Analysis Of Rare Cells Research Articles

Optofluidic time-stretch imaging flow cytometry with a real-time storage rate beyond 5.9 GB/s.

Optofluidic time-stretch imaging flow cytometry (OTS-IFC) provides a suitable solution for high-precision cell analysis and high-sensitivity detection of rare cells due to its high-throughput and continuous image acquisition. However, transferring and storing continuous big data streams remains a challenge. In this study, we designed a high-speed streaming storage strategy to store OTS-IFC data in real-time, overcoming the imbalance between the fast generation speed in the data acquisition and processing subsystem and the comparatively slower storage speed in the transmission and storage subsystem. This strategy, utilizing an asynchronous buffer structure built on the producer-consumer model, optimizes memory usage for enhanced data throughput and stability. We evaluated the storage performance of the high-speed streaming storage strategy in ultra-large-scale blood cell imaging on a common commercial device. The experimental results show that it can provide a continuous data throughput of up to 5891 MB/s.

Precision Phenotypic Profiling and Capture of Circulating Tumor Cells via a Vertical Laminar Flow-Stacked Microfluidic Chip.

The heterogeneity of circulating tumor cells has a significant impact on the diagnosis, treatment, and monitoring of cancer. Research on the subtypes of circulating tumor cells can bring better treatment outcomes for cancer patients. Here, we proposed a microfluidic chip for the magnetic capture of subtypes of circulating tumor cells from the whole blood and phenotypic profiling by stacking laminar flow vertically. Circulating tumor cells were sorted and captured by the three-dimensional regulation of both magnetic fields in the vertical direction and flow fields in the lateral direction. Using EpCAM-magnetic beads, we achieved sorting and sectional capture of target cells in whole blood and analyzed the surface expression levels of the captured cells, confirming the functionality of the microfluidic chip in sorting and capturing subtypes of circulating tumor cells. This microfluidic chip can also aid in the subsequent subtype analysis of other rare cells.

True volumetric counting of CD34+ cells using flow cytometry

While the single-platform flow cytometric CD34+ cell counting method is the preferred choice to predict the yield of mobilized peripheral blood stem cells, most flow cytometers lack the ability of hematology counter analyzers to perform volumetric counting. However, one of the problems using reference microbeads is the vanishing counting bead phenomenon. This phenomenon results in a drop in microbeads concentration and reduces the total and relative number of beads in calibration procedures. In the last years, flow cytometers including a volumetric system to quantify cells have been developed and may represent a promising alternative to enumerate CD34+ cells avoiding the use of beads.In this study we have used a direct true volumetric counting of CD34+ cells under continuous flow pump to overcome potential drawbacks with impact in rare cell analysis. To confirm this hypothesis, we have compared the results of CD34+ cell enumeration using non-volumetric vs. volumetric systems with FC500 (Beckman Coulter) and Attune NxT (ThermoFisher) flow cytometers, respectively, in mobilized peripheral blood samples.No statistically significant differences were observed between measurements of CD34+ cells using beads, when the FC500 and Attune NxT absolute counting values were compared, or when CD34+ counts were compared on the Attune NxT, either using or not using beads. Linear regressions to study the relationship between volumetric and non-volumetric CD34+ counts confirmed the accuracy of each method. Bland-Altman test showed agreement between both methods.Our data showed that CD34+ cell enumeration using a volumetric system is comparable with current counting systems. This method represents an alternative with the advantage of the simplification of sample preparation and the reduction of the analysis subjectivity.

Multiparameter flow cytometric detection and analysis of rare cells in in vivo models of cancer metastasis.

Rapid and reliable circulating tumor cell (CTC) and disseminated tumor cell (DTC) detection are critical for rigorous evaluation of in vivo metastasis models. Clinical data show that each step of the metastatic cascade presents increasing barriers to success, limiting the number of successful metastatic cells to fewer than 1 in 1,500,000,000. As such, it is critical for scientists to employ approaches that allow for the evaluation of metastatic competency at each step of the cascade. Here, we present a flow cytometry-based method that enables swift and simultaneous comparison of both CTCs and DTCs from single animals, enabling evaluation of multiple metastatic steps within a single model system. We present the necessary gating strategy and optimized sample preparation conditions necessary to capture CTCs and DTCs using this approach. We also provide proof-of-concept experiments emphasizing the appropriate limits of detection of these conditions. Most importantly, we successfully recover CTCs and DTCs from murine blood and bone marrow. In Supplemental materials, we expand the applicability of our method to lung tissue and exemplify a potential multi-plexing strategy to further characterize recovered CTCs and DTCs. This approach to multiparameter flow cytometric detection and analysis of rare cells in in vivo models of metastasis is reproducible, high throughput, broadly applicable, and highly adaptable to a wide range of scientific inquiries. Most notably, it simplifies the recovery and analysis of CTCs and DTCs from the same animal, allowing for a rapid first look at the comparative metastatic competency of various model systems throughout multiple steps of the metastatic cascade.

SPPUSM: An MS/MS spectra merging strategy for improved low-input and single-cell proteome identification

Single and rare cell analysis provides unique insights into the investigation of biological processes and disease progress by resolving the cellular heterogeneity that is masked by bulk measurements. Although many efforts have been made, the techniques used to measure the proteome in trace amounts of samples or in single cells still lag behind those for DNA and RNA due to the inherent non-amplifiable nature of proteins and the sensitivity limitation of current mass spectrometry. Here, we report an MS/MS spectra merging strategy termed SPPUSM (same precursor-produced unidentified spectra merging) for improved low-input and single-cell proteome data analysis. In this method, all the unidentified MS/MS spectra from multiple test files are first extracted. Then, the corresponding MS/MS spectra produced by the same precursor ion from different files are matched according to their precursor mass and retention time (RT) and are merged into one new spectrum. The newly merged spectra with more fragment ions are next searched against the database to increase the MS/MS spectra identification and proteome coverage. Further improvement can be achieved by increasing the number of test files and spectra to be merged. Up to 18.2% improvement in protein identification was achieved for 1 ng HeLa peptides by SPPUSM. Reliability evaluation by the “entrapment database” strategy using merged spectra from human and E. coli revealed a marginal error rate for the proposed method. For application in single cell proteome (SCP) study, identification enhancement of 28%–61% was achieved for proteins for different SCP data. Furthermore, a lower abundance was found for the SPPUSM-identified peptides, indicating its potential for more sensitive low sample input and SCP studies.

Digital-scRRBS: A Cost-Effective, Highly Sensitive, and Automated Single-Cell Methylome Analysis Platform via Digital Microfluidics.

Single-cell DNA methylation sequencing is highly effective for identifying cell subpopulations and constructing epigenetic regulatory networks. Existing methylome analyses require extensive starting materials and are costly, complex, and susceptible to contamination, thereby impeding the development of single-cell methylome technology. In this work, we report digital microfluidics-based single-cell reduced representation bisulfite sequencing (digital-scRRBS), the first microfluidics-based single-cell methylome library construction platform, which is an automatic, effective, reproducible, and reagent-efficient technique to dissect the single-cell methylome. Using our digital microfluidic chip, we isolated single cells in 15 s and successfully constructed single-cell methylation sequencing libraries with a unique genome mapping rate of up to 53.6%, covering up to 2.26 million CpG sites. Digital-scRRBS demonstrates a high capacity for distinguishing cell identity and tracking DNA methylation during drug administration. Digital-scRRBS expands the applicability of single-cell methylation methods as a versatile tool for epigenetic analysis of rare cells and populations with high levels of heterogeneity.

Micro-Magnetofluidic System for Rare Cell Analysis: From Principle to Translation

Rare cells play essential roles in the initiation and progression of diseases and therefore their analysis is of great interest. The micro-magnetofluidic system is one of the emerging platforms that have been proposed for the rapid, sensitive, and cost-effective analysis of rare cells. Given its unprecedented throughput, micro-magnetofluidic systems have attracted substantial research interest in the last decade—multiple designs have been proposed, validated, and even advanced to the stage of clinical trials. This mini review aims to provide a timely summary of the relevant progress in the field thus far. We reviewed the concepts and realizations of micro-magnetofluidic devices based on the interaction between nanoparticles and on-chip micro-magnets. Their real-world applications in rare cell analysis were also highlighted and explained. In addition, we discussed the major challenges in the development and translation of micro-magnetofluidic into the clinic, including multi-marker capability and large-scale manufacturability.

Highly stable integration of graphene Hall sensors on a microfluidic platform for magnetic sensing in whole blood

The detection and analysis of rare cells in complex media such as blood is increasingly important in biomedical research and clinical diagnostics. Micro-Hall detectors (μHD) for magnetic detection in blood have previously demonstrated ultrahigh sensitivity to rare cells. This sensitivity originates from the minimal magnetic background in blood, obviating cumbersome and detrimental sample preparation. However, the translation of this technology to clinical applications has been limited by inherently low throughput (<1 mL/h), susceptibility to clogging, and incompatibility with commercial CMOS foundry processing. To help overcome these challenges, we have developed CMOS-compatible graphene Hall sensors for integration with PDMS microfluidics for magnetic sensing in blood. We demonstrate that these graphene μHDs can match the performance of the best published μHDs, can be passivated for robust use with whole blood, and can be integrated with microfluidics and sensing electronics for in-flow detection of magnetic beads. We show a proof-of-concept validation of our system on a silicon substrate and detect magnetic agarose beads, as a model for cells, demonstrating promise for future integration in clinical applications with a custom CMOS chip.

Tumor Microenvironment-Inspired Glutathione-Responsive Three-Dimensional Fibrous Network for Efficient Trapping and Gentle Release of Circulating Tumor Cells

Detection of circulating tumor cells (CTCs) is important for early cancer diagnosis, prediction of postoperative recurrence, and individualized treatment. However, it is still challenging to achieve efficient capture and gentle release of CTCs from the complex peripheral blood due to their rarity and fragility. Herein, inspired by the three-dimensional (3D) network structure and high glutathione (GSH) level of the tumor microenvironment (TME), a 3D stereo (3D-G@FTP) fibrous network is developed by combining the liquid-assisted electrospinning method, gas foaming technique, and metal-polyphenol coordination interactions to achieve efficient trapping and gentle release of CTCs. Compared with the traditional 2D@FTP fibrous scaffold, the 3D-G@FTP fibrous network could achieve higher capture efficiency (90.4% vs 78.5%) toward cancer cells in a shorter time (30 min vs 90 min). This platform showed superior capture performance toward heterogeneous cancer cells (HepG2, HCT116, HeLa, and A549) in an epithelial cell adhesion molecule (EpCAM)-independent manner. In addition, the captured cells with high cell viability (>90.0%) could be gently released under biologically friendly GSH stimulus. More importantly, the 3D-G@FTP fibrous network could sensitively detect 4-19 CTCs from six kinds of cancer patients' blood samples. We expect this TME-inspired 3D stereo fibrous network integrating efficient trapping, broad-spectrum recognition, and gentle release will promote the development of biomimetic devices for rare cell analysis.

T-RHEX-RNAseq – a tagmentation-based, rRNA blocked, random hexamer primed RNAseq method for generating stranded RNAseq libraries directly from very low numbers of lysed cells

BackgroundRNA sequencing has become the mainstay for studies of gene expression. Still, analysis of rare cells with random hexamer priming – to allow analysis of a broader range of transcripts – remains challenging.ResultsWe here describe a tagmentation-based, rRNA blocked, random hexamer primed RNAseq approach (T-RHEX-RNAseq) for generating stranded RNAseq libraries from very low numbers of FACS sorted cells without RNA purification steps.ConclusionT-RHEX-RNAseq provides an easy-to-use, time efficient and automation compatible method for generating stranded RNAseq libraries from rare cells.

Abstract 2292: Novel method for the detection and evaluation of disease biomarkers on white blood cells from liquid biopsies

Abstract Purpose: Blood based liquid biopsies of circulating tumor cells (CTCs) are a non-invasive tool with proven effectiveness in the prognosis, treatment selection, and stratification of cancer patients. In this study we demonstrate a novel application of the RareCyte platform, which was developed to find and analyze rare CTCs from blood samples, in evaluating white blood cell (WBC) sub-populations for disease biomarkers. Experimental Procedures: Using AccuCyte®, an unbiased density-based method for collecting nucleated cells from whole blood, we transferred the nucleated cells to slides and stained for CD14, CD66b, and CK/EpCAM to identify monocytes (CD14+/CD66b-), granulocytes (CD14-/CD66b+), and CTCs (CK/EpCAM+), as well as a drug-target biomarker to evaluate its expression across all cells. These slides were then imaged with CyteFinder®, an automated multiparameter immunofluorescent (IF) microscopy system that applies machine learning algorithms for cell identification. Each slide contains millions of cells, so we developed novel scanning and analysis algorithms to quantify WBC sub-populations in addition to rare CTCs. Results: In addition to detection and enumeration of CTCs, the RareCyte system was able to detect individual white blood cells and quantify monocyte and granulocyte subpopulations. Our results correlated very well with standard CBC (Complete Blood Count) readings from multiple donors. Additionally, the monocyte and granulocyte populations were evaluated for expression of a drug-target biomarker. While expression was observed on both granulocytes and monocytes, monocytes had a significantly higher expression of the drug-target biomarker, a critical finding for future implementation of this drug. Conclusions: Our results show that through modifications to the RareCyte platform we can identify rare and non-rare cell populations simultaneously and evaluate expression of biomarkers and drug-targets across multiple cells of interest. This new application combines the benefits of high throughput data analysis of flow cytometry with the specificity and sensitivity of rare cell detection. Additionally, high-resolution multiplexed immunofluorescent imaging provides cell morphology and biomarker localization information. Our ability to add multiple biomarkers in conjunction with CTC and other rare cell detection provides multiple avenues for future research. One possible application is characterizing white blood cells that cluster with CTCs, which are known to portend a worse prognosis for patients. Our modified workflow could be implemented to precisely characterize these WBCs that contribute to the metastatic process. Utilization of the RareCyte platform for both rare and non-rare cell analysis presents an exciting opportunity for a deeper evaluation of patients and their prognosis through liquid biopsies. Citation Format: Erin Bayer, Jon Ladd, Joshua Nordberg, Arturo B. Ramirez, Jessica A. Baker, Thomas Daly. Novel method for the detection and evaluation of disease biomarkers on white blood cells from liquid biopsies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2292.

Lossless Immunocytochemistry Based on Large-Scale Porous Hydrogel Pellicle for Accurate Rare Cell Analysis.

Rare cells, such as circulating tumor cells or circulating fetal cells, provide important information for the diagnosis and prognosis of cancer and prenatal diagnosis. Since undercounting only a few cells can lead to significant misdiagnosis and incorrect decisions in subsequent treatment, it is crucial to minimize cell loss, particularly for rare cells. Moreover, the morphological and genetic information on cells should be preserved as intact as possible for downstream analysis. The conventional immunocytochemistry (ICC), however, fails to meet these requirements, causing unexpected cell loss and deformation of the cell organelles which may mislead the classification of benign and malignant cells. In this study, a novel ICC technique for preparing lossless cellular specimens was developed to improve the diagnostic accuracy of rare cell analysis and analyze intact cellular morphology. To this end, a robust and reproducible porous hydrogel pellicle was developed. This hydrogel encapsulates cells to minimize cell loss from the repeated exchange of reagents and prevent cell deformation. The soft hydrogel pellicle allows stable and intact cell picking for further downstream analysis, which is difficult with conventional ICC methods that permanently immobilize cells. The lossless ICC platform will pave the way for robust and precise rare cell analysis toward clinical practice.

Impact of red blood cell lysing on rare event analysis.

The challenges associated with analyzing rare cells are dependent on a series of factors, which usually require large numbers of cells per sample for successful resolution. Among these is determining the minimum number of total events needed to be acquired as defined by the expected frequency of the target cell population. The choice of markers that identify the target population, as well as the event rate and the number of aborted events/second, will also determine the statistically significant detection of rare cell events. Sample preparation is another important but often overlooked factor in rare cell analysis, and in this study we examine Poisson theory and methods to determine the effect of sample manipulation on rare cell detection. After verifying the applicability of this theory, we have evaluated the potential impact of red cell lysis on rare cell analysis, and how cell rarity can be underestimated or overestimated based on erythrolytic sensitivity or resistance of healthy leukocytes and pathological rare cells.

Clinical significance of circulating tumor cells testing in auxiliary diagnosis ofgastrointestinal cancer

Routine diagnostic tools and serum biomarkers of gastrointestinal (GI) cancer has limits in detecting early and micrometastasis,and circulating tumor cells (CTCs) had emerged as a promising metrics to complement this gap. The presentstudy is designed to explore technical feasibility of using CTCs as an auxiliary diagnostic tool in GI cancer. Over all 70inpatients with GI cancer and 30 healthy volunteers were recruited, and 10 mL of peripheral venous blood was collectedfrom all subjects. CTCs were detected by microfluidic blood rare cell analysis technique, and the sensitivity and specificityof CTCs in GI cancer diagnosis were derived from comparison with the pathological diagnosis results and tumor serummarker results. Compared with the healthy volunteers, the CTCs levels of the patients in gastrointestinal cancer group weresignificantly increased. Advanced stage subjects demonstrated higher level of CTCs, yet without statistical significance. Thesensitivity of CTCs to diagnose stage I to IV disease were 84.62%, 94.12%, 94.44%, and 100.00% respectively, yieldingcomprehensive sensitivity was 92.56% and specificity was 89.66%. Combined detection of CTCs and four tumor serummarkers was helpful in detecting positive rate, but without statistical signifiance compared with detecting CTCs alone. Ourstudy demonstrates the value of CTCs as an auxiliary diagnostic method for gastrointestinal cancer, and is expected to makeup for the deficiency of routine tissue biopsy, which can be used alone or in combination with conventional serum tumormarkers that can greatly promote the clinical diagnosis/prognosis of gastrointestinal cancer

Abstract 3375: Molecular subtyping of circulating tumor cells in patients with small cell lung cancer

Abstract Background: Recently, a new molecular classification of small cell lung cancer (SCLC), defined by expression of four key transcription regulators - ASCL1, NEUROD1, YAP1 and POU2F3 - has been proposed. These molecular subtypes may confer differential biology and therapeutic vulnerabilities, however, studies to date have been constrained by the paucity of available longitudinal tumor biopsy samples obtained from patients with SCLC. Herein, we utilized circulating tumor cells (CTCs) to characterize subtype marker heterogeneity at the time of diagnosis and evaluate the dynamic nature of marker expression during treatment in patients with SCLC. Methods: Informed consent was obtained from patients with SCLC using an IRB approved protocol (IRB#030763). We have collected blood samples from 32 patients, including treatment naïve samples from the entire cohort as well as on-treatment samples (median of 4 collections per patient). We utilized the RareCyte rare-cell analysis platform to isolate and quantify CTCs, which were defined as CK/EpCAM positive and CD45 negative. Each sample was also evaluated for expression of neuroendocrine markers (ASCL1, NEUROD1) and non-neuroendocrine markers (YAP1, POU2F3). Each set of neuroendocrine and non-neuroendocrine markers were co-stained along with the CTC markers. Mean fluorescence intensity of each marker was extracted using a python program. Results: To date, we have analyzed 22/32 (69%) treatment naïve samples, including n=6 patients with early stage disease and n=16 patients with advanced/metastatic disease. We detected CTCs from 16/22 (73%) patients, with quantities ranging from 1 - 3406 (median = 66) per 7.5ml of blood. CTCs were positive for the neuroendocrine markers ASCL1 in 16/16 (100%) and NEUROD1 in 10/16 (62%); all NEUROD1 positive CTCs were also positive for ASCL1. The non-neuroendocrine markers YAP1 and POU2F3 were detected in 6/16 (37%) and 11/16 (69%) samples, respectively, including YAP1/POU2F3 double positives in 6/16 (37%). Analysis of on-treatment samples is ongoing. Conclusion: We have developed a protocol for monitoring expression of subtype markers on CTCs isolated from patients with SCLC. We found CTCs expressing both neuroendocrine and non-neuroendocrine markers at the time of diagnosis. Ongoing work will attempt to delineate the dynamic nature of subtype marker expression during therapy, to correlate with clinical outcomes. These studies have the potential to offer critical insights regarding the heterogeneity and evolution of SCLC, a tumor type in which novel disease insights and innovative treatment strategies are urgently needed to improve patient survival. Citation Format: Prasad R. Kopparapu, Melinda Duplessis, Edward Lo, Yingjun Yan, Wade T. Iams, Josh Nordberg, Arturo Ramirez, Tad George, Christine Lovly. Molecular subtyping of circulating tumor cells in patients with small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3375.

Nanoparticle Amplification Labeling for High-Performance Magnetic Cell Sorting.

Magnetic cell sorting is an enabling tool for the isolation of specific cellular subpopulations for downstream applications and requires the cells to be labeled by a sufficient number of magnetic nanoparticles to leverage magnetophoresis for efficient separation. This requirement makes it challenging to target weakly expressed biomarkers. Here, we developed a new approach that selectively and efficiently amplifies the magnetic labeling on cells through sequentially connected antibodies and nanoparticles delivered to the surface or interior of the cell. Using this approach, we achieved amplification up to 100-fold for surface and intracellular markers. We also demonstrated the utility of this assay for enabling high-performance magnetic cell sorting when it is applied to the analysis of rare tumor cells for cancer diagnosis and the purification of transfected CAR T cells for immunotherapy. The data presented demonstrate a useful tool for the stratification of rare cell subpopulations.

Selective, user-friendly, highly porous, efficient, and rapid (SUPER) filter for isolation and analysis of rare tumor cells

Rapid, efficient, and selective separation of tumor cells from complex body fluids is urgently needed for clinical application of tumor-cell-based liquid biopsy. Herein, a size-selective affinity filtration system, named selective, user-friendly, highly porous, efficient, and rapid filter (SUPER Filter), was developed for high-performance tumor cell isolation and analysis. SUPER Filter enabled selective interaction of tumor cells with size-optimized and antibody-coated micropore walls during filtration, achieving a high efficiency of 91.0 ± 6.1% in buffer and 83.7 ± 6.4% in whole blood. Meanwhile, its larger micropore size than those of traditional filtration devices greatly reduced the nonspecific capture of background cells (55-126 cells per mL blood) with enrichment factors of 1.1 × 104-1.0 × 105 and a purity of 52.7 ± 4.2%. Moreover, its high porosity enabled ultra-fast (<5 s for 1 mL of blood or 10 mL of buffer samples) and user-friendly gravity-driven filtration. Finally, SUPER Filter demonstrated rapid, efficient, and selective separation of tumor cells from blood and large-volume pleural and ascetic fluid samples from cancer patients for morphological and molecular analysis. We expect that this size-selective affinity filtration strategy facilitates the clinical application of tumor-cell-based liquid biopsy.

Antibody-engineered red blood cell interface for high-performance capture and release of circulating tumor cells

Circulating tumor cells (CTCs), as important liquid biopsy target, can provide valuable information for cancer progress monitoring and individualized treatment. However, current isolation platforms incapable of balancing capture efficiency, specificity, cell viability, and gentle release have restricted the clinical applications of CTCs. Herein, inspired by the structure and functional merits of natural membrane interfaces, we established an antibody-engineered red blood cell (RBC-Ab) affinity interface on microfluidic chip for high-performance isolation and release of CTCs. The lateral fluidity, pliability, and anti-adhesion property of the RBC microfluidic interface enabled efficient CTCs capture (96.5%), high CTCs viability (96.1%), and high CTCs purity (average 4.2-log depletion of leukocytes). More importantly, selective lysis of RBCs by simply changing the salt concentration was utilized to destroy the affinity interface for efficient and gentle release of CTCs without nucleic acid contamination. Using this chip, CTCs were successfully detected in colon cancer samples with 90% sensitivity and 100% specificity (20 patients and 10 healthy individuals). After the release process, KRAS gene mutations of CTCs were identified from all the 5 cancer samples, which was consistent with the results of tissue biopsy. We expect this RBC interface strategy will inspire further biomimetic interface construction for rare cell analysis.

Novel Stable Isotope-Resolved Metabolomics Method for a Small Number of Cells Using Chip-Based Nanoelectrospray Mass Spectrometry.

Stable isotope-resolved metabolomics (SIRM) can provide metabolic conversion information of specific targets; it is a powerful tool for cell metabolism studies. The common analytical platform for SIRM is chromatography-mass spectrometry, which requires a large number of cells and is not suitable for precious rare cell analysis. To study a small number of cells, we established a novel SIRM method using chip-based nanoelectrospray mass spectrometry (MS). 13C-glutamine was taken as an example; the unlabeled and 13C-labeled cells were cultured and extracted in a 96-well plate and then directly injected into MS and analyzed in full scan mode and parallel reaction monitoring (PRM) mode targeting 44 glutamine-derived metabolites and their isotopologues. To define focused metabolite-related MS2 fragments produced in the PRM, a new strategy was proposed including MS2 exact m/z matching, MS2 false positive filtering, and MS2 fragment grouping to remove the interfering MS2 ions. In total, 292 and 349 pairs of paired MS2 ions were obtained in positive and negative ionization modes, respectively. By searching spectra databases, 31 targeted metabolites with their isotopologues were identified and their characteristic product ions were confirmed for MS2 quantification. The relative quantification was achieved by MS2 quantification, which showed better sensitivity and accuracy than common MS1-based quantification. Finally, this method was applied to isocitrate dehydrogenase I-mutated glioma cells for revealing the effects of triptolide on glioma cell metabolism using U-13C-glutamine as a labeling substrate.

POS0424 DETERMINING CIRCULATING ENDOTHELIAL CELLS USING CELLSEARCH SYSTEM IN SYSTEMIC SCLEROSIS PATIENTS

Background:Endothelial damage and fibroproliferative vasculopathy of small vessels are pathological hallmarks of Systemic Sclerosis (SSc). Detection and analysis of circulating endothelial cells (CECs) detached from affected blood vessels may be an informative tool to study vascular dysfunction and could be considered a novel biomarker of scleroderma vasculopathy. Our group first showed the presence of CECs in SSc by fluorescence-activated cell sorting (FACS), demonstrating that a raised counts of active CECs may represent direct evidence of active vascular disease in SSc. Despite these interesting data, issues related to difficulties in CEC counting through FACS analysis, due their very low concentration in peripheral blood, prevented further investigations in this field. Recently, a specific kit for the detection of CECs has been developed through the CellSearch System (CS), a semi-automated device for the standardized analysis of rare cells, such as CECs, in peripheral blood.Objectives:To assess the counts of CECs determined by the CS in SSc patients and to evaluate their clinical implication and potential as vascular biomarker in SSc.Methods:10mL of blood samples were collected from 29 subjects (19 SSc patients and 10 healthy donors - HDs) and stored in tubes containing a specific preservative, to allow the analysis of 4mL of blood within 72 hours, according to manufacturer instructions. Out of 19 SSc patients, 18 were female, 10 had the limited form and 9 the diffuse cutaneous variant of SSc. CS uses a proprietary kit containing a ferrofluid-based reagent, that target CD146 to magnetically capture CECs, and the immunofluorescent reagents to stain the CECs, defined as CD146+, CD105-PE+, DAPI+ and CD45-APC-. Clinical, laboratoristic and demographic data were also collected.Results:The mean number of CECs in patients with SSc was significantly higher in comparison to HDs (554/4mL vs. 53.5/4 mL, p=0.0042). When analyzed according to disease subset, both lcSSc and dcSSc showed significantly increased levels of CECs in comparison with HDs (p=0.003 and p=0.005, respectively). No statistical difference was observed in the mean number of CECs in patients with lcSSc compared to those with dcSSc. Regarding vascular involvement, the CECs counts strictly correlated with the presence of digital ulcers (DUs) (p=0.0001) showing a median of 863cells/4mL for the SSc patients with DUs versus a median of 276.2/4mL for the SSc patients without DUs. No statistical correlation was found between CECs and serological autoantibody pattern, skin parameters, or joint and muscle involvement. Patients with active disease, according to the EUSTAR Activity Index, showed a higher CECs value than those with inactive disease (p=0.0012).Conclusion:The amount of CECs detectable in peripheral blood has been recently proposed as a marker of endothelial damage in different vascular diseases, including SSc. However, currently no standardized method is available to determine CEC counts, which makes reported data on CECs reliable and suitable. The CS system is a commercially available semi-automated system that enables standardized determination of CECs. Thus, we examined clinical utility of CECs count by this system in SSc patients. Our results confirm that baseline CEC counts, evaluated by a new standardized method, may represent direct evidence of endothelial damage in SSc and could be a promising tool for monitoring active disease and evaluating therapeutic responses to vascular and immunosuppressive treatments.