interest of the scientific community dealing with bioanalysis. This technique is based on a permeable and flexible substrate made of fabric, coated with a sol-gel organic-inorganic sorbent. Among the benefits of FPSE are its tunable selectivity, adjustable porosity, minimized sample preparation workflow, substantially reduced organic solvent consumption, rapid extraction kinetics and superior extraction efficiency, many of which are well-known criteria for Green Analytical Chemistry. As such, FPSE has established itself as a leading green sample preparation technology of 21st century. In this review, we discuss the principal steps for the development of an FPSE method, the main method optimization strategies, as well as the applications of FPSE in bioanalysis for the extraction of a wide range of analytes (e.g., estrogens, benzodiazepines, androgens and progestogens, penicillins, anti-inflammatory drugs, parabens etc.).

Ultramicrotomy preparation of magnetic nanoparticles for transmission electron microscopy

Transmission electron microscopy (TEM) is one of the most important methods for the morphological characterization and structure analysis of nanomaterials. However, the characterization of magnetic materials has always been a challenge due to limitations arising from the design of electron microscopes. To tackle this problem, advanced sample preparation technology is needed, especially for magnetic materials. Here in this work ultrathin sectioning technology (ultramicrotomy) is used for the sample preparation of magnetic Fe3O4 nanoparticles embedded into a resin, where the loaded resin can be sliced into nanoscale sheets. By the optimization of the embedding method and the slicing process, nano-sheets with uniform thickness and exceptional flatness were prepared, where the nanoparticles exhibited uniform dispersion. It is shown that this technology also helps reducing the degree of pollution of the electron microscope by the magnetic nanoparticles under different electron beam irradiation intensities. Generally, the magnetic nanoparticles are more resistant to electron beam bombardment when embedded into a resin.

Research progress of microextraction by packed sorbent and its application in microvolume sample extraction

微萃取技术是分析化学领域发展迅速,且已经得到广泛应用的样品前处理技术。填充吸附剂微萃取(MEPS)是一种微量固相萃取技术,使用微量的吸附剂填充于微量注射器,通过反复抽推方式使样品多次流经吸附剂以完成样品吸附萃取过程,萃取后的样品可直接用于色谱分析。典型的MEPS萃取设备包括MEPS注射器和MEPS吸附床(BIN)。MEPS优化的主要因素为MEPS处理过程的参数,包括样品流速、样品量与样品萃取循环次数,吸附剂及淋洗、洗脱溶剂的种类和体积,还需要考虑样品基质对MEPS性能的影响和样品残留和重复使用问题。MEPS中最重要的部分是吸附剂,主要有商品化的MEPS吸附剂,包括硅基的Silica、C18、C8等,碳材料的Hypercarb和聚苯乙烯聚合物类的SDVB、HDVB吸附剂等。研究用的吸附剂包括分子印迹材料、限进分子印迹材料、碳基材料、导电聚合物类材料、改性硅基材料及共价-有机骨架材料等。MEPS结合多种分析仪器已经成功应用于从不同基质中提取单一或多种分析物,所涵盖基质包括生物样品(尿液、唾液、血浆或血液)、河流水体或生活污水以及几种食品和饮料。MEPS处理复杂生物基质样品时,通常需要稀释样品、除蛋白质等预处理。MEPS具有需要样品体积小、操作快速等特点,在生物基质样品分析中有望得到更广泛的使用。在环境样品中,该技术可与现场便携仪器联用,未来将有望在现场进行快速检测,并于易分解样品等方面发挥作用。

Fully Integrated and Multiplexed Sample Preparation Technology for Sensitive Interactome Profiling.

Affinity purification coupled to mass spectrometry (AP-MS) is a popular approach for deciphering the architecture of protein interaction networks. Protein lysates (100 μg) are typically required for multistep sample processing in large volumes, which often causes sample loss and reduces the MS analysis sensitivity. Herein, we reported a fully integrated spintip-based AP-MS technology, termed FISAP, for multiplexed and sensitive interactome profiling. The FISAP device can be easily employed for routine use by introducing AP beads into a C18 StageTip. Taking advantage of the switchable functionalization of the C18 matrix by sodium dodecyl sulfate, all the sample preparation steps encompassing peptide or antibody-based AP, reduction, alkylation, tryptic digestion, tandem mass tag (TMT) labeling, and desalting can be performed in a single tip with a benchtop centrifuge in 4 h. Using a biotinylated tyrosine phosphorylated (pTyr) peptide as an affinity ligand, we mapped the pTyr-dependent interactome of the pY191 motif on the immune receptor CD28 cytoplasmic domain. When processing 50 μg of protein lysates, FISAP showed a comparable interactome identification performance but better quantification performance and lower background interference compared to the traditional tube-based method. Furthermore, a cost-effective on-column TMT labeling protocol was established and integrated into the FISAP pipeline with increased sensitivity. Compared to the tube-based method, the usage of a synthetic peptide probe and a TMT reagent was both reduced by 20 times. As low as 1 μg of protein lysates could be applied for interactome profiling. Finally, we expanded the applicability of the FISAP technology to epitope tag-based AP-MS for profiling the ILK/PINCH/Parvin complex using 100 times less protein lysate than a previous report. Collectively, FISAP is an easy-to-use and sensitive technology for quantitatively profiling protein complexes when the starting material and affinity reagent are the limitation, especially for applications in biomedical research and chemical biology.

Recent advances in sample preparation technologies for analysis of harmful substances in aquatic products

水产品含有丰富的蛋白、维生素和多种微量元素,是人们摄取动物性蛋白质的重要来源之一,我国是世界上最大的水产品消费国,其质量安全问题一直备受关注。但水产样品基质复杂,有害物质的含量低,须对其进行分离富集后才能进行检测,传统的液-液萃取、固相萃取和快速固相分散萃取等样品前处理技术在水产品分析中得到广泛应用,同时针对一些挥发性和超痕量有害物质检测时,固相微萃取同样体现出巨大优势。这些样品前处理技术可以有效去除基体对分析对象的干扰,提高检测方法的灵敏度和准确度。根据目标分析物性质的不同,选择合适的样品前处理技术,是水产品中有害物质分析的关键步骤。该文以水产品中有害物的来源不同,将其分为3类:(1)水产品中环境污染物的分析;(2)养殖运输和加工过程中有害物的分析;(3)水产品中生物毒素的分析。以这3类有害物质的分析为主线,综述了近10年水产品中有害物质分析的样品前处理技术,包括液-液萃取、固相萃取、固相微萃取、快速固相分散萃取和磁性固相萃取等。此外,还对各种技术的优缺点进行了探讨,并对其未来发展方向进行了展望。

A Review: Sample Preparation and Chromatographic Technologies for Detection of Aflatoxins in Foods.

As a class of mycotoxins with regulatory and public health significance, aflatoxins (e.g., aflatoxin B1, B2, G1 and G2) have attracted unparalleled attention from government, academia and industry due to their chronic and acute toxicity. Aflatoxins are secondary metabolites of various Aspergillus species, which are ubiquitous in the environment and can grow on a variety of crops whereby accumulation is impacted by climate influences. Consumption of foods and feeds contaminated by aflatoxins are hazardous to human and animal health, hence the detection and quantification of aflatoxins in foods and feeds is a priority from the viewpoint of food safety. Since the first purification and identification of aflatoxins from feeds in the 1960s, there have been continuous efforts to develop sensitive and rapid methods for the determination of aflatoxins. This review aims to provide a comprehensive overview on advances in aflatoxins analysis and highlights the importance of sample pretreatments, homogenization and various cleanup strategies used in the determination of aflatoxins. The use of liquid-liquid extraction (LLE), supercritical fluid extraction (SFE), solid phase extraction (SPE) and immunoaffinity column clean-up (IAC) and dilute and shoot for enhancing extraction efficiency and clean-up are discussed. Furthermore, the analytical techniques such as gas chromatography (GC), liquid chromatography (LC), mass spectrometry (MS), capillary electrophoresis (CE) and thin-layer chromatography (TLC) are compared in terms of identification, quantitation and throughput. Lastly, with the emergence of new techniques, the review culminates with prospects of promising technologies for aflatoxin analysis in the foreseeable future.

Spatial proteome profiling by immunohistochemistry-based laser capture microdissection and data-independent acquisition proteomics

Understanding the tumor heterogeneity through spatially resolved proteome profiling is important for biomedical research and clinical application. Laser capture microdissection (LCM) is a powerful technology for exploring local cell populations without losing spatial information. Conventionally, tissue sections are stained with hematoxylin and eosin (H&E) for cell-type identification before LCM. However, it generally requires experienced pathologists to distinguish different cell types, which limits the application of LCM to broad cancer research field. Here, we designed an immunohistochemistry (IHC)-based workflow for cell type-resolved proteome analysis of tissue samples. Firstly, targeted cell type was marked by IHC using antibody targeting cell-type specific marker to improve accuracy and efficiency of LCM. Secondly, to increase protein recovery from chemically crosslinked IHC tissues, we optimized a decrosslinking procedure to seamlessly combine with the integrated spintip-based sample preparation technology SISPROT. This newly developed approach, termed IHC-SISPROT, has comparable performance as H&E staining-based proteomic analysis. High sensitivity and reproducibility of IHC-SISPROT were achieved by combining with data independent acquisition proteomics. More than 3500 proteins were identified from only 0.2 mm2 and 12 μm thickness of hepatocellular carcinoma (HCC) tissue section. Furthermore, using 5 mm2 and 12 μm thickness of HCC tissue section, 6660 and 6052 protein groups were quantified from cancer cells and cancer-associated fibroblasts (CAFs) by the IHC-SISPROT workflow. Bioinformatic analysis revealed the enrichment of cell type-specific ligands and receptors and potentially new communications between cancer cells and CAFs by these signaling proteins. Therefore, IHC-SISPROT is a sensitive and accurate proteomic approach for spatial profiling of cell type-specific proteome from tissues.

Electron Microscopy Views of Dimorphic Chloroplasts in C4 Plants.

C4 plants enhance photosynthesis efficiency by concentrating CO2 to the site of Rubisco action. Chloroplasts in C4 plants exhibit structural dimorphism because thylakoid architectures vary depending on energy requirements. Advances in electron microscopy imaging capacity and sample preparation technologies allowed characterization of thylakoid structures and their macromolecular arrangements with unprecedented precision mostly in C3 plants. The thylakoid is assembled during chloroplast biogenesis through collaboration between the plastid and nuclear genomes. Recently, the membrane dynamics involved in the assembly process has been investigated with 3D electron microscopy, and molecular factors required for thylakoid construction have been characterized. The two classes of chloroplasts in C4 plants arise from common precursors, but little is known about how a single type of chloroplasts grow, divide, and differentiate to mature into distinct chloroplasts. Here, we outline the thylakoid structure and its assembly processes in C3 plants to discuss ultrastructural analyses of dimorphic chloroplast biogenesis in C4 plant species. Future directions for electron microscopy research of C4 photosynthetic systems are also proposed.

Recent progress in the analysis of protein deamidation using mass spectrometry.

Deamidation is a nonenzymatic and spontaneous posttranslational modification (PTM) that introduces changes in both structure and charge of proteins, strongly associated with aging proteome instability and degenerative diseases. Deamidation is also a common PTM occurring in biopharmaceutical proteins, representing a major cause of degradation. Therefore, characterization of deamidation alongside its inter-related modifications, isomerization and racemization, is critically important to understand their roles in protein stability and diseases. Mass spectrometry (MS) has become an indispensable tool in site-specific identification of PTMs for proteomics and structural studies. In this review, we focus on the recent advances of MS analysis in protein deamidation. In particular, we provide an update on sample preparation, chromatographic separation, and MS technologies at multi-level scales, for accurate and reliable characterization of protein deamidation in both simple and complex biological samples, yielding important new insight on how deamidation together with isomerization and racemization occurs. These technological progresses will lead to a better understanding of how deamidation contributes to the pathology of aging and other degenerative diseases and the development of biopharmaceutical drugs.

Abstract B40: Analytical validation and initial clinical utility of multianalyte transcriptomic biomarker profiling of circulating tumor cells using automated exclusion-based sample preparation technology

Abstract The efficacy of androgen receptor (AR) signaling inhibitors (ARSIs) in prostate cancer is limited by both de novo and acquired resistance. Two well-described mechanisms of resistance to ARSIs are the development of constitutively activated AR splice variants (AR-Vs) or neuroendocrine prostate cancer (NEPC). Expression of NEPC markers is traditionally evaluated in solid tumor biopsies while AR-Vs can be detected in circulating tumor cells (CTCs) and have prognostic relevance in prostate cancer. Unlike solid tumor biopsy-based detection strategies, liquid biopsies can be performed periodically over the course of treatments, enabling early identification of resistance mechanisms. To evaluate for ARSI resistance, we developed a multiplex gene expression panel of AR-Vs and NEPC markers. We evaluated potential clinical utility of this assay in CTCs captured using an exclusion-based sample preparation technology. This test was evaluated with a cohort of 46 patients, showing 39% sensitivity and 94% specificity at detecting ARSI resistance. To develop a clinical-grade liquid biopsy assay with this multiplexed gene expression panel, we utilized a high-throughput automated exclusion-based sample preparation (ESP) technology for CTC capture, mRNA extraction, and gene expression analysis using RT-qPCR. Cell lines were used to evaluate the analytical performance of the methodology, and spike-in control materials were designed to monitor the efficiency of mRNA extraction for each individual patient sample. Serial dilutions of cell lines demonstrated the sensitivity to extract and quantify mRNA at the single-cell level with high precision between evaluations performed on three different days. Increasing numbers of cells resulted in the quantification of proportionally higher amounts of both AR-Vs and NEPC markers, demonstrating the analytical accuracy of the methodology (R2 values of 0.8 and above). To monitor for RNA degradation, a grape mRNA amplicon material was designed as an internal spike-in control for daily use in testing patient samples. Healthy donor PBMCs were used to confirm primers were specific for cells of cancer origin. ESP-mRNA extraction followed by RT-qPCR demonstrates single-cell resolution for the detection of AR-V/NEPC transcriptomic expression, with high precision in evaluating actual patient samples. The high analytical performance, and the promising preliminary correlation with ARSI resistance, warrant further efforts towards implementation into clinical laboratory testing environments and testing in prospective clinical trials. Citation Format: Zachery D. Schultz, Jennifer L. Schehr, Rory M. Bade, Molly M. Morgan, Madalyn S. Gill, Hannah M. Pezzi, Jaime M. Sperger, Charlotte N. Stahlfeld, Anupama Singh, Jay W. Warrick, David J. Beebe, Joshua M. Lang. Analytical validation and initial clinical utility of multianalyte transcriptomic biomarker profiling of circulating tumor cells using automated exclusion-based sample preparation technology [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr B40.

Abstract PR10: Analytical validation and preliminary clinical utility of PD-L1 and HLA I expression profiling of circulating tumor cells using automated exclusion-based sample preparation technology

Abstract Only a subset of patients with non-small cell lung cancer (NSCLC) respond to PD-L1 targeted therapy, even after solid tumor biopsy screening for PD-L1 expression. Downregulation of HLA I may contribute to this high prevalence of resistance, warranting its inclusion in attempts to increase response rates. While it is unclear if loss of HLA I occurs as a de novo or acquired resistance mechanism to immunotherapy, liquid biopsies could detect dynamic changes in expression levels, enable early detection of resistance, and accelerate the shift to alternate, more effective treatments. To quantify the expression of both PD-L1 and HLA I on circulating tumor cells (CTCs), we have developed automated exclusion-based sample preparation technology (ESP), which leverages the forces of surface tension and magnetism at the microscale for high-efficiency, high-throughput cell capture and manipulation. On-chip fluorescent antibody staining is performed for CTC identification, enumeration, and analysis of protein expression. A quantitative readout of protein expression is performed using a refined fluorescence microscopy approach wherein images of cells are acquired and analyzed with automated algorithms for uniformity and objectivity. To evaluate precision, accuracy, and analytical sensitivity of both ESP processing and quantitative microscopy, we used standardized cell lines, synthetic beads, and patient sample replicates. Calibration beads demonstrate high precision between multiple users for the quantitative microscopy (0.9% CV max of all wavelengths between 3 different analysts). ELISA beads with serial dilutions of protein standards demonstrate the accuracy of the quantitative microscopy (R2 values of 0.99 for both proteins). Cell lines demonstrate the precision of ESP for protein staining (3.4% CV max of two different cell lines tested on three different days). Patient sample replicates demonstrate precision for patients with as few as 3 CTCs per sample (6% CV max between triplicates). Early clinical utility of this assay was demonstrated on a preliminary patient cohort of 21 patient time points from 8 unique patients with NSCLC treated with immunotherapies. Phenotypic analysis for CTC expression of PD-L1 and HLA I with this assay demonstrated a sensitivity of 94% and specificity of 75% for detecting either resistant or stable disease during treatment with immunotherapies. Preliminary evaluation of patients with clear cell renal cell carcinoma demonstrates the feasibility of applying this approach to other cancer types. By confirming high performance, we ensure compatibility for clinical laboratory implementation and eventual application to better predict and detect resistance to PD-L1 targeted therapy in patients with NSCLC. The preliminary patient cohort suggests this diagnostic approach can detect fluctuations in expression of PD-L1 and HLA I in CTCs that also correspond to patient response to immunotherapy and warrants its pursuance in larger cohorts of patients with NSCLC and other tumor types. This abstract is also being presented as Poster B38. Citation Format: Jennifer L. Schehr, Zachery D. Schultz, Camila I. Hernandez, Matthew C. Mannino, Jay W. Warrick, Ticiana A. Leal, David J. Beebe, Joshua M. Lang. Analytical validation and preliminary clinical utility of PD-L1 and HLA I expression profiling of circulating tumor cells using automated exclusion-based sample preparation technology [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr PR10.

Centrosome amplification is a frequent event in circulating tumor cells from subjects with metastatic breast cancer.

Centrosome amplification (CA) is a common phenomenon in cancer, promotes genomic stability and cancer evolution, and has been reported to promote metastasis. CA promotes a stochastic gain/loss of chromosomes during cell division, known as chromosomal instability (CIN). However, it is unclear whether CA is present in circulating tumor cells (CTCs), the seeds for metastasis. Here, we surveyed CA in CTCs from human subjects with metastatic breast cancer. CTCs were captured by CD45 exclusion and selection of EpCAM‐positive cells using an exclusion‐based sample preparation technology platform known as VERSA (versatile exclusion‐based rare sample analysis). Centriole amplification (centrin foci> 4) is the definitive assay for CA. However, determination of centrin foci is technically challenging and incompatible with automated analysis. To test if the more technically accessible centrosome marker pericentrin could serve as a surrogate for centriole amplification in CTCs, cells were stained with pericentrin and centrin antibodies to evaluate CA. This assay was first validated using breast cancer cell lines and a nontransformed epithelial cell line model of inducible CA, then translated to CTCs. Pericentrin area and pericentrin area x intensity correlate well with centrin foci, validating pericentrin as a surrogate marker of CA. CA is found in CTCs from 75% of subjects, with variability in the percentage and extent of CA in individual circulating cells in a given subject, similar to the variability previously seen in primary tumors and cell lines. In summary, we created, validated, and implemented a novel method to assess CA in CTCs from subjects with metastatic breast cancer. Such an assay will be useful for longitudinal monitoring of CA in cancer patients and in prospective clinical trials for assessing the impact of CA on response to therapy.

Investigation of Sensitive Structures of Nanostructured Silicon – Melanin

The goal of this work is to study the current-voltage characteristics of structures of porous silicon – melanin and sensitivity of structures to glucose, to compare the characteristics between the samples obtained by different techniques. Object of study – structures of porous silicon – melanin. The subject of the study is the change of electrophysical parameters depending on the change in the analyte concentration. Organic-inorganic structures have recently attracted considerable interest in electronics due to the ability to create cheaper and simpler devices than traditional inorganic semiconductors. Melanin is an organic pigment that is present in many living organisms and has semiconductor properties. Melanin interacts with most biological substances, making it a promising material in the field of biosensors. Measuring glucose levels in the world today is very popular in the health and food sectors, so there is a constant need for easy-to-use and low-cost sensors. The purpose of this work was to investigate the heterojunction of melanin-porous silicon and the possibility of creating glucose sensors on such structures. For sample preparation metal-assisted chemical etching with three different types of metal nanoparticles (silver, copper, gold) was used. This technique allow formation of arrays of silicon nanowires with high aspect ratio. After porous silicon etching metal residuals were removed and thin layer of water-solube melanin was deposited at the surface and polymerized. Silver-based dot contacts was used for measurements. The I-V curves of the studied samples were measured in the work, such parameters as: resistance of structure, coefficient of rectification at contact of porous silicon – melanin, coefficient of rectification at contact of substrate – melanin, sensitivity to glucose were calculated. It was shown that I-V curves of both melanin/porous silicon and melanin/porous silicon/bulk silicon structures are diode-like with rectification factor at 5 V up to 80 for melanin/porous silicon heterojunction and up to 800 for melanin/porous silicon/bulk silicon structure. Resistor-like sencitive structures of melanin – nanostructured silicon are sensitive to glucose with a sensitivity coefficient of up to 1.83 mA /%. It was noticed that samples prepared with copper nanoparticles have opposite dependence (decreasing of current with increase of glucose concentration). No clear correlation was observed between the sensitivity, the rectifying properties and the technology of sample preparation, but the most stable results were obtained for structures made with silver nanoparticles.