Coulter Counter Multisizer Research Articles

An Investigation of Size Distribution and Calcium Signaling in Human Platelets.

Background Platelets are thin disc-shaped blood cells that play a major role in hemostasis, maintenance of vascular integrity, and blood coagulation. Large platelets are more reactive and seen in patients with cardiovascular disease. This study aims to analyze the changes in platelet size of ex vivo activated platelets which phenotypically simulates that of a patient at risk of cardiovascular disease and elucidate the calcium signaling pathway responsible for this change. Methodology Platelets were isolated from adult human blood by differential centrifugation. Calcium was mobilized into platelets by treatment with calcium ionophore A23187 in the presence of Ca2+. Platelet size distribution was analyzed using Coulter Counter Multisizer 4. The following signaling parameters were studied: intracellular Ca2+ measurement (using Fura-2/AM by fluorescence spectrophotometry), Ca2+-dependent thiol protease calpain assay (using fluorogenic substrate t-butoxycarbonyl-Leu-metchloromethylcoumarin in fluorescence microplate reader), platelet-derived microparticles (using FACS Calibur flow cytometry), and cytoskeletal protein talin expression (by western immunoblotting). Results When adult platelets were treated with A23187 and Ca2+, two subcellular populations (<2 µm and between 2-4 µm) were noted. The mean size of the second cell population was significantly higher than that of resting platelets (2.94 ± 0.13 µm vs. 2.82 ± 0.15 µm, t = 4.605, p = 0.00). A23187 treatment led to elevated intracellular Ca2+, release of platelet-derived microparticles, increase in calpain activity, and cytoskeletal talin degradation. These events were inhibited by calpeptin (a specific calpain inhibitor). Conclusions Elevated calcium caused talin degradation by calpain activity. Breakdown of this cytoskeletal protein leads to relative swelling of cells reflected by the increase in platelet size.

In Vitro and In Vivo Behavioral Evaluation of Condensed Lipid-Coated Perfluorocarbon Nanodroplets

BackgroundPhase-shift contrast agents consist of a liquid perfluorocarbon core that can be vaporized by ultrasound to generate echogenic contrast with excellent spatiotemporal control. The purpose of the present work was to evaluate the in vitro and in vivo behavior of condensed lipid-shelled nanodroplets using different analytical procedures. MethodsIn this study, perfluorobutane nanodroplets were prepared by condensation of precursor fluorescently labeled lipid shelled microbubbles and were characterized in terms of size distribution, gas core content, in vitro stability in blood as well as for their acoustic vaporization behavior using a custom-made set-up. In particular, their in vivo behavior was thoroughly investigated after intravenous bolus injection in rats. To this end, we report, for the first time, the efficient use of three complementary detection procedures to assess the NDs in vivo persistence namely 1) Ultrasound contrast imaging of vaporized NDs, 2) Gas chromatography-mass spectrometry to determine the perfluorobutane core content and 3) Fluorescence intensity measurement in the collected blood samples. ResultsThe Coulter Counter Multisizer results confirmed the size distribution shift post-condensation. Furthermore, similar PFB concentration from MBs and NDs suspensions was obtained, thus demonstrating an exceptionally low rate of microbubbles breaking and spontaneous nanodroplets vaporization. As expected, these nanoscale droplets display longer circulation time compared to clinically approved microbubbles and only slight variations of half-life were observed between the three monitoring procedures. Finally, echogenic signal observed in focal area of the liver and spleen after vaporization was confirmed by accumulation of fluorescent nanodroplets in these organs. ConclusionThese results further contribute to deepen our understanding of both in vitro and in vivo behavior of sonoresponsive nanodroplets, which is key to enable efficient clinical translation.

Analysis of common pre-treatments in grain-size analysis (using a grain-size standard)

ABSTRACT This study investigates the impacts of sample preparation procedures on grain-size measurements to determine comparability of data collected using differing methodologies. Grain-size distributions of marine and terrestrial sediments contain important information about the depositional environment. For example, the “sortable-silt index” (or mean grain size between 10 and 63 μm in marine sediments, is used as an indicator of flow speed and has been applied to the reconstruction of ocean current strength before the instrumental period. Similarly, the mean grain size of a sediment is used to classify it (e.g., silt versus sand). Accurate measurements of grain-size distributions often require chemical pre-treatments in order to remove sedimentary components of biogenic origin (e.g., shells), and multiple ways to perform these pre-treatments exist. This study tests whether the choice of pre-treatment introduces variability into grain-size distributions. We simulate multiple commonly used pre-treatments on a well-characterized internal standard (“Sillikers”) and compare the resulting mean size and sortable-silt index in each treatment group to untreated samples using ANOVA. Two instruments, a Coulter Counter Multisizer III and a Coulter LS 230 Laser Diffraction Analyzer are used. Results from the Multisizer III suggest that the choice of pre-treatment method does not significantly impact the final grain-size distributions but underlines the importance of replicates. Results from the laser sizer suggest that oven-drying leads to a small but statistically significant difference of ∼ 0.3 μm in the sortable-silt index, and drying samples via hot plate leads to another small but statistically significant difference of ∼ 0.29 μm. While it is unclear what causes these differences in the laser sizer data, they are smaller than the observed variations in sortable-silt index used to infer changes in current speed in a typical paleoclimate study. In conclusion, grain-size measurements are a robust tool for sediment analysis and are resistant to changes from differing pre-treatment methods tested here.

INJECTION OF DISPERSED OIL-IN-WATER EMULSION TO IMPROVE VOLUMETRIC SWEEP EFFICIENCY DURING WATER FLOODING OF OIL RESERVOIRS

During water flooding of heterogeneous oil reservoirs, early breakthrough of water can occur and lead to poor volumetric sweep efficiency. Macro-emulsions with oil droplets dispersed in a continuous water phase [oil-in-water (o/w) emulsions], have the potential to block high-permeability zones in these reservoirs, and thereby improve the volumetric sweep by diverting the water flow from high-permeability layers toward lower-permeability layers. In the present work, the potential for produced water with dispersed oil to block sandstone rocks (with permeability of 150-2000 mD) was investigated. Different o/w emulsions with similar oil concentrations as in the produced water were prepared using synthetic oil and crude oil. Water-soluble surfactant was dissolved in synthetic formation water and homogenized with oil using a high-shear rate mixer at different speeds and mixing times. The prepared emulsions were injected into sandstone cores. A Coulter Counter Multisizer was used to measure the droplet size distributions in the injected emulsions and the produced effluent. The droplet size distributions in the emulsions and the pore throat size distributions in rock were compared. The flooding experiments demonstrated that injection of emulsions can significantly reduce the permeability, and depending on the initial permeability of the rock the permeability reduction was up to 47%. Emulsion has the potential to accelerate and improve oil production and reduce the water cut at low-cost investment. Further work is required to qualify this method for field applications.

Sizing biological cells using a microfluidic acoustic flow cytometer

We describe a new technique that combines ultrasound and microfluidics to rapidly size and count cells in a high-throughput and label-free fashion. Using 3D hydrodynamic flow focusing, cells are streamed single file through an ultrasound beam where ultrasound scattering events from each individual cell are acquired. The ultrasound operates at a center frequency of 375 MHz with a wavelength of 4 μm; when the ultrasound wavelength is similar to the size of a scatterer, the power spectra of the backscattered ultrasound waves have distinct features at specific frequencies that are directly related to the cell size. Our approach determines cell sizes through a comparison of these distinct spectral features with established theoretical models. We perform an analysis of two types of cells: acute myeloid leukemia cells, where 2,390 measurements resulted in a mean size of 10.0 ± 1.7 μm, and HT29 colorectal cancer cells, where 1,955 measurements resulted in a mean size of 15.0 ± 2.3 μm. These results and histogram distributions agree very well with those measured from a Coulter Counter Multisizer 4. Our technique is the first to combine ultrasound and microfluidics to determine the cell size with the potential for multi-parameter cellular characterization using fluorescence, light scattering and quantitative photoacoustic techniques.

Effect of microplastics on the toxicity of chlorpyrifos to the microalgae Isochrysis galbana, clone t-ISO

It is highly likely that phytoplanktonic organisms will interact with MPs in the ocean, and consequently with the pollutants sorbed onto their surfaces. Microalgae play an essential role in maintaining the balance of the marine ecosystem due to the fact that they are a primary producer and the base of marine trophic chains. Therefore, their fitness represents an important index in the assessment of water quality. The objectives of this study were i) to assess the toxicity of MPs and the pesticide chlorpyrifos (CPF) to the microalgae, Isochrysis galbana, clone t-ISO and ii) to ascertain whether the presence of MPs affects the toxicity of CPF. Microalgae growth rate was selected as the endpoint and a commercial virgin PE micronized powder was chosen as a micro-plastic model, with mean size ranging from 2 to 6 µm, assayed until 25 mg L−1. CPF was tested at concentrations ranging from 0 to 4 mg L−1. A constant concentration of MPs (5 mg L−1) was loaded with increasing doses of CPF (0–3 mg L−1) with a 2 h incubation period. Bioassays were performed at 20 °C, in glass tubes of 50 ml, with air and constant light and an exposure time of 72 h. Cell counts were performed using a Coulter Counter Multisizer III and HPLC was used to quantify the partition of this pollutant among MPs and water. Although microalgae growth was not impacted by MPs, growth was clearly affected by exposure to CPF from 2 mg L−1 and above, with a total growth inhibition at concentrations over 3 mg L−1. Subsequent to incubation, 80% of CPF was sorbed onto MP surfaces. Two different dose-response curves resulted from CPF bioassays depending on the presence of MP, with lower percentages of inhibition when CPF was presented through MP. Thus, the adsorption of CPF onto MP surfaces modulates the toxicity of CPF on I. galbana growth through a reduction in its toxicity, as CPF is adsorbed onto MP surfaces which are less bio-available to the algal cells.

Fecundidad en gónadas maduras de Haliotis fulgens y Haliotis corrugata (Vetigastropoda)

Background: The term fecundity in abalone is defined according to an estimate of its absolute fecundity, which is the number of mature oocytes present in the gonad, or as potential fecundity, i.e., the total number of oocytes present in the gonad. In Mexico, few studies have estimated the fecundity of mollusks and these have been performed on gonads in different phases of gonadal development, obtaining values that are very different or overestimated, for example, values ranging from 80000 to 22 million oocytes in a gonad. Goals: Estimate the fecundity of Haliotis fulgens (blue abalone) and Haliotis corrugata (yellow abalone), using ma­ture gonads and a particle counter (Multisizer 3) to count only mature oocytes. Methods: Each month from October 2011 to February 2013, 30 adult organisms of each species were collected in two localities of Baja California Sur, Mexico (La Bocana and Bahia Tortugas). A histological analysis identified those that were mature females. To obtain the number of eggs per gram, triplicate oocyte counts were performed in a coulter counter Multisizer 3, in a solution of 4 g wet weight of mature oocytes diluted to 50 mL of filtered seawater (μm). Absolute fecundity was calculated by multiplying the total weight of the gonad by the number of mature oocytes present in one gram of oocytes. Results: The fecundity in La Bocana was found to be 2x106 eggs in H. corrugata and H. fulgens, while in Bahia Tortugas it was 4x106 eggs in H. fulgens. Conclusions: This study suggests that estimating absolute fecundity in species with partial spawning, such as H. fulgens and H. corrugata, should be per­formed on all females regardless of the degree of gonadal development. However, only mature oocytes should be considered as an indicator of absolute fecundity. Therefore, the use of an apparatus that counts and measures mature oocytes, such as a particle counter, facilitates and guarantees a good estimate of fecundity.

Visible and subvisible particles in the BCG immunotherapeutic product ImmuCyst®

Bacille Calmette–Guerin, BCG, is a live attenuated bovine tubercle bacillus used for the treatment of non-muscle invasive bladder cancer. In this study, an Electrical Sensing Zone (ESZ) method was developed to measure the particle count and the size of BCG immunotherapeutic (BCG IT), or ImmuCyst® product using a Coulter Counter Multisizer 4® instrument. The focus of this study was to establish a baseline for reconstituted lyophilized BCG IT product using visible and sub-visible particle concentration and size distribution as reportable values. ESZ method was used to assess manufacturing process consistency using 20 production scale lots of BCG IT product. The results demonstrated that ESZ can be used to accumulate product and process knowledge of BCG IT.

Experimental determination of multiple thermodynamic and kinetic risk factors for nephrolithiasis in the urine of healthy controls and calcium oxalate stone formers: does a universal discriminator exist?

Nephrolithiasis is thought to be governed by urinary thermodynamic and kinetic risk factors. However, identification of one or more of these factors which consistently and unambiguously differentiates between healthy subjects (N) and calcium oxalate (CaOx) renal stone patients (SF) remains elusive. The present study addresses this challenge. 24 h urines were collected from 15 N and 10 SF. Urine compositions were used to compute thermodynamic risk indices including urinary ratios, quotients and supersaturation (SS) values, while CaOx metastable limits (MSL) were determined experimentally. Crystallisation kinetics was determined by measuring rates of particle formation (number, volume, size) using a Coulter counter multisizer (CC) and a Coulter flow cytometer (FC). Particle shapes were qualitatively differentiated by FC and were viewed directly by scanning electron microscopy. Several urinary composition ratios and risk quotients were significantly different between the groups. However, there were no significant differences between CaOx MSL or SS values. Using transformed FC data, the rate of CaOx crystallisation in SF was significantly greater than in N. This was not supported by CC measurements. There were no significant differences between the groups with respect to particle size or CaOx crystal growth rates. Single and aggregated CaOx dihydrate crystals were observed in both groups with equal frequency and there were no differences in the kinetic properties of these deposits. A few CaOx monohydrate crystals were observed in SF. Although several risk factors were found to be significantly different between the groups, none of them were consistently robust when compared to other cognate factors. Arguments were readily invoked which demonstrated inter-factor inconsistencies and conflicts. We suspect that a unique discriminatory factor, such as any of those which we investigated in the present study, may not exist.

Quantifying silicone oil emulsification in patients: are we only seeing the tip of the iceberg?

Emulsification of silicone oil in the eye is a difficult problem. In an effort to find an objective way to quantify emulsification, we used the Coulter principle to measure silicone oil emulsified droplets from the washings of a series of patients. Aqueous washouts after silicone oil removal were obtained from nine patients (nine eyes). We used the Coulter counter Multisizer® 4 to obtain the size distribution of the oil droplets. Over 65 % of the emulsified silicone oil droplets in the clinical samples had a diameter smaller than is detectable by light microscopy (2 μm). The median size of the droplets was between 1.1 and 1.9 μm. Based on the Spearman's correlation coefficient (r), there was a strong correlation between the number of the droplets that cannot be seen (between 1 and 2 μm) and those that can be seen (7-30 μm) (r = 0.817, p = 0.007). Once emulsification was detected clinically in the anterior chamber, extensive emulsification would have already occurred in the posterior chamber, with most of the emulsified droplets that were too small in size to be seen on clinical examination. Ostwald ripening might explain why there were so many small droplets. The predominance of small droplets might account for some of the clinical complications associated with silicone oil use.

Influence of crystallization conditions on crystal morphology and size of CaCO3 and their effect on pressure filtration

AbstractThe temperature, supersaturation, seeding procedure, stirring speed and other parameters were varied in crystallization experiments of calcium carbonate performed in aqueous solutions to control size, particle size distribution and morphology of the particles. Particle size information was obtained by focused beam reflectance measurements and the Coulter Counter Multisizer. Crystals of CaCO3 could be crystallized as spherical polycrystalline particles of the vaterite polymorph, needle‐like crystals of aragonite and both cube‐like and novel plate‐like crystals of calcite. Filtration experiments for calcium carbonate, performed at a constant pressure difference of 2 bar, show that spherical particles with a larger size show better filterability and that spheres with a wider size distribution, as a result of high supersaturation and nucleation, give higher average cake resistance values. Comparing different particle morphologies, plate‐like crystals and needle‐like crystals show worse filterability than spherical particles and cube‐like particles. © 2011 American Institute of Chemical Engineers AIChE J, 2012

Getting more from cell size distributions: Establishing more accurate biovolumes by estimating viable cell populations

Current approaches for cell size distribution modeling are attempting to describe the behavior of the entire distribution with respect to time. Although some advances have been made in this area, the modeling process requires a large number of culture-specific parameters and an a priori assumption of the distribution nature (Poisson, Gaussian, etc.). In this work, we propose a deconvolution of the distribution into size ranges and an iterative regression process with respect to a single culture variable, such as viability. Following this approach, two example applications are outlined using data collected with a Coulter Counter Multisizer. In the first, traditional biovolume measurements are corrected to account for the noneven distribution of nonviable cells. These corrections amount to an average increase of 7-65% in the calculated biovolume from 24 to 72 h postinfection and are expected to aid in the development of a new basis for nutrient consumption postinfection. In the second example, viability is predicted from the cell size distribution using both linear and exponential regressions. Differences between predicted and measured viabilities were found to be normally distributed with means of 0.4% and 0% as well as standard deviations of 7.6% and 8.1% for linear and exponential regression, respectively. Although only viability relationships were tested, our approach yielded significant results for both applications, allowing the possibility for further development.

Performance evaluation of six different aerosol samplers in a particulate matter generation chamber

The present study was carried out with the aim of evaluating the performance of six different aerosol samplers in terms of mass concentration, particle size distribution, and mass fraction for the international size-sampling conventions. The international size-sampling criteria were defined as inhalable, thoracic, and respirable mass fractions with 50% cutoff at an aerodynamic equivalent diameter of 100 μm, 10 μm, and 4 μm, respectively. Two Andersen, four total suspended particulate (TSP), two RespiCon, four PM 10, two DustTrak, and two SidePak samplers were selected and tested to quantitatively estimate human exposure in a carefully controlled particulate matter (PM) test chamber. The overall results indicate that (1) Andersen samplers underestimate total suspended PM and overestimate thoracic and respirable PM due to particle bounce and carryover between stages, (2) TSP samplers provide total suspended PM as reference samplers, (3) TSP samplers quantified by a coulter counter multisizer provide no information below an equivalent spherical diameter of 2 μm and therefore underestimate respirable PM, (4) RespiCon samplers are free from particle bounce as inhalable samplers but underestimate total suspended PM, (5) PM 10 samplers overestimate thoracic PM, and (6) DustTrak and SidePak samplers provide relative PM concentrations instead of absolute PM concentrations.

Properties of protease-treated maize starches

Commercially produced maize starches were treated with protease (Promod 25P) and their composition and properties were compared with untreated controls. It was found that, although protease treatment reduced the starch protein contents by 41%, 21% and 37% for the waxy, normal and amylomaize starches, respectively, it also caused some pits on the granule surfaces, which were evident by scanning electron microscopy (SEM), but no obvious decrease in granule dimensions (Coulter Counter Multisizer). The protein extraction was associated with decreases in starch lipid content by 42%, 40% and 45% (waxy, normal and amylomaize starches, respectively) and a decrease in total amylose content (30.7–26.0%) for the normal maize starch. The gelatinisation parameters of the starches by differential scanning calorimetry (DSC) in water, 0.001M HCl or NaOH were less obviously affected by protease treatment in common with the swelling factors at 80°C. The amount of α-glucan leached by the swollen (80°C) granules was, however, increased by the protease treatment by factors of 3.8, 1.4, and 1.1, for the waxy, normal and amylomaize starches, respectively. Although proteases provide a useful tool for the purification of native starches, commercial protease preparations need to be controlled in terms of amylase content to prevent modifications to starch structure and properties during industrial processing.

Purinergic signaling underlies CFTR control of human airway epithelial cell volume

Background: Loss of cystic fibrosis transmembrane conductance regulator (CFTR) function in cystic fibrosis (CF) causes dysregulation of multiple ion channels, water channels, and acid–base transporters in epithelia. As such, we hypothesized that dysregulation of many critical ion channels and transporters may cause defects in human airway epithelial cell volume regulation. Methods: Cell volume, regulatory volume decrease, and its regulation was assessed in real-time via Coulter Counter Multisizer III-driven electronic cell sizing in non-CF, CF, and CFTR-complemented CF human airway epithelial cells. SPQ halide fluorescence assay of hypotonicity-induced chloride efflux provided indirect validation of the cell volume assays. Results: CFTR, via autocrine ATP signaling, governs human airway epithelial cell volume regulation. Non-CF cells and wild-type (WT)-CFTR-transfected CF cells had normal regulatory volume decrease (RVD) responses that were attenuated by blockade of autocrine and paracrine purinergic signaling. In contrast, parental IB3-1 CF cells or IB3-1 cells expressing CFTR mutants (ΔF508, G551D, and S1455X) failed to RVD. CF cell RVD was rescued by agonists to P2Y G protein-coupled receptors and, more robustly, by agonists to P2X purinergic receptor channels. Conclusions: Loss of CFTR and CFTR-driven autocrine ATP signaling may underlie defective cell volume regulation and dysregulated ion, water, and acid–base transport in CF airway epithelia.

Regulatory volume decrease in human esophageal epithelial cells.

In vivo human esophageal epithelial cells are regularly exposed to hyposmolal stress. This stress, however, only becomes destructive when the surface epithelial cell (barrier) layers are breached and there is contact of the hyposmolal solution with the basolateral cell membranes. The present investigation was designed to examine the effects of hyposmolal stress in the latter circumstance using as a model for human esophageal epithelial cells the noncancer-derived HET-1A cell line. Cell volume and the response to hyposmolal stress in suspensions of HET-1A cells were determined by cell passage through a Coulter Counter Multisizer II. HET-1A cells behaved as osmometers over the range of 280 to 118 mosmol/kg H(2)O with rapid increases in cell volume < or = 15-20% above baseline. Following swelling, the cells exhibited regulatory volume decrease (RVD), restoring baseline volume within 30 min, despite continued hyposmolal stress. With the use of pharmacologic agents and ion substitutions, RVD appeared to result from rapid activation of parallel K(+) and Cl(-) conductance pathways and this was subsequently joined by activation of a KCl cotransporter. Exposure to hyposmolal stress in an acidic environment, pH 6.6, inhibited, but did not abolish, RVD. These data indicate that human esophageal epithelial cells can protect against hyposmolal stress by RVD and that the redundancy in mechanisms may, to some extent, serve as added protection in patients with reflux disease when hyposmolal stress may occur in an acidic environment.

Analysis of Dispersed Phase of Coconut Milk Emulsion

Experiments were conducted to study the dispersed phase of coconut milk emulsion. They were optical microscopy analysis using a Nikon Microscope and particle size analysis using a Coulter Counter Multisizer. Particle size analysis using a Coulter Counter Multisizer on both original coconut milk and homogenized coconut milk at T = 19 °C indicated that they had a wide range of particle size with average value of 5.988 + 1 .0 pm and 6.696 + 1 . 1 pm in diameter respectively. Optical microscopy analysis showed that homogenization of coconut milk after it was heated in a water bath at T = 35 °C for about 15 minutes resulted in changes of particle size, the particle size became smaller. The result lead to a conclusion that the coconut milk emulsion may be considered as a polydisperse emulsion and it indicates that the system should not be sensitive to small variations in preparation or subsequent handling.

Swelling-activated amino acid efflux in the human neuroblastoma cell line CHP-100.

1. The effects of hypoosmotic stress on cell volume and amino acid efflux were evaluated in the human neuroblastoma cell line CHP-100 with the Coulter Counter Multisizer and radiolabeled amino acid efflux, respectively. 2. CHP-100 cells swelled by approximately 35 +/- 5% (means +/- SE) when the osmolarity of the solution was decreased from 290 to 190 mOsm/kg H2O. The rapid swelling was followed by a biphasic regulatory volume decrease (RVD). 3. In cells loaded with 14C-taurine, hypoosmotic stress induced a 300 +/- 22% (n = 23, P < 0.05) increase in taurine efflux compared with controls. This efflux was inhibited by the chloride channel blockers 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), 4,4'-diisothio-cyanostilbene-2,2'-disulfonic acid (DIDS), niflumic acid and by the volume-activated anion channel blocker tamoxifen. In addition, the swelling-activated taurine efflux was dependent upon extracellular calcium. 4. Similarly, in cells loaded with 14C-glycine, hypoosmotic stress significantly increased glycine efflux, which was also sensitive to NPPB. In contrast, efflux of 3H-glutamate was not significantly altered after hypoosmotic stress. 5. With the use of patch clamp recording techniques, Cl- channels were activated in cell attached patches after exposure to hypoosmotic solutions. 6. In nystatin perforated patches, permeability of the hypoosmotically activated anion channel was observed to be SCN- > I- > Br- > Cl- >> Glutamate. 7. It is concluded that in CHP-100 cells, anion channels are activated during hypoosmotic stress and these channels represent a pathway for efflux of amino acids.