Low-volume Method Research Articles

An automated method for thrombocyte counting in capillary microsamples.

We aimed to develop an automated, low-volume method for thrombocyte counting in capillary blood using the Sysmex predilution (PD) mode. Microsamples were prepared by resuspension of 50 μL blood in 300 μL DCL CellPack. Thrombocyte counting was done in the impedance (PLT-I) and fluorescence (PLT-F) channels. The imprecision and bias was evaluated in >394 microsamples from adult blood. Preanalytical factors (skin-piercing, storage, and transportation in our pneumatic tube system) was assessed, and studies on pediatric microsamples were made for comparison. The improvement in analytical quality and turnaround time was examined. For PLT-F, the imprecision was 1.1%-3.7%, and the bias was 10.1% (95% CI: 8.8-11.3). After skin-piercing, the bias was 8.1% (95% CI: 5.6-10.6) and the imprecision 1.9% (95% CI: 1.3-2.5). Thrombocyte counts kept stable after 4 h at room temperature (94.8% [95% CI: 93.2-96.4]) and after pneumatic tube transportation [6.7% (95% CI: 4.8-8.6)]. The bias of the PD mode for pediatric microsamples was 13.0% (95% CI: -8.4-34.4) in the PLT-F channel. The automated method had a considerably lower imprecision than the existing manual thrombocyte counting method and reduced turnaround times. The automated microsample method offers a low-volume alternative for measurement of thrombocytes. The method appears useful also in pediatric samples.

Алгоритмизация технологических режимов автоматизированной системы комбинированного орошения в теплицах с использованием электрохимически активированной воды

Irrigation is one of the main technological processes in greenhouse farming, which essentially provides qualitative and volumetric indicators of produced products. Thus, the combined irrigation technology which combines low-volume methods of drip and aerosol irrigation is widespread. We consider the combined irrigation system which includes an installation for water electro-chemical activation (ECA). Extended abilities of this system are caused by the reactivity of catholyte and anolyte, which are metastable products of water ECA. Catholyte and anolyte are used as growth stimulator and antiseptic respectively in the plant cultivation. Automation of the combined irrigation system, in addition to resource-saving, is also aimed to prevent technological modes violation due to the subjective (“human”) factor. Control algorithms for drip and aerosol irrigation with ordinary water, drip and aerosol irrigation with catholyte and aerosol irrigation with anolyte are described in the article. When the algorithm starts, the control testing is performed to detect possible failures in the irrigation system (for example, rupture of drip tubes or salting of drippers). In case of a detected issue, a diagnostic message is shown and accompanied by a sound signal. More complex technological irrigation modes associated with the use of catholyte and anolyte also include the performance monitoring of the water electrochemically activation unit. The control of the irrigation technological parameters (soil moisture, air humidity, soil pH) is carried out by retrieving the relevant sensors data and comparing the obtained values with the set ones. When the set points are reached the relevant technological links (electric pumps and electromagnetic valves) are switched off by the signal of the microcontroller. The presented algorithms are designed for developing the microcontroller control unit software for the combined irrigation system.

A Simple Microextraction Method for Toxic Industrial Dyes Using a Fatty-Acid Solvent Mixture

A mixture of dodecanoic and hexanoic fatty acids was used to perform a simple and efficient microextraction method for industrial dyes such as methylene blue (MB), methyl violet (MV), and malachite green (MG) in aqueous solution. The fatty-acid microextractants were simply mixed and heated until the mixture became homogeneous before adding it to the dye solutions. The fatty-acid solvent and its components were characterized with Fourier transform infrared spectroscopy (FTIR) and proton nuclear magnetic resonance (1H NMR) measurements, while the dye concentrations were measured using UV-Vis spectroscopy. The performance of the extracting mixture was observed to vary across different dye contaminants, dosages of the extractant, concentrations of the dyes, and contact times. High extraction efficiencies of up to ~99% were obtained for MG as well as MV, and ~73% efficiency was achieved for MB. The study shows how a mixture of fatty acids can be used as a simple, efficient, green, and sustainable low-volume method for the removal of toxic industrial dyes in aqueous solutions.

Quantification of Nonpersistent Pesticides in Small Volumes of Human Breast Milk with Ultrahigh Performance Liquid Chromatography Coupled to Tandem Mass Spectrometry.

Existing methods for the analysis of pesticides in human breast milk involve multiple extraction steps requiring large sample and solvent volumes, which can be a major obstacle in large epidemiologic studies. Here, we developed a simple, low-volume method for extracting organophosphates, pyrethroids, carbamates, atrazine, and imidacloprid from 100 to 200 μL of human breast milk. Multiple extraction protocols were tested including microwave-assisted acid/base digestion and double-solvent extraction with 2 or 20 mL of 2:1 (v/v) dichloromethane/hexane, with or without subsequent solid-phase extraction (SPE) cleanup. Samples were analyzed by liquid chromatography tandem mass spectrometry. Analyte recoveries and reproducibility were highest when 100-200 μL of milk were extracted with 2 mL of dichloromethane/hexane without subsequent SPE steps. Analysis of 79 breast milk samples using this method revealed the presence of carbamates, organophosphates, pyrethroids, and imidacloprid at detection frequencies of 79-96, 53-90, 1-7, and 61%, respectively. This study demonstrates the feasibility of a simple low-volume extraction method for measuring pesticides in human breast milk.

Whole-Body High-Pitch CT Angiography: Strategies to Reduce Radiation Dose and Contrast Volume.

The purpose of this study was to assess the noninferiority of dual-source high-pitch CT angiography (CTA) performed with high-concentration (iopamidol 370) low-volume (60 mL) iodinated contrast material at low voltage (100 kVp) in comparison with dual-source high-pitch CTA with standard-of-care low-concentration (iopamidol 300) standard-volume (75 mL) iodinated contrast material at high voltage (120 kVp) to determine whether use of the high-concentration low-volume method would afford a reduction in radiation dose and contrast volume without negatively affecting vascular opacification. This study had three arms. A phantom was used to assess vascular contrast enhancement at different iodine and saline solution dilutions with iopamidol 300 or 370 to compare lower-iodination (iopamidol 300) high-voltage (120 kVp) high-pitch (120 kVp, 250 mAs) imaging with higher-iodination (iopamidol 370) low-voltage (100 kVp) high-pitch (100 kVp, 100-240 mAs) acquisition. Metal-oxide-semiconductor field-effect transistors were placed in an anthropomorphic phantom to extract organ-based radiation profiles, and ANOVA was performed. The study prospectively enrolled 150 patients: 50 patients received 75 mL iopamidol 300, and image acquisition was performed at 120 kVp and 250 mAs; 50 patients received 75 mL iopamidol 370, and acquisition was performed at 100 kVp and 240 mAs; and 50 patients received 60 mL iopamidol, and acquisition was performed at 370 at 100 kVp and 240 mAs. Vascular signal-to-noise ratio was evaluated at 18 anatomic locations. Longitudinal signal-to-noise ratio was used to assess homogeneity of contrast enhancement. Size-specific dose estimates were calculated. Statistical analyses were performed by ANOVA. Noninferiority of high-concentration (iopamidol 370) low-voltage (100 kVp) high-pitch acquisitions compared with low-concentration (iopamidol 300) high-voltage (120 kVp) high-pitch acquisition was achieved at 170 mAs in vitro. Radiation assessment showed significant decreases in radiation dose for the 100-kVp 240-mAs protocol (p < 0.0001). Noninferior vascular contrast (p > 0.280) and luminal homogeneity (p > 0.191) were found for all high-pitch protocols. Significantly decreased radiation dose was observed for the two groups that received 60 and 75 mL of iopamidol 370 at 100 kVp and 240 mAs (p < 0.0001). Dual-source high-pitch CTA with high-concentration (iopamidol 370) low-volume (60 mL) iodinated contrast medium and low-voltage acquisition (100 kVp) is noninferior to dual-source high-pitch CTA with low-concentration (iopamidol 300) standard-volume (75 mL) iodinated contrast material at high voltage (120 kVp) and affords simultaneous reduction in radiation dose and contrast volume without negatively affecting vascular contrast enhancement.

Validation of Reduced Reagent Volumes in the Implementation of the Quantifiler® Trio Quantification Kit.

The Quantifiler® Trio Quantification Kit has been developed to quantify the total amount of amplifiable and human male DNA in samples and to estimate the extent of DNA degradation. To minimize the cost of DNA quantification, we evaluated kit performance using a reduced volume of reagents (1/10-volume) using DNA samples of varying types and concentrations. Our results demonstrated concordance between the manufacturer's method and the low-volume method for DNA quantification, DNA degradation index estimation, and human male DNA quantification. We confirmed the practical utility of the low-volume method with 109 casework samples by evaluating short tandem repeat (STR) profiling success with respect to DNA quantity and quality. We also defined a cutoff value for DNA quantity to ensure reliable STR results. Using a reduced volume of reagents, 10 times more reactions per kit are possible; accordingly, this method reduces the cost of DNA quantification, while maintaining performance.

Evaluation Effective Low-Volume Cardioplegia By «Custodiol» Solution

Objectives. The study presents the results of quality assessment of antiischemic myocardial protection by low-volume method of cardioplegia by Custodiol solution during operations with cardiopulmonary bypass. Material and Methods. The study involved 57 patients who underwent different cardiosurgery operations with cardiopulmonary bypass. The patients were divided into two groups on the basis of the volume of cardioplegic solution. Research group (group 1) consists of 33 patients who were administered Custodiol in low volume limited by 1000 ml. The control group (group 2) consists of 24 patients who were administered standart volume of the solution corresponding to the manufacturer’s instructions: 1 ml per 1 g of myocardial mass during 6–8 minutes. Results. The volume of Custodiol administered per 1 kg of body weight was 12.7 ± 2.0 and 22.7 ± 4 ml and 4.0 ± 1.3 and 6.3 ± 1.9 ml per 1 g of myocardial mass in groups 1 and 2, respectively (p &lt; 0.001). The frequency of catecholamines usage, temporary cardiac pacing and other adverse events with a small-volume cardioplegia technique with Custodiol solution did not differ significantly. The results has indicated that the use of low volume of cardioplegic Custodiol in a single administration manner provides a complete antiischemic protection of the myocardium during the correction of valvular heart disease, including combination with coronary artery bypass grafting. Conclusion. Usage of low volume Custodiol solution method does not increase the need of inotropic and vasopressor usage and pacing time.

Downscaling procedures reduce chemical use in androgen receptor reporter gene assay

Bioactivity screening studies often face sample amount limitation with respect to the need for reliable, reproducible and quantitative results. Therefore approaches that minimize sample use are needed. Low-volume exposure and chemical dilution procedures were applied in an androgen receptor reporter gene human cell line assay to evaluate environmental contaminants and androgen receptor modulators, which were the agonist 5α-dihydrotestosterone (DHT); and the antagonists flutamide, bisphenol A, 1-hydroxypyrene and triclosan. Cells were exposed in around 1/3 of the medium volume recommended by the protocol (70μL/well). Further, chemical losses during pipetting steps were minimized by applying a low-volume method for compound dilution in medium (250μL for triplicate wells) inside microvolume glass inserts. Simultaneously, compounds were evaluated following conventional procedures (200μL/well, dilution in 24-well plates) for comparison of results. Low-volume exposure tests produced DHT EC50 (3.4–3.7×10−10M) and flutamide IC50 (2.2–3.3×10−7M) values very similar to those from regular assays (3.1–4.2×10−10 and 2.1–3.3×10−7M respectively) and previous studies. Also, results were within assay acceptance criteria, supporting the relevance of the downscaling setup for agonistic and antagonistic tests. The low-volume exposure was also successful in determining IC50 values for 1-hydroxypyrene (2.1–2.8×10−6M), bisphenol A (2.6–3.3×10−6M), and triclosan (1.2–1.9×10−6M) in agreement with values obtained through high-volume exposure (2.3–2.8, 2.5–3.4 and 1.0–1.3×10−6M respectively). Finally, experiments following both low-volume dosing and exposure produced flutamide and triclosan IC50 values similar to those from regular tests. The low-volume experimental procedures provide a simple and effective solution for studies that need to minimize bioassay sample use while maintaining method reliability. The downscaling methods can be applied for the evaluation of samples, fractions or chemicals which require minimal losses during the steps of pipetting, transference to medium and exposure in bioassays.

Pharmaceutical feasibility of sub-visible particle analysis in parenterals with reduced volume light obscuration methods

The draft for a new United States Pharmacopoeia (USP) monograph 〈787〉 “Sub-visible Particulate Matter in Therapeutic Protein Injections” describes the analysis of sub-visible particles by light obscuration at much lower sample volumes as so far required by the European Pharmacopoeia (Ph. Eur.) and the USP for parenterals in general. Our aim was to show the feasibility of minimizing the sample expenditure required for light obscuration similar to the new USP settings for standards and pharmaceutically relevant samples (both proteins and small molecules), without compromising the data quality. The light obscuration method was downscaled from >20ml volume as so far specified in Ph. Eur./USP to 1ml total sample volume. Comparable results for the particle concentration in all tested size classes were obtained with both methods for polystyrene standards, stressed BSA solutions, recombinant human IgG1 formulations, and pantoprazol i.v. solution. An additional advantage of the low volume method is the possibility to detect vial-to-vial variations, which are leveled out when pooling several vials to achieve sufficient volume for the Ph. Eur./USP method. This is in particular important for biotech products where not only the general quality aspect, but also aggregate formation of the drug substance is monitored by light obscuration.

A Novel, Low-Volume Method for Organ Culture of Embryonic Kidneys That Allows Development of Cortico-Medullary Anatomical Organization

Here, we present a novel method for culturing kidneys in low volumes of medium that offers more organotypic development compared to conventional methods. Organ culture is a powerful technique for studying renal development. It recapitulates many aspects of early development very well, but the established techniques have some disadvantages: in particular, they require relatively large volumes (1–3 mls) of culture medium, which can make high-throughput screens expensive, they require porous (filter) substrates which are difficult to modify chemically, and the organs produced do not achieve good cortico-medullary zonation. Here, we present a technique of growing kidney rudiments in very low volumes of medium–around 85 microliters–using silicone chambers. In this system, kidneys grow directly on glass, grow larger than in conventional culture and develop a clear anatomical cortico-medullary zonation with extended loops of Henle.

Heat-treatment method for producing fatty acid-bound alpha-lactalbumin that induces tumor cell death

HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells), which was identified in human breast milk as an alpha-lactalbumin (LA)–oleic acid complex, kills tumor cells, selectively. Although it may have potential as a therapeutic agent against various tumor cells, only low-volume methods for its production exist. In this study, heat treatment was used to produce complexes from LAs and oleic acid using a simple method. In the case of human LA and oleic acid, heat-treated samples apparently showed much stronger activities than those treated at room temperature, with cytotoxicities equal to that of HAMLET. Furthermore, circular dichroism spectroscopy revealed that heat-treated samples lost their tertiary structure, suggesting a molten globule as oleic acid-bound LA. BLA samples also showed strong activities by heat treatment. Batch production with heat treatment can efficiently convert LAs into tumoricidal complexes.

Development of a low volume plasma sample precipitation procedure for liquid chromatography/tandem mass spectrometry assays used for drug discovery applications

The demand for high sensitivity bioanalytical methods has dramatically increased in the drug discovery stage; in addition, there has been a growing trend of reducing the sample volume that is required for these assays. A sensitive high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) procedure has been developed and tested to meet these needs. The assay requires only a low plasma sample volume (10 microL) and employs a protein precipitation procedure using a 1:6 plasma/acetonitrile ratio. The supernatant is injected directly into the LC/MS/MS system using the selected reaction monitoring (SRM) procedure for detection. A generic HPLC gradient based on a methanol/water mobile phase with a flow rate set to 0.8 mL/min was used. The test method showed very good linearity between 0.1-1000 ng/mL (R2 = 0.9737), precision (%RSD = 6-9), accuracy (%RE = -2) and reproducibility (%RSD = 11). A drug discovery IV/PO study was assayed using both the new low volume method and our standard volume (50 microL) method. The correlation of the two sets of data from the two methods was excellent (R2 = 0.9287). This new assay procedure has been successfully used in our laboratory for over 100 different rat or mouse discovery PK studies.

The use of low-volume dosing in the eye irritation test

The Draize rabbit eye test was developed to provide a method for assessing the irritation potential of materials that might come in contact with human eyes. The method involves the instillation of 0.1 ml of a test liquid (100 mg solid) into the conjunctival sac of an animal's eye. A refinement of the Draize test is the low-volume eye test in which 0.01 ml of a substance is placed directly on the cornea of the eye. Studies indicate that the low-volume method provides a better correlation to human eye irritation experience for some substances. The Interagency Regulatory Alternatives Group (IRAG) proposes that the low-volume eye test can be used to substantiate the irritancy of suspect severe ocular irritants that have not been eliminated by various pre-eye test ‘screens’. A substance testing positive by the low-volume method can be classified as an irritant; one that tests negative will require further testing by the use of the 0.1-ml volume procedure. For all other definitive testing, the Draize test (0.1 ml) should be used. Results from a questionnaire distributed at the IRAG workshop showed that many workshop participants thought that the low-volume test should be used as an eye irritation screening procedure.

Correlation of Animal Eye Test Data with Human Experience for Household Products: An Update

AbstractAccidental consumer eye exposures to household products provide useful data for determining whether animal tests adequately predict human response. Reports of accidental consumer exposures from mid-1983 through 1984 were compared to two animal methods and to the results of a similar analysis for the period 1979–1980. The new comparison confirmed the earlier analysis in showing that the low-volume rabbit eye test is more predictive of human eye response than the Federal Health and Safety Administration (modified Draize) test. Furthermore, the data show that rigorous follow-up after consumer accidents can provide useful information for assessing the validity of animal studies used to estimate human safety. Finally, because the low-volume method correlates better with human eye effects than the Draize method does for household products, it is the method that should be used to validate any new in vitro eye testing procedures. In fact, any laboratory test developed to assess a health or safety effect m...

An Appraisal of High Medium and Low Volume Methods of Spray Application

An Appraisal of High Medium and Low Volume Methods of Spray Application