Optimisation of chemical lysis protocol for point-of-care leukocyte differentiation

The full blood cell (FBC) count is the most common indicator of diseases. At present hematology analyzers are used for the blood cell characterization, but, recently, there has been interest in using techniques that take advantage of microscale devices and intrinsic properties of cells for increased automation and decreased cost. Microfluidic technologies offer solutions to handling and processing small volumes of blood (2-50 uL taken by finger prick) for point-of-care(PoC) applications. Several PoC blood analyzers are in use and may have applications in the fields of telemedicine, out patient monitoring and medical care in resource limited settings. They have the advantage to be easy to move and much cheaper than traditional analyzers, which require bulky instruments and consume large amount of reagents. The development of miniaturized point-of-care diagnostic tests may be enabled by chip-based technologies for cell separation and sorting. Many current diagnostic tests depend on fractionated blood components: plasma, red blood cells (RBCs), white blood cells (WBCs), and platelets. Specifically, white blood cell differentiation and counting provide valuable information for diagnostic purposes. For example, a low number of WBCs, called leukopenia, may be an indicator of bone marrow deficiency or failure, collagen- vascular diseases, disease of the liver or spleen. The leukocytosis, a high number of WBCs, may be due to anemia, infectious diseases, leukemia or tissue damage. In the laboratory of hybrid biodevices, at the University of Southampton,it was developed a functioning micro impedance cytometer technology for WBC differentiation and counting. It is capable to classify cells and particles on the base of their dielectric properties, in addition to their size, without the need of labeling, in a flow format similar to that of a traditional flow cytometer. It was demonstrated that the micro impedance cytometer system can detect and differentiate monocytes, neutrophils and lymphocytes, which are the three major human leukocyte populations. The simplicity and portability of the microfluidic impedance chip offer a range of potential applications in cell analysis including point-of-care diagnostic systems. The microfluidic device has been integrated into a sample preparation cartridge that semi-automatically performs erythrocyte lysis before leukocyte analysis. Generally erythrocytes are manually lysed according to a specific chemical lysis protocol, but this process has been automated in the cartridge. In this research work the chemical lysis protocol, defined in the patent US 5155044 A, was optimized in order to improve white blood cell differentiation and count performed by the integrated cartridge.

Comparative study of human erythrocytes by digital holographic microscopy, confocal microscopy, and impedance volume analyzer.

Red blood cell (RBC) parameters such as morphology, volume, refractive index, and hemoglobin content are of great importance for diagnostic purposes. Existing approaches require complicated calibration procedures and robust cell perturbation. As a result, reference values for normal RBC differ depending on the method used. We present a way for measuring parameters of intact individual RBCs by using digital holographic microscopy (DHM), a new interferometric and label-free technique with nanometric axial sensitivity. The results are compared with values achieved by conventional techniques for RBC of the same donor and previously published figures. A DHM equipped with a laser diode (lambda = 663 nm) was used to record holograms in an off-axis geometry. Measurements of both RBC refractive indices and volumes were achieved via monitoring the quantitative phase map of RBC by means of a sequential perfusion of two isotonic solutions with different refractive indices obtained by the use of Nycodenz (decoupling procedure). Volume of RBCs labeled by membrane dye Dil was analyzed by confocal microscopy. The mean cell volume (MCV), red blood cell distribution width (RDW), and mean cell hemoglobin concentration (MCHC) were also measured with an impedance volume analyzer. DHM yielded RBC refractive index n = 1.418 +/- 0.012, volume 83 +/- 14 fl, MCH = 29.9 pg, and MCHC 362 +/- 40 g/l. Erythrocyte MCV, MCH, and MCHC achieved by an impedance volume analyzer were 82 fl, 28.6 pg, and 349 g/l, respectively. Confocal microscopy yielded 91 +/- 17 fl for RBC volume. In conclusion, DHM in combination with a decoupling procedure allows measuring noninvasively volume, refractive index, and hemoglobin content of single-living RBCs with a high accuracy.

On-chip non-invasive and label-free cell discrimination by impedance spectroscopy

OBJECTIVES: Many flow-cytometric cell characterization methods require costly markers and colour reagents. We present here a novel device for cell discrimination based on impedance measurement of electrical cell properties in a microfluidic chip, without the need of extensive sample preparation steps and the requirement of labelling dyes. MATERIALS AND METHODS, RESULTS: We demonstrate that in-flow single cell measurements in our microchip allow for discrimination of various cell line types, such as undifferentiated mouse fibroblasts 3T3-L1 and adipocytes on the one hand, or human monocytes and in vitro differentiated dendritic cells and macrophages on the other hand. In addition, viability and apoptosis analyses were carried out successfully for Jurkat cell models. Studies on several species, including bacteria or fungi, demonstrate not only the capability to enumerate these cells, but also show that even other microbiological life cycle phases can be visualized. CONCLUSIONS: These results underline the potential of impedance spectroscopy flow cytometry as a valuable complement to other known cytometers and cell detection systems.

Flow cytometry as a tool in the evaluation of blood leukocyte function in Chelonia mydas (Linnaeus, 1758) (Testudines, Cheloniidae)

Chelonia mydas is a sea turtle that feeds and nests on the Brazilian coast and a disease called fibropapillomatosis is a threat to this species. Because of this, it is extremely necessary to determine a methodology that would enable the analysis of blood leukocyte function in these sea turtles. In order to achieve this aim, blood samples were collected from C. mydas with or without fibropapillomas captured on the São Paulo north coast. Blood samples were placed in tubes containing sodium heparin and were transported under refrigeration to the laboratory in sterile RPMI 1640 cell culture medium. Leukocytes were separated by density gradient using Ficoll-PaqueTM Plus, Amershan Biociences®. The following stimuli were applied in the assessment of leukocyte function: Phorbol Miristate-Acetate (PMA) for oxidative burst activity evaluation and Zymosan A (Saccharomyces cerevisiae) Bio Particles®, Alexa Fluor® 594 conjugate for phagocytosis evaluation. Three cell populations were identified: heterophils, monocytes and lymphocytes. Monocytes were the cells responsible for phagocytosis and oxidative burst.

The influence of adsorption phenomena on the impedance spectroscopy of an electrolytic cell

In this work we investigate the influence of the adsorption of ions on the impedance spectroscopy of an electrolytic cell. We consider that the positive and negative ions present in a dielectric liquid are adsorbed in the electrode surfaces with different adsorption energies. This difference in adsorption energies causes an additional plateaux in the limit of the low-frequency range of the real part of the impedance Z. In the same frequency range, a second minimum in the imaginary part of Z is predicted. The theory is illustrated with measurements of the impedance of an electrolytic solution in the frequency range from 10(-2) Hz to 1 KHz. A comparison between the present model and others from the literature to describe the experimental results is also made.

Further improvements on impedance-based fault location for power distribution systems

In this study, further improvements regarding the fault location problem for power distribution systems are presented. The proposed improvements relate to the capacitive effect consideration on impedance-based fault location methods, by considering an exact line segment model for the distribution line. The proposed developments, which consist of a new formulation for the fault location problem and a new algorithm that considers the line shunt admittance matrix, are presented. The proposed equations are developed for any fault type and result in one single equation for all ground fault types, and another equation for line-to-line faults. Results obtained with the proposed improvements are presented. Also, in order to compare the improvements performance and demonstrate how the line shunt admittance affects the state-of-the-art impedance-based fault location methodologies for distribution systems, the results obtained with two other existing methods are presented. Comparative results show that, in overhead distribution systems with laterals and intermediate loads, the line shunt admittance can significantly affect the state-of-the-art methodologies response, whereas in this case the proposed developments present great improvements by considering this effect.

Dynamic Imaging in Electrical Impedance Tomography of the Human Chest With Online Transition Matrix Identification

One of the electrical impedance tomography objectives is to estimate the electrical resistivity distribution in a domain based only on electrical potential measurements at its boundary generated by an imposed electrical current distribution into the boundary. One of the methods used in dynamic estimation is the Kalman filter. In biomedical applications, the random walk model is frequently used as evolution model and, under this conditions, poor tracking ability of the extended Kalman filter (EKF) is achieved. An analytically developed evolution model is not feasible at this moment. The paper investigates the identification of the evolution model in parallel to the EKF and updating the evolution model with certain periodicity. The evolution model transition matrix is identified using the history of the estimated resistivity distribution obtained by a sensitivity matrix based algorithm and a Newton-Raphson algorithm. To numerically identify the linear evolution model, the Ibrahim time-domain method is used. The investigation is performed by numerical simulations of a domain with time-varying resistivity and by experimental data collected from the boundary of a human chest during normal breathing. The obtained dynamic resistivity values lie within the expected values for the tissues of a human chest. The EKF results suggest that the tracking ability is significantly improved with this approach.

Void fraction measurement and signal analysis from multiple-electrode impedance sensors

Void fraction sensors are important instruments not only for monitoring two-phase flow, but for furnishing an important parameter for obtaining flow map pattern and two-phase flow heat transfer coefficient as well. This work presents the experimental results obtained with the analysis of two axially spaced multiple-electrode impedance sensors tested in an upward air-water two-phase flow in a vertical tube for void fraction measurements. An electronic circuit was developed for signal generation and post-treatment of each sensor signal. By phase shifting the electrodes supplying the signal, it was possible to establish a rotating electric field sweeping across the test section. The fundamental principle of using a multiple-electrode configuration is based on reducing signal sensitivity to the non-uniform cross-section void fraction distribution problem. Static calibration curves were obtained for both sensors, and dynamic signal analyses for bubbly, slug, and turbulent churn flows were carried out. Flow parameters such as Taylor bubble velocity and length were obtained by using cross-correlation techniques. As an application of the void fraction tested, vertical flow pattern identification could be established by using the probability density function technique for void fractions ranging from 0% to nearly 70%.

Recycling Krylov subspaces for efficient large-scale electrical impedance tomography

Electrical impedance tomography (EIT) captures images of internal features of a body. Electrodes are attached to the boundary of the body, low intensity alternating currents are applied, and the resulting electric potentials are measured. Then, based on the measurements, an estimation algorithm obtains the three-dimensional internal admittivity distribution that corresponds to the image. One of the main goals of medical EIT is to achieve high resolution and an accurate result at low computational cost. However, when the finite element method (FEM) is employed and the corresponding mesh is refined to increase resolution and accuracy, the computational cost increases substantially, especially in the estimation of absolute admittivity distributions. Therefore, we consider in this work a fast iterative solver for the forward problem, which was previously reported in the context of structural optimization. We propose several improvements to this solver to increase its performance in the EIT context. The solver is based on the recycling of approximate invariant subspaces, and it is applied to reduce the EIT computation time for a constant and high resolution finite element mesh. In addition, we consider a powerful preconditioner and provide a detailed pseudocode for the improved iterative solver. The numerical results show the effectiveness of our approach: the proposed algorithm is faster than the preconditioned conjugate gradient (CG) algorithm. The results also show that even on a standard PC without parallelization, a high mesh resolution (more than 150,000 degrees of freedom) can be used for image estimation at a relatively low computational cost. (C) 2010 Elsevier B.V. All rights reserved.

Electrochemical impedance spectroscopy investigation of the electrochemical behaviour of copper coated with artificial patina layers and submitted to wet and dry cycles

Due to rain events historical monuments exposed to the atmosphere are frequently submitted to wet and dry cycles. During drying periods wetness is maintained in some confined regions and the corrosion product layer, generally denominated patinas, builds up and gets thicker. The aim of this study is to use electrochemical impedance spectroscopy (EIS) to investigate the electrochemical behaviour of pure copper coated with two artificial patina layers and submitted either to continuous or to intermittent immersion tests, this latter aiming to simulate wet and dry cycles. The experiments were performed in 0.1 mol dm(-3) NaCl solution and in artificial rainwater containing the most significant pollutants of the city of Sao Paulo. The results of the continuous immersion tests in the NaCl solution have shown that the coated samples behave like a porous electrode with finite pore length. On the other hand, in the intermittent tests a porous electrode response with semi-infinite pore length can be developed. The results were interpreted based on the model of de Levie and a critical comparison with previous interpretations reported in the literature for similar systems is presented. (C) 2011 Elsevier Ltd. All rights reserved.

The relationship of flow cytometry results with classical measures of bacterial counts in raw refrigerated milk

Bulk milk was collected from 100 farms throughout the year and analysed after storage for either 24, 48 or 72 h, using flow cytometry. The total bacterial counts obtained by two methods - flow cytometry and standard plate count were compared and the conversion relationship between them was assessed: the results showed no effect of the age of the samples relationship between these two methods.

Bioelectrical impedance analysis for the estimation of body composition in rats

Bioelectrical impedance analysis (BIA) was used to assess body composition in rats fed on either standard laboratory diet or on a high-fat diet designed to induce obesity. Bioelectrical impedance analysis predictions of total body water and thus fat-free mass (FFM) for the group mean values were generally within 5% of the measured values by tritiated water ((H2O)-H-3) dilution. The limits of agreement for the procedure were, however, large, approximately +/-25%, limiting the applicability of the technique for measurement of body composition in individual animals.

Evaluation of multifrequency bioelectrical impedance data for predicting lean tissue mass in beef cattle

Multifrequency bioimpedance analysis has the potential to provide a non-invasive technique for determining body composition in live cattle. A bioimpedance meter developed for use in clinical medicine was adapted and evaluated in 2 experiments using a total of 31 cattle. Prediction equations were obtained for total body water, extracellular body water, intracellular body water, carcass water and carcass protein. There were strong correlations between the results obtained through chemical markers and bioimpedance analysis when determined in cattle that had a wide range of liveweights and conditions. The r(2) values obtained were 0.87 and 0.91 for total body water and extracellular body water respectively. Bioimpedance also correlated with carcass water, measured by chemical analysis (r(2) = 0.72), but less well with carcass protein (r(2) = 0.46). These correlations were improved by inclusion of liveweight and sex as variables in multiple regression analysis. However, the resultant equations were poor predictors of protein and water content in the carcasses of a group of small underfed beef cattle, that had a narrow range of liveweights. In this case, although there was no statistical difference between the predicted and measured values overall, bioimpedance analysis did not detect the differences in carcass protein between the 2 groups that were apparent following chemical analysis. Further work is required to determine the sensitivity of the technique in small underfed cattle, and its potential use in heavier well fed cattle close to slaughter weight.

Potential errors in the application of mixture theory to multifrequency bioelectrical impedance analysis

Potential errors in the application of mixture theory to the analysis of multiple-frequency bioelectrical impedance data for the determination of body fluid volumes are assessed. Potential sources of error include: conductive length; tissue fluid resistivity; body density; weight and technical errors of measurement. Inclusion of inaccurate estimates of body density and weight introduce errors of typically < +/-3% but incorrect assumptions regarding conductive length or fluid resistivities may each incur errors of up to 20%.

Prediction of the chemical composition of lamb carcasses from multi-frequency impedance data

Multi-frequency bioimpedance analysis (MFBIA) was used to determine the impedance, reactance and resistance of 103 lamb carcasses (17.1-34.2 kg) immediately after slaughter and evisceration. Carcasses were halved, frozen and one half subsequently homogenized and analysed for water, crude protein and fat content. Three measures of carcass length were obtained. Diagonal length between the electrodes (right side biceps femoris to left side of neck) explained a greater proportion of the variance in water mass than did estimates of spinal length and was selected for use in the index L-2/Z to predict the mass of chemical components in the carcass. Use of impedance (Z) measured at the characteristic frequency (Z(c)) instead of 50 kHz (Z(50)) did not improve the power of the model to predict the mass of water, protein or fat in the carcass. While L-2/Z(50) explained a significant proportion of variation in the masses of body water (r(2) 0.64), protein (r(2) 0.34) and fat (r(2) 0.35), its inclusion in multi-variate indices offered small or no increases in predictive capacity when hot carcass weight (HCW) and a measure of rib fat-depth (GR) were present in the model. Optimized equations were able to account for 65-90 % of the variance observed in the weight of chemical components in the carcass. It is concluded that single frequency impedance data do not provide better prediction of carcass composition than can be obtained from measures of HCW and GR. Indices of intracellular water mass derived from impedance at zero frequency and the characteristic frequency explained a similar proportion of the variance in carcass protein mass as did the index L-2/Z(50).