Immunomagnetic Capture Research Articles

Paramagnetic Structures within a Microfluidic Channel for Enhanced Immunomagnetic Isolation and Surface Patterning of Cells.

In this report, we demonstrate a unique method for embedding magnetic structures inside a microfluidic channel for cell isolation. We used a molding process to fabricate these structures out of a ferrofluid of cobalt ferrite nanoparticles. We show that the embedded magnetic structures significantly increased the magnetic field in the channel, resulting in up to 4-fold enhancement in immunomagnetic capture as compared with a channel without these embedded magnetic structures. We also studied the spatial distribution of trapped cells both experimentally and computationally. We determined that the surface pattern of these trapped cells was determined by both location of the magnet and layout of the in-channel magnetic structures. Our magnetic structure embedded microfluidic device achieved over 90% capture efficiency at a flow velocity of 4 mm/s, a speed that was roughly two orders of magnitude faster than previous microfluidic systems used for a similar purpose. We envision that our technology will provide a powerful tool for detection and enrichment of rare cells from biological samples.

Mass Barcode Signal Amplification for Multiplex Allergy Diagnosis by MALDI-MS.

A highly sensitive method based on mass-barcoded gold nanoparticles (AuNPs) and immunomagnetic separation has been developed for multiplex allergy diagnosis by MALDI mass spectrometry in a component-resolved manner. Different analytical probes were prepared by coating AuNPs with individual allergenic proteins and mass barcode, represented by polyethylene glycol molecules of various chain lengths. Magnetic beads (MBs) functionalized with antihuman IgE antibodies (Abs) were used as immunomagnetic capture probes. IgE Abs were extracted from a patient's blood serum by the formation of a sandwich structure between the AuNPs and MBs. Multiple specific IgE Abs were simultaneously identified by mass spectrometry detection of the mass barcodes, providing an efficient component-resolved allergy diagnosis. Because of the signal amplification provided by the mass barcodes, the developed diagnosis method is very sensitive, with a limit of detection down to picograms per milliliter level for specific IgE Abs. The method can be potentially useful when the sample amount is highly limited and a multiplex diagnostic procedure is required.

Abstract OT1-02-01: Pilot study of prognostic utility of circulating tumor cells (CTCs) assessed by AdnaGen technology and clinical outcome of patients with stage III breast cancer who completed locoregional and systemic treatment

Abstract Background: Detection of high number of CTCs (>5) before initiation of first-line therapy in patients with metastatic breast cancer is associated with shorter progression free survival and overall survival. The most widely used method is CellSearch (Veridex, Raritan, NJ). It relies on immunomagnetic capture of CTCs, using antibodies against the epithelial cell adhesion molecule (EpCAM). Although the US Food and Drug Administration approved CellSearch assay for clinical use. In addition to isolation and enumeration, a promising area of research is genomic CTCs characterization which entails phenotyping and molecular expression profiling of CTC subsets consisting of those of epithelial origin (CTC-Epi), others undergoing epithelial to mesenchymal transition (CTC-EMT), or expressing cancer stem cell-like phenotype (CTC-CSC; CD44+ CD24low, ALDH+), respectively. EMT is a molecular process to acquire the traits needed to execute the multiple steps of metastasis. Through the EMT process, epithelial cells lose cell-cell contacts and cell polarity, downregulate epithelial-associated genes, acquire mesenchymal gene expression and undergo major changes in their cytoskeleton. Currently, a CTC detection kit is available to detect CTCs expressing EMT-associated genes by semiquantitative RT-PCR (Adna EMT2/Stem Cell test). EMT will be detected by measuring EMT-inducing transcription factors such as TWIST1, SNAIL1, SLUG, ZEB1 and FOXC2) by RT-PCR Objectives. Primary objective: To investigate if activated pathways in CTCs are correlated with clinical outcome of patient with stage III breast cancer. Secondary objective: To prospectively determine if assessment of the pathways profiling in CTCs can be used to stratify NED breast cancer patients Patients Eligibility: Inclusion: histologically confirmed invasive breast cancer (any subtype), clinical stage III, no evidence of distant metastasis by PET-CT or CT scan of chest and abdomen, and body scan, age 18 years or older, pts must be scheduled to start neoadjuvant/adjuvant therapy, ECOG PS 0-2. Pts must sign a written informed consent. Exclusion: distant metastasis, investigational therapy, prior history of other malignancies within the last 2 years, except non-melanoma skin cancer. This study (PA12-0097) was approved by IRB of UT MD Anderson Cancer Center. Trial Design. This is a pilot, international, multicenter, prospective, blood sample collection from 200 patients with clinical or pathologic stage III breast cancer. Statistical Analysis: This study is a 7-year study (84 months). Pts will be classified as to the presence [negative (neg) vs. positive (pos)] of CTC and as to the expression of a biomarker (neg vs. pos). The primary endpoint of the study is breast cancer recurrence. Time to recurrence curves for the four breast cancer patient groups (neg/neg, neg/pos, pos/neg, or pos/pos) will be estimated using the Kaplan-Meier method and differences in the recurrence rates will be evaluated by the log-rank test at the end of the study (84 months). The confidence intervals for the quantiles of the recurrence distribution will be based on the sign test as described by Brookmeyer and Crowley. Citation Format: Alvarez RH, Gao H, Ensor JE, Gomez HL, Ruiz-Garcia EB, Arce C, Sun H, Willey JS, Ueno NT, Valero V, Reuben JM. Pilot study of prognostic utility of circulating tumor cells (CTCs) assessed by AdnaGen technology and clinical outcome of patients with stage III breast cancer who completed locoregional and systemic treatment. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr OT1-02-01.

A portable immunomagnetic cell capture system to accelerate culture diagnosis of bacterial infections.

Bacterial infections continue to be a major cause of deaths globally, particularly in resource-poor settings. In the absence of rapid and affordable diagnostic solutions, patients are mostly administered broad spectrum antibiotics leading to antibiotics resistance and poor recovery. Culture diagnosis continues to be a gold standard for diagnosis of bacterial infection, despite its long turnaround time of 24 to 48 h. We have developed a portable immunomagnetic cell capture (iMC(2)) system that allows rapid culture diagnosis of bacterial pathogens. Our approach involves the culture growth of the blood samples in broth media for 6 to 8 h, followed by immunomagnetic enrichment of the target cells using the iMC(2) device. The device comprises a disposable capture chip that has two chambers of 5 ml and 50 μl volume connected through a channel with a manual valve. Bacterial cells bound to antibody coated magnetic nanoparticles are swept from the 5 ml sample chamber into the 50 μl recovery chamber by moving an external magnetic field with respect to the capture chip using a linear positioner. This enables specific isolation and up to 100× enrichment of the target cells. The presence of bacteria in the recovered sample is confirmed visually using a lateral flow immunoassay. The system is demonstrated in buffer and blood samples spiked with S. typhi. The method has high sensitivity (10 CFU ml(-1)), specificity and a rapid turnaround time of less than 7 h, a significant improvement over conventional methods.

Immunomagnetic capture and colorimetric detection of malarial biomarker Plasmodium falciparum lactate dehydrogenase

We report a sensitive, magnetic bead-based colorimetric assay for Plasmodium falciparum lactate dehydrogenase (PfLDH) in which the biomarker is extracted from parasitized whole blood and purified based on antigen binding to antibody-functionalized magnetic particles. Antigen-bound particles are washed, and PfLDH activity is measured on-bead using an optimized colorimetric enzyme reaction (limit of detection [LOD] = 21.1 ± 0.4 parasites/μl). Enhanced analytical sensitivity is achieved by removal of PfLDH from the sample matrix before detection and elimination of nonspecific reductases and species that interfere with the optimal detection wavelength for measuring assay development. The optimized assay represents a simple and effective diagnostic strategy for P. falciparum malaria with time-to-result of 45 min and detection limits similar to those of commercial enzyme-linked immunosorbent assay (ELISA) kits, which can take 4–6 h. This method could be expanded to detect all species of malaria by switching the capture antibody on the magnetic particles to a pan-specific Plasmodium LDH antibody.

Polymer-based microfluidic chip for rapid and efficient immunomagnetic capture and release of Listeria monocytogenes.

Infections caused by foodborne pathogens such as Listeria monocytogenes pose a threat to public health while timely detection is challenging due to pathogen low numbers. The development of robust and efficient sample preparation techniques is crucial to improve detection sensitivity and workflow. Immunomagnetic separation using magnetic nanoparticles (MNPs) is attractive, as it can efficiently capture target cells. For food safety applications, a platform is needed to rapidly process large sample volumes, allowing capture and release of target bacteria conjugated to immunomagnetic nanoparticles (IMNPs). Herein, we demonstrate a method for magnetic capture and release of bacteria-IMNPs complex based on a 3D magnetic trap integrated on a polymeric microfluidic device. The 3D magnetic capture region consist of a dense array of high-aspect ratio (3 : 1) cylindrical pillars embossed in thermoplastic polymer and coated with soft ferromagnetic nickel by an electroless deposition technique. This allows the generation of strong and switchable magnetic capture regions due to the very low remanence of the nickel shell. We propose and validate an optimized configuration of capture regions for efficient localized capture and rapid release of MNPs and IMNPs conjugated to L. monocytogenes. A maximum recovery rate for MNPs corresponded to 91% while a maximum capture efficiency of 30% was obtained for live bacteria, with a minimum detectable sample concentration of ~10 cfu ml(-1) in 1 ml volume using plate-culture method. We believe that the flexible design and low-cost fabrication process of the proposed system will allow rapid sample preparation for applications beyond food and water safety, including point-of-care diagnosis.

Development of a specific immunomagnetic capture-PCR for rapid detection of viable Mycoplasma agalactiae in sheep milk samples.

To develop an immunomagnetic capture (IMC) to detect viable Mycoplasma agalactiae in routine ovine milk samples. Polyclonal antibodies against two M.agalactiae membrane surface proteins (P80 and P55) were covalently conjugated to magnetic beads (MBs) to form MB-Ab80 and MB-Ab55. Mycoplasma agalactiae cells were captured by a specific antigen-antibody reaction and magnetic separation. Immunomagnetic capture (IMC) was used to isolate and concentrate M.agalactiae in serial decimal dilutions and in artificially contaminated milk to facilitate subsequent detection by PCR. A 375-bp fragment of M.agalactiae was amplified using a pair of M.agalactiae-specific primers in PCR. The limit of detection of IMC-PCR method ranged from 10 to 10(2) CCUml(-1) when mycoplasmas were resuspended in PBS and from 10(2) to 10(3) CCUml(-1) when mycoplasmas were resuspended in uncontaminated ovine milk. This study also describes the application of IMC-PCR method to test for M.agalactiae in 516 milk samples collected from sheep with suspected contagious agalactia. Its performance was evaluated relative to culture. This report has demonstrated for the first time, the effective use of rapid and reliable IMC combined with PCR assay for the detection of viable M.agalactiae. The method IMC-PCR provides an alternative to conventional microbiological detection, method and it could be applied to quick detection of M.agalactiae in routine sheep milk samples.

Colorimetric detection of Shewanella oneidensis based on immunomagnetic capture and bacterial intrinsic peroxidase activity.

Rapid detection and enumeration of target microorganisms is considered as a powerful tool for monitoring bioremediation process that typically involves cleaning up polluted environments with functional microbes. A novel colorimetric assay is presented based on immunomagnetic capture and bacterial intrinsic peroxidase activity for rapidly detecting Shewanella oneidensis, an important model organism for environmental bioremediation because of its remarkably diverse respiratory abilities. Analyte bacteria captured on the immunomagnetic beads provided a bacterial out-membrane peroxidase-amplified colorimetric readout of the immunorecognition event by oxidizing 3, 3′, 5, 5′-tetramethylbenzidine (TMB) in the present of hydrogen peroxide. The high-efficiency of immunomagnetic capture and signal amplification of peroxidase activity offers an excellent detection performance with a wide dynamic range between 5.0 × 103 and 5.0 × 106 CFU/mL toward target cells. Furthermore, this method was demonstrated to be feasible in detecting S. oneidensis cells spiked in environmental samples. The proposed colorimetric assay shows promising environmental applications for rapid detection of target microorganisms.

Acute changes in endothelial function with cisplatin among germ cell tumor (GCT) patients (Pts).

9587 Background: We hypothesized that the increased risk of cardiovascular disease (CVD) in GCT pts following cisplatin-based chemotherapy (CBCT) may be due to endothelial damage. Methods: In this pilot study, men age ≥18 with GCT, no prior chemotherapy (CT) or radiation, and no history of CVD were enrolled to the CT group (CG) if CBCT was planned and to the surgery group (SG) if only surgical management (orchiectomy +/- RPLND) was planned. Endo-PAT2000, a noninvasive method of quantifying endothelial function, was used to measure the peripheral arterial tonometry reactive hyperemia index (PAT-RHI). Lower values correlate with endothelial dysfunction. Circulating endothelial cells (CECs) were quantified by immunomagnetic capture as a secondary endpoint. Covariates included hypertension, smoking status, dyslipidemia, diabetes, and fasting lipids, glucose, HbA1C, and testosterone. Measurements were obtained at time points T1-T5 for CG pts and T1, T2 and T5 for SG pts (Table). Nonparametric tests were used t...

Rapid colorimetric detection of Salmonella typhimuriumusing a selective filtration technique combined with antibody–magnetic nanoparticle nanocomposites

Detection of pathogenic bacteria that pose a great risk to human health requires a rapid, convenient, reliable, and sensitive detection method. In this study, we developed a selective filtration method using monoclonal antibody (MAb)-magnetic nanoparticle (MNP) nanocomposites for the rapid and sensitive colorimetric detection of Salmonella typhimurium. The method contains two key steps: the immunomagnetic separation of the bacteria using MAb-MNP nanocomposites and the filtration of the nanocomposite-bound bacteria. Color signals from the nanocomposites remaining on the membrane were measured, which reflected the amount of bacteria in test samples. Immunomagnetic capture efficiencies of 8 to 90% for various concentrations of the pathogen (2 × 10(4)-2 × 10(1) cells) were obtained. After optimization of the method, 2 × 10(1) cells of S. typhimurium in pure culture solution was detectable as well as in artificially inoculated vegetables (100 cells/g). The method was confirmed to be highly specific to S. typhimurium without cross-reaction to other pathogenic bacteria and could be concluded within 45min, yielding results in a shorter or similar time period as compared with recently reported antibody immobilized on magnetic-particle-based methods. This study also demonstrated direct application of MAb-MNP nanocomposites without a dissociation step of bacteria from magnetic beads in colorimetric assays in practice.

A hollow nanogold/meso-magnetite composite: pulsed laser synthesis, properties, and biosensing application

A facile and template-free UV (Excimer) laser photolysis process is demonstrated to transform a Fe-complex into unique hollow Fe3O4 nanosphere morphology with each sphere having nanometric pores and an opening. Depending on the Fe-complex concentration and processing time interesting configurations are seen to evolve. When an identical process is applied to a mixture of Fe-complex and chloroauric acid an enthrallingly decorated Au–Fe3O4 nanostructure evolves, with Au nanoparticles surface-loaded on mesospheric Fe3O4. This room temperature process implemented under normal laboratory conditions is clearly versatile and applicable to heterojunction nanomaterials synthesis in a single-step process. The potential application of these gold-decorated magnetic nanostructures was also investigated for immuno-magnetic capture of E. coli in biosensing and these were found to be sensitive even below 1,000 cfu/ml. The test results demonstrate linear sensing response in the range of 103–105 cfu/ml. We also show that these nanostructures can be used for simple electrical conductivity-based biosensing since they show dramatic conductivity change in a simple drop-cast test. A new laser-based approach to the synthesis of unique hollow sphere morphology of magnetite (Fe3O4) without and with Au nanoparticle decoration is presented. The potential application of these gold-decorated magnetic nanostructures was also investigated for immuno-magnetic capture of E. coli in biosensing and these were found to be sensitive even below 1,000 cfu/ml.

Mutational Analysis of Circulating Tumor Cells Using a Novel Microfluidic Collection Device and qPCR Assay

Circulating tumor cells (CTCs) provide a readily accessible source of tumor material from patients with cancer. Molecular profiling of these rare cells can lead to insight on disease progression and therapeutic strategies. A critical need exists to isolate CTCs with sufficient quantity and sample integrity to adapt to conventional analytical techniques. We present a microfluidic platform (IsoFlux) that uses flow control and immunomagnetic capture to enhance CTC isolation. A novel cell retrieval mechanism ensures complete transfer of CTCs into the molecular assay. Improved sensitivity to the capture antigen was demonstrated by spike-in experiments for three cell lines of varying levels of antigen expression. We obtained spike-in recovery rates of 74%, 75%, and 85% for MDA-MB-231 (low), PC3 (middle), and SKBR3 (high) cell lines. Recovery using matched enumeration protocols and matched samples (PC3) yielded 90% and 40% recovery for the IsoFlux and CellSearch systems, respectively. In matched prostate cancer samples (N = 22), patients presenting more than four CTCs per blood draw were 95% and 36% using IsoFlux and CellSearch, respectively. An assay for detecting KRAS mutations was described along with data from patients with colorectal cancer, of which 87% presented CTCs above the assay's limit of detection (four CTCs). The CTC KRAS mutant rate was 50%, with 46% of patients displaying a CTC KRAS mutational status that differed from the previously acquired tissue biopsy data. The microfluidic system and mutation assay presented here provide a complete workflow to track oncogene mutational changes longitudinally with high success rates.

Isolation of circulating tumor cells by immunomagnetic enrichment and fluorescence-activated cell sorting (IE/FACS) for molecular profiling

Circulating tumor cells (CTCs) are cells shed by the primary tumor into the blood stream capable of initiating distant metastasis. In the past decade, numerous assays have been developed to reliably detect these extremely rare cells. However, methods for purification of CTCs with little or no contamination of normal blood cells for molecular profiling are limited. We have developed a novel protocol to isolate CTCs by combining immunomagnetic enrichment and fluorescence-activated cell sorting (IE/FACS). The two-part assay includes (1) immunomagnetic capture using magnetic beads conjugated to monoclonal antibody against an epithelial cell adhesion marker (EpCAM) to enrich for tumor cells; and (2) FACS analysis using EpCAM to purify tumor cells away from mononuclear cells of hematopoietic lineage. Downstream molecular analyses of single and pooled cells confirmed the isolation of highly pure CTCs with characteristics typical that of malignant cells.

Fast immunosensing technique to detect Legionella pneumophila in different natural and anthropogenic environments: comparative and collaborative trials

BackgroundLegionellosis is an uncommon form of pneumonia. After a clinical encounter, the necessary antibiotic treatment is available if the diagnosis is made early in the illness. Before the clinical encounter, early detection of the main pathogen involved, Legionella pneumophila, in hazardous environments is important in preventing infectious levels of this bacterium. In this study a qualitative test based on combined magnetic immunocapture and enzyme-immunoassay for the fast detection of Legionella pneumophila in water samples was compared with the standard method, in both comparative and collaborative trials. The test was based on the use of anti-Legionella pneumophila antibodies immobilized on magnetic microspheres. The final protocol included concentration by filtration, resuspension and immunomagnetic capture. The whole assay took less than 1 hour to complete.ResultsA comparative trial was performed against the standard culture method (ISO 11731) on both artificially and naturally contaminated water samples, for two matrices: chlorinated tap water and cooling tower water. Performance characteristics of the test used as screening with culture confirmation resulted in sensitivity, specificity, false positive, false negative, and efficiency of 96.6%, 100%, 0%, 3.4%, and 97.8%, respectively. The detection limit at the level under which the false negative rate increases to 50% (LOD50) was 93 colony forming units (CFU) in the volume examined for both tested matrices. The collaborative trial included twelve laboratories. Water samples spiked with certified reference materials were tested. In this study the coincidence level between the two methods was 95.8%.ConclusionResults demonstrate the applicability of this immunosensing technique to the rapid, simple, and efficient detection of Legionella pneumophila in water samples. This test is not based on microbial growth, so it could be used as a rapid screening technique for the detection of L. pneumophila in waters, maintaining the performance of conventional culture for isolation of the pathogen and related studies.

A Simple Point-of-Care Microfluidic Immunomagnetic Fluorescence Assay for Pathogens

In this work, we reported a simple rapid and point-of-care magnetic immunofluorescence assay for avian influenza virus (AIV) and developed a portable experimental setup equipped with an optical fiber spectrometer and a microfluidic device. We achieved the integration of immunomagnetic target capture, concentration, and fluorescence detection in the microfluidic chip. By optimizing flow rate and incubation time, we could get a limit of detection low up to 3.7 × 10(4) copy/μL with a sample consumption of 2 μL and a total assay time of less than 55 min. This approach had proved to possess high portability, fast analysis, high specificity, high precision, and reproducibility with an intra-assay variability of 2.87% and an interassay variability of 4.36%. As a whole, this microfluidic system may provide a powerful platform for the rapid detection of AIV and may be extended for detection of other viral pathogens; in addition, this portable experimental setup enables the development of point-of-care diagnostic systems while retaining adequate sensitivity.

439 Comparison of Dielectrophoretic Field Flow Fractionation (DEP-FFF) with ApoStream™, an Antibody Independent Platform, with Immunomagnetic Capture Using CellSearch™ for Enumeration of Circulating Tumor Cells in Patients with Metastatic Prostate Cancer

439 Comparison of Dielectrophoretic Field Flow Fractionation (DEP-FFF) with ApoStream™, an Antibody Independent Platform, with Immunomagnetic Capture Using CellSearch™ for Enumeration of Circulating Tumor Cells in Patients with Metastatic Prostate Cancer

O1-023 - Sensitivity of Microcavity Array System for Circulating Tumor Cells in Lung Cancer Patients

ABSTRACT Background Epithelial cell adhesion molecule (EpCAM)-based enumeration of circulating tumor cells (CTCs) has a prognostic value in solid tumors such as advanced breast, colon and prostate cancers. However, poor sensitivity has been reported in non-small-cell lung cancer (NSCLC). We have developed a microcavity array (MCA) system integrated with a microfluidic device for recovery and enumeration of CTsC, regardless of EpCAM expression level. This system can isolate tumor cells on the basis of differences in size and deformability between tumor and hematologic cells. Methods Paired peripheral blood samples were collected from metastatic lung cancer patients. CTCs were enumerated by EpCAM-based immunomagnetic capture (CellSearch, Veridex) and by the MCA system. In the MCA system, trapped cells were stained with Hoechst 33342, FITC-labeled anti-pan cytokeratin antibodies and PE-labeled anti-CD45 antibodies for subsequent imaging analysis. CTCs were defined as cells with round to oval morphology, a visible nucleus, positive staining for pan-cytokeratin and negative staining for CD45. We evaluated the sensitivity of the MCA system for detecting CTCs in lung cancer patients compared with the CellSearch system. Results Twenty-two metastatic NSCLC patients and 13 small-cell-lung cancer (SCLC) patients were enrolled into this study between April 2011 and January 2012. CTCs were detected using the MCA system in 17 of 22 NSCLC patients (count >1 per 7.5 ml) compared with 9 of 22 patients using CellSearch (P = 0.013). On the other hand, CTCs were detected using MCA in all 13 SCLC patients compared with just 9 of 13 patients using CellSearch (P = 0.012). More CTCs from NSCLC patients were detected by the MCA system (median 13, range 0–313 cells/7.5 ml) than by the CellSearch system (median 0, range 0–37 cells/7.5 ml) demonstrating statistical superiority (P = 0.002, Wilcoxon test). Conclusion Our results suggest that the MCA system is potentially superior to the CellSearch system for detecting CTCs in lung cancer patients and further clinical development should be considered.

Immunomagnetic capture of Bacillus anthracis spores from food.

Food is a vulnerable target for potential bioterrorist attacks; therefore, a critical mitigation strategy is needed for the rapid concentration and detection of biothreat agents from food matrices. Magnetic beads offer a unique advantage in that they have a large surface area for efficient capture of bacteria. We have demonstrated the efficient capture and concentration of Bacillus anthracis (Sterne) spores using immunomagnetic beads for a potential food application. Magnetic beads from three different sources, with varying sizes and surface chemistries, were functionalized with monoclonal antibodies and polyclonal antibodies from commercial sources and used to capture and concentrate anthrax spores from spiked food matrices, including milk, apple juice, bagged salad, processed meat, and bottled water. The results indicated that the Pathatrix beads were more effective in the binding and capture of anthrax spores than the other two bead types investigated. Furthermore, it was observed that the use of polyclonal antibodies resulted in a more efficient recovery of anthrax spores than the use of monoclonal antibodies. Three different magnetic capture methods, inversion, the Pathatrix Auto system, and the new i CropTheBug system, were investigated. The i CropTheBug system yielded a much higher recovery of spores than the Pathatrix Auto system. Spore recoveries ranged from 80 to 100% for the i CropTheBug system when using pure spore preparations, whereas the Pathatrix Auto system had recoveries from 20 to 30%. Spore capture from food samples inoculated at a level of 1 CFU/ml resulted in 80 to 100% capture for milk, bottled water, and juice samples and 60 to 80% for processed meat and bagged salad when using the i CropTheBug system. This efficient capture of anthrax spores at very low concentrations without enrichment has the potential to enhance the sensitivity of downstream detection technologies and will be a useful method in a foodborne bioterrorism response.

ApoStream, a novel device for antibody-independent capture of circulating cancercells (CCCs) from blood of patients with various types of cancer.

e13500 Background: The detection of circulating tumor cells (CTCs) using immunomagnetic EpCAM-based capture methods has been conceptually accepted as a “liquid tumor biopsy”. However, these methods have limited the recovery of CTCs for molecular profiling applications. We developed a novel continuous flow dielectrophoresis field-flow fractionation (DEP-FFF) device, ApoStream for antibody-independent capture of circulating cancer cells (CCCs), with improved recovery across multiple cancer types and preserved viability of CCCs for downstream characterization. Methods: The performance of ApoStream was demonstrated using a low EpCAM expressing cell line, SKOV3. ApoStream was further used to enrich CCCs from various cancer patient blood. Prostate, breast and NSCLC CCCs were stained for cytokeratin (CK), CD45, and DAPI; melanoma CCCs were stained with S100, CD45 and DAPI. CCC enumeration was performed using laser scanning cytometry. Results: In system precision performance studies, average inter-day recovery on the ApoStream was 80.3 ± 3.5%, CV = 4.3% when cancer cells were spiked into buffer, and 78.5 ± 3.0%, CV = 3.3% when cancer cells spiked into ~10 million healthy peripheral blood mononuclear cells (PBMCs). Linearity performance was demonstrated with spiking 5-2600 SKOV3 cells into 10 million PBMCs (R2=1). Cell viability was not affected by processing through ApoStream device. High CCC recovery from metastatic cancer patient blood samples was obtained with counts ranging from 0 - 2630 (NSCLC, n=66), 0 - 3490 (prostate, n=29), 10 - 968 (breast, n=11), and 3 - 3120 (melanoma, n=13) CCCs per 7.5 mL blood. Positive CCC counts were obtained in 90% of NSCLC samples, 93% of prostate cancer samples, 100% breast cancer and melanoma specimens. There were no CK+ cells detected in healthy donor blood controls. Conclusions: Improved CCC recovery from various cancer types was demonstrated with the ApoStream device. ApoStream provides an antibody-independent method for capture of viable CCCs that enables further downstream molecular characterization of rare cells for use in clinical applications. Acknowledgements: Funded by NCI Contract No. HHSN261200800001E.

Sensitivity of microcavity array system for circulating tumor cells in lung cancer patients.

e21007 Background: Epithelial Cell Adhesion Molecule (EpCAM)-based enumeration of circulating tumor cells (CTCs) has prognostic value in solid tumors such as advanced breast, colon and prostate cancers. However, poor sensitivity has been reported in non-small cell lung cancer (NSCLC). We have developed a microcavity array (MCA) system integrated with a microfluidic device for recovery and enumeration of CTsC, regardless of EpCAM expression level. This system can isolate tumor cells on the basis of differences in size and deformability between tumor and hematologic cells. Methods: Paired peripheral blood samples were collected from metastatic lung cancer patients. CTCs were enumerated by EpCAM-based immunomagnetic capture (CellSearch, Veridex) and by the MCA system. In the MCA system, trapped cells were stained with Hoechst 33342, FITC-labeled anti-pan cytokeratin antibodies and PE-labeled anti-CD45 antibodies for subsequent imaging analysis. CTCs were defined as cells with round to oval morphology, a visible nucleus, positive staining for pan-cytokeratin and negative staining for CD45. We evaluated the sensitivity of the MCA system for detecting CTCs in lung cancer patients compared with the CellSearch system. Results: Twenty-two metastatic NSCLC patients and 13 small-cell lung cancer (SCLC) patients were enrolled into this study between April 2011 and January 2012. CTCs were detected using the MCA system in 17 of 22 NSCLC patients (count ≥1 per 7.5 ml) compared with 9 of 22 patients using CellSearch (p=0.013). On the other hand, CTCs were detected using MCA in all 13 SCLC patients compared with just 9 of 13 patients using CellSearch (p=0.012). More CTCs from NSCLC patients were detected by the MCA system (median 13, range 0-313 cells/7.5ml) than by the CellSearch system (median 0, range 0-37 cells/7.5ml) demonstrating statistical superiority (p=0.002, Wilcoxon test). Conclusions: Our results suggest that the MCA system is potentially superior to the CellSearch system for detecting CTCs in lung cancer patients and further clinical development should be considered.