Track-etched Filters Research Articles

Effects of substrate (track-etched filter) properties on the performance of forward osmosis membranes

Highly permeable membranes can improve the validity of water treatment using forward osmosis (FO) membranes. This study aims to identify the effects of substrate (track-etched (TE) filter) properties on the permeability of FO membranes. The polyamide active layer properties of three types of TE filters with similarly-sized pore diameters (0.2 μm) were observed to be equivalent regardless of the substrate porosity (13.7%–15.8 %) and substrate thickness (10–25 μm). Under the same fabrication protocol using 1.5 wt% m-phenylenediamine (MPD) solution with 0.25 wt% sodium dodecyl sulfate (SDS) addition, the increased porosity of the TE filter increased water flux. This suggested that further reduction in thickness and increase in porosity can enhance the water flux. The addition of SDS to an MPD solution was crucial in maximizing the water flux of TE filter-based FO membranes, and the other approaches (no surfactant addition or the addition of Tween80 or hexadecyltrimethylammonium bromide) produced lower water flux. The optimized fabrication protocol presented the highest water flux of 43 L/m2h and reverse salt flux of 10 g/m2h with the draw (1.0 M NaCl) and feed (pure water) solutions. This study determined the best approach for forming a highly permeable PA active layer on TE filters.

Platelet removal from human blood plasma improves detection of extracellular vesicle-associated miRNA.

Human blood plasma prepared by centrifugation contains not only extracellular vesicles (EVs) but also platelets and erythrocyte ghosts (ery-ghosts). Here we studied whether analysis of miRNA associated with plasma EVs (EV-miRNA) is affected by the presence of platelets and ery-ghosts. EDTA blood was collected from healthy donors (n=3), and plasma was prepared by the centrifugation protocol recommended by the International Society on Thrombosis and Haemostasis (ISTH), and by a centrifugation protocol from an EV-miRNA expert lab (non-ISTH protocol). EVs were isolated from plasma by size-exclusion chromatography CL-2B (SEC2B), and concentrations of platelets, activated platelets, ery-ghosts and EVs (150-1000nm) were measured by calibrated flow cytometry. Two EV-associated miRNAs (let7a-5p and miR-21-5p), and one platelet-associated miRNA (miR-223-3p), were measured by qRT-PCR. Measurements were performed with and without filtration using 0.8μm track-etched filters to remove platelets and ery-ghosts from plasma and EV-enriched SEC fractions. Plasma prepared by both centrifugation protocols contained platelets and ery-ghosts, which co-migrated with EVs into the EV-enriched SEC2B fractions. Filtration removed platelets and ery-ghosts (>97%; p≤0.05) and did not affect the EV concentrations (p>0.17). The miRNA concentrations were 2-4-fold overestimated due to the presence of platelets but not ery-ghosts. Thus, filtration of human plasma is expected to improve comparability and reproducibility of quantitative EV-miRNA studies. Therefore, we recommend to measure and report the plasma concentration of platelets for EV-miRNA studies, and to filter plasma before downstream analyses or storage in biobanks.

Circulating tumor cell isolation: the assets of filtration methods with polycarbonate track-etched filters.

Circulating tumor cells (CTCs) arise from primary or secondary tumors and enter the bloodstream by active or passive intravasation. Given the low number of CTCs, enrichment is necessary for detection. Filtration methods are based on selection of CTCs by size using a filter with 6.5 to 8 µm pores. After coloration, collected CTCs are evaluated according to morphological criteria. Immunophenotyping and fluorescence in situ hybridization techniques may be used. Selected CTCs can also be cultivated in vitro to provide more material. Analysis of genomic mutations is difficult because it requires adapted techniques due to limited DNA materials. Filtration-selected CTCs have shown prognostic value in many studies but multicentric validating trials are mandatory to strengthen this assessment. Other clinical applications are promising such as follow-up, therapy response prediction and diagnosis. Microfluidic emerging systems could optimize filtration-selected CTCs by increasing selection accuracy.

Long duration, room temperature preservation of filtered eDNA samples

The use of environmental DNA is a rapidly evolving approach for surveillance and detection of species. Often water samples are collected in the field and then immediately cooled, filtered, and the resulting filters are stored in freezers to preserve the DNA for subsequent analyses. Recently it was shown that filtered samples could be stored at room temperature for 14 days without any discernable loss in the total DNA. However, for many conservation applications, particularly in remote settings with limited capacity to freeze samples, it would be advantageous to store samples at room temperature for longer periods of time. Here we test for significant loss of DNA yield from storage of polycarbonate track etched filters in Longmire’s lysis buffer at room temperature (20 °C) for 150 days.

A microfluidic chip integrated with a high-density PDMS-based microfiltration membrane for rapid isolation and detection of circulating tumor cells.

Isolation of circulating tumor cells (CTCs) by size exclusion is a widely researched technique that offers the advantage of capturing tumor cells without reliance on cell surface expression markers. In this work, we report the development of a novel polydimethylsiloxane (PDMS) membrane filter-based microdevice for rapid and highly efficient isolation of CTCs from peripheral blood. A precise and highly porous PDMS microfilter was fabricated and integrated into the microfiltration chip by combining a sacrificial transferring film with a sandwich molding method. We achieved >90% recovery when isolating lung cancer cells from spiked blood samples, with a relatively high processing throughput of 10 mL/h. In contrast to existing CTC filtration systems, which rely on low-porosity track-etch filters or expensive lithography-based filters, our microfiltration chip does not require complex e-beam lithography or the reactive ion etching process, therefore it offers a low-cost alternative tool for highly efficient CTC enrichment and in situ analysis. Thus, this new microdevice has the potential for use in routine monitoring of cancer development and cancer therapy in a clinical setting.

Filtration parameters influencing circulating tumor cell enrichment from whole blood.

Filtration can achieve circulating tumor cell (CTC) enrichment from blood. Key parameters such as flow-rate, applied pressure, and fixation, vary largely between assays and their influence is not well understood. Here, we used a filtration system, to monitor these parameters and determine their relationships. Whole blood, or its components, with and without spiked tumor cells were filtered through track-etched filters. We characterize cells passing through filter pores by their apparent viscosity; the viscosity of a fluid that would pass with the same flow. We measured a ratio of 5·104∶102∶1 for the apparent viscosities of 15 µm diameter MDA-231 cells, 10 µm white cells and 90 fl red cells passing through a 5 µm pore. Fixation increases the pressure needed to pass cells through 8 µm pores 25-fold and halves the recovery of spiked tumor cells. Filtration should be performed on unfixed samples at a pressure of ∼10 mbar for a 1 cm2 track-etched filter with 5 µm pores. At this pressure MDA-231 cells move through the filter in 1 hour. If fixation is needed for sample preservation, a gentle fixative should be selected. The difference in apparent viscosity between CTC and blood cells is key in optimizing recovery of CTC.

Filter Characteristics Influencing Circulating Tumor Cell Enrichment from Whole Blood

A variety of filters assays have been described to enrich circulating tumor cells (CTC) based on differences in physical characteristics of blood cells and CTC. In this study we evaluate different filter types to derive the properties of the ideal filter for CTC enrichment. Between 0.1 and 10 mL of whole blood spiked with cells from tumor cell lines were passed through silicon nitride microsieves, polymer track-etched filters and metal TEM grids with various pore sizes. The recovery and size of 9 different culture cell lines was determined and compared to the size of EpCAM+CK+CD45−DNA+ CTC from patients with metastatic breast, colorectal and prostate cancer. The 8 µm track-etched filter and the 5 µm microsieve had the best performance on MDA-231, PC3-9 and SKBR-3 cells, enriching >80% of cells from whole blood. TEM grids had poor recovery of ∼25%. Median diameter of cell lines ranged from 10.9–19.0 µm, compared to 13.1, 10.7, and 11.0 µm for breast, prostate and colorectal CTC, respectively. The 11.4 µm COLO-320 cell line had the lowest recovery of 17%. The ideal filter for CTC enrichment is constructed of a stiff, flat material, is inert to blood cells, has at least 100,000 regularly spaced 5 µm pores for 1 ml of blood with a ≤10% porosity. While cell size is an important factor in determining recovery, other factors must be involved as well. To evaluate a filtration procedure, cell lines with a median size of 11–13 µm should be used to challenge the system.

Abstract 2396: Key factors for filtration of tumor cells from whole blood

Abstract Introduction: Circulating tumor cells (CTC) in patients with metastatic carcinomas are associated with poor survival. The current standard CTC enrichment method employs antibodies, which misses cells not expressing the target antigen. Several alternative methods enrich CTC by filtration, exploiting differences in size between blood cells and CTC. Between different filtration methods large variations in material, pore size, operating pressure and sample fixation exist. As their influence for CTC enrichment is poorly understood we have investigated these parameters. Methods: Track etched polycarbonate filters with pore diameters of: 5, 8 and 10 µm and microfabricated silicon nitride filters with pore diameters of 5, 6, 7, 8, 9 and 10 µm were used. 300 cells from the cell lines SKBR3, PC3-9, MDA-231, MDA-468, MCF-7, SW480, HL-60 or K-586 were spiked in 1 ml of blood from healthy volunteers. Samples were filtered while both pressure across the filter membrane and the flow speed was measured. The pressure across the filter provides the force driving a cell through a filter pore. Cells were identified on the filters by fluorescence microscopy. Results: MDA-231, a 15 µm sized epithelial cell line, can readily pass through a 5 µm pore at low pressure (< 50 mbar). We show that the number of red and white blood cells contribute most to the pressure across a filter and at least 10e5 8 µm pores are needed to process 0.5 mL of whole blood per minute at 10 mbar of pressure. The influence of buffers such as PBS or serum was found to be negligible. Fixation using paraformaldehyde increases the pressure needed to push white, red and culture cells through a pore by 25 fold and recovery with spiked samples decreases from 85% to 40% at the same flow rate. Different filter types were compared and evaluated for cell line recovery using unfixed blood. Optimal recovery of cell lines was between 80-90% with low WBC contamination for track etch filters with 8 µm pores and 5 µm pores for silicon nitride chips. Only a weak correlation was found between recovery and size for the eight different cell lines. Conclusions: There are big differences in the ease with which different culture cell lines of similar size pass a filter pore; a challenging cell line such as MDA-231 should be selected for evaluation of filtration methods. Pressure across the filter is an important parameter in determining whether a cell passes a filter pore. Enrichment without fixation is best and should be performed at low pressures (< 50 mbar). A sufficiently small pore retains most of the tumor cells, but passes most white and all red cells. The optimal pore size was different for the different filter types evaluated, possibly due to surface of the filter or filter thickness. In the optimal conditions recoveries of at least 80% were found for most cell lines across a wide range of CTC concentrations and blood volumes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2396. doi:1538-7445.AM2012-2396

Abstract 2369: Isolation of circulating tumor cells by size exclusion using lithography fabricated precision microfilters

Abstract Background: We present a novel precision microfilter that achieves rapid and highly efficient isolation of circulating tumor cells (CTCs) from peripheral blood. Isolation of CTCs by size exclusion is a widely researched technique with the advantage of capturing cells without reliance on cell surface expression markers. For many years CTC filtration technology has relied on track-etch microfilters with randomly located pores and low-porosity. More recently, precision microfilters with high porosity have been tested for CTC isolation, but they are not widely available. We have developed a new method of fabricating precision, high porosity microfilters that are capable of high volume manufacturing. These microfilters are strong, clear, non-fluorescent, and have high CTC capture efficiencies. Methods: We describe an assay to capture and enumerate prefixed and live CTCs. MCF-7 human breast adenocarcinoma cells (ATCC) were cultured in DMEM until confluent. Cells were stained in a fixative/staining solution containing paraformaldehyde, acridine orange, and DAPI dilacetate. After incubation, a cell sample was placed on a microscope slide and cells counted to obtain the exact input. Cells were then washed into 7.5 mL whole human blood with 7.5 mL PBS and placed into a syringe. An 8 µm pore precision microfilter was placed into Swinnex filter holder, and the sample drawn by negative pressure through the filter at ∼10 mL/min. The microfilter was mounted onto a microscope slide and counted using a fluorescence microscope under FITC and DAPI settings. Cells were defined by their shape and coloration under FITC fluorescence and nuclear signature under DAPI fluorescence. The procedure was then repeated using a staining solution without fixative to capture live MCF-7 cells. Both live and fixed MCF-7 cell isolation by microfiltration were performed in triplicate. Results: Precision microfilters were able to capture MCF-7 cells spiked into 7.5 mL human blood with a recovery rate of 98±3% for fixed cells, and 90±2% for live cells. The filtration was performed in 2 minutes, and cell counts were obtained in 10 minutes. By contrast, track-etched microfilters captured only 73% of fixed cells and cell counts required 20 minutes. Conclusions: Creatv's precision microfilters are shown to recover MCF-7 cells more efficiently than track-etch filters. The pore uniformity and low fluorescent background allowed for faster and more accurate counts of spiked CTC in human peripheral blood compared to track-etch microfilters. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2369. doi:10.1158/1538-7445.AM2011-2369

Particulate fouling of surface microfilters with slotted and circular pore geometry

During microfiltration it is possible to obtain a permeate rate equal to that of the clean liquid permeation rate under certain critical conditions. This occurs when a deposit does not foul the surface of the membrane and internal deposition of material within the filter does not occur. Surface filters do not possess an internal structure, therefore, the only particulate fouling possible is that on the surface. Experiments in a stirred cell compared the surface fouling of two types of true surface filters: a commercially available track-etched filter with circular pores 10 μm in diameter and a filter with slotted pores 10 μm×420 μm in size. When using a challenge suspension containing dilute latex particles of diameters 15 μm, and less, the circular pore membrane exhibited a critical flux at 260 l m −2 h −1. Under the same conditions, the critical flux for the slotted membrane was in excess of 1500 l m −2 h −1. Surface fouling was removed by back-flushing for both filters. These results indicate that slotted pores are less likely to suffer from particles bridging the pores leading to cake deposition, or secondary membrane formation, and that further development of filters with slotted pores of smaller slot widths would be worthwhile.

Virus passage through track-etch membranes modified by salinity and a nonionic surfactant.

Why do viruses sometimes not pass through larger pores in track-etch filters? Increasing the salinity (0.8 to 160 mM Na+) decreased phiX174 and PRD1 passage through track-etch polycarbonate membranes (sodium dodecyl sulfate coated but not polyvinylpyrrolidone coated) and PRD1 passage through polyester membranes. Undiminished passage when 0.1% Tween 80 was added implied that nonionic virus adsorption occurred and indicated that high levels of salinity decreased virus passage by decreasing electrostatic repulsion that prevented adsorption.

Pore Size Distributions in Microporous Membranes II. Bulk Characterization of Track-Etched Filters by Air Porometry and Mercury Porosimetry

The structure and morphology of several Cyclopore filters, consisting of thin track-etched sheets of polycarbonate, is analyzed here through an extended bubble point method and mercury intrusion porometry. Relevant morphological parameters have been studied for six types of filters (C01, C02, C04, C06, C08, and C10). The bulk pore size distributions are directly obtained for each filter. The statistical distributions are studied thoroughly and those obtained from both methods are compared. Specific surface areas are also obtained from both methods and compared with those directly measured in BET experiments with fair agreement. Scanning electron microscopy (SEM) and computerized image analysis (CIA) were previously used to characterize and analyze the surface pore size distributions. They are also compared with those obtained here, confirming the previous assumption of inner widenings in the pores.

Pore Size Distributions in Microporous Membranes: I. Surface Study of Track-Etched Filters by Image Analysis

The surface morphology of several Cyclopore filters, consisting of thin track-etched sheets of polycarbonate, is analyzed here. Scanning electron microscopy and computerized image analysis have been applied. The surface porosity and the pore density or number of pores per surface unit are directly obtained for each filter, while the statistical distribution of the pore areas, pore perimeters, equivalent pore diameters, and pore shape factors are studied as well. These pore size distributions have been studied for six types of filters (C01, C02, C04, C06, C08, and C10) and the existence of a relevant portion of double and other multiple pores has been revealed. The fraction of these multiple pores are correlated with the nominal pore radii. The results on pore size have been used to predict the volume flows of the membranes studied. This can be done only by assuming that the surface characteristics remain unchanged in the internal volume of the filters, which leads to hydrodynamic radii well in accordance with the experimental ones, within the error range. Nevertheless, it seems that some of the pores should have internal widenings with inner radii close to 120% of the external ones.

Analysis of Biogenic Silica Fibers from Rice Farming Operations

Abstract A provisional method has been developed for the quantitation of biogenic silica fibers. Particles were sampled onto track-etched filters prepared by the direct-clearing method for transmission electron microscopy analysis. Fibers were identified by their length/width ratio, X-ray spectrum, and electron diffraction pattern. Biogenic silica fibers were found to contain mainly silicon and no aluminum, and to have an amorphous crystal structure. Approximately 100 samples from rice farming operations were analyzed. Cyclones were used to exclude large, nonrespirable particles when sampling near farming operations. The optimum fiber loading was found to be 100 to 1000 fibers/mm2. To prevent overloading, in most cases the sampled volume was held to 30 L using 25-mm filters, giving a limit of detection of 0.1 fibers/cc. Higher volumes were practical when sampling away from farming operations, giving limits of detection ranging from 0.02 to 0.004 fibers/cc. For a given microscopist, the coefficient of vari...

Nuclear track-etched filters as model pores for biological membranes

Ion current through individual pores in track-etched polyethyleneterephthalate filters can have the following properties which are shared by a number of biological ion channels: 1. 1) selectivity for cations over anions, or vice versa depending on the surface charge of the filter and the diameter of the pore; 2. 2) fluctuations between high- and low-conductance states; 3. 3) inhibition by protons and divalent cations with efficacy H + > Zn 2+ > Ca 2+ ∼ Mg 2+. Pore topography in differing states can be imaged by scanning probe microscopy. We conclude that track-etched filters provide a useful model system for studying the physical and chemical basis for some of the characteristic behaviour of biological channels.

The hydrodynamic resistance of filter cakes

In calculating the hydrodynamic resistance of a filter cake or polarized layer the influence of the rejecting membrane itself on the permeate flow field is usually ignored. However, because permeate is constrained to pass through the reduced cross-sectional area offered by the membrane's pores, fluid velocities must increase in the vicinity of the membrane surface. Rejected material near the membrane will therefore cause a proportionally larger pressure drop than material further upstream. A model taking account of this phenomenon is presented, and a solution for the simple case of a filter cake or gel layer of constant concentration (and hence constant permeability) is given. Sample calculations for 0.2 μm track-etched filters are also shown. Generally, this effect is of greatest importance when: (i) the thickness of the layer formed by the rejected material is small compared with the rejecting membrane's pore separation distance, or (ii) when the filter porosity is small. Implications for classical boundary layer filtration models are discussed.