Array Of Microfluidic Channels Research Articles

Stretchable Sensors for Soft Robotic Grippers in Edge-Intelligent IoT Applications.

The rapid development of electronic material and sensing technology has enabled research to be conducted on liquid metal-based soft sensors. The application of soft sensors is widespread and has many applications in soft robotics, smart prosthetics, and human-machine interfaces, where these sensors can be integrated for precise and sensitive monitoring. Soft sensors can be easily integrated for soft robotic applications, where traditional sensors are incompatible with robotic applications as these types of sensors show large deformation and very flexible. These liquid-metal-based sensors have been widely used for biomedical, agricultural and underwater applications. In this research, we have designed and fabricated a novel soft sensor that yields microfluidic channel arrays embedded with liquid metal Galinstan alloy. First of all, the article presents different fabrication steps such as 3D modeling, printing, and liquid metal injection. Different sensing performances such as stretchability, linearity, and durability results are measured and characterized. The fabricated soft sensor demonstrated excellent stability and reliability and exhibited promising sensitivity with respect to different pressures and conditions.

A Microfluidic Flip-Chip Combining Hydrodynamic Trapping and Gravitational Sedimentation for Cell Pairing and Fusion.

Cancer cell–immune cell hybrids and cancer immunotherapy have attracted much attention in recent years. The design of efficient cell pairing and fusion chips for hybridoma generation has been, subsequently, a subject of great interest. Here, we report a three-layered integrated Microfluidic Flip-Chip (MFC) consisting of a thin through-hole membrane sandwiched between a mirrored array of microfluidic channels and saw-tooth shaped titanium electrodes on the glass. We discuss the design and operation of MFC and show its applicability for cell fusion. The proposed device combines passive hydrodynamic phenomenon and gravitational sedimentation, which allows the transportation and trapping of homotypic and heterotypic cells in large numbers with pairing efficiencies of 75~78% and fusion efficiencies of 73%. Additionally, we also report properties of fused cells from cell biology perspectives, including combined fluorescence-labeled intracellular materials from THP1 and A549, mixed cell morphology, and cell viability. The MFC can be tuned for pairing and fusion of cells with a similar protocol for different cell types. The MFC can be easily disconnected from the test setup for further analysis.

Note: Multi-sheet light enables optical interference lithography

We propose and demonstrate a modified spatial filter-based single-shot lithography technique for fabricating an array of microfluidic channels. This is achieved by illuminating the photopolymer specimen with a multiple light sheet (MLS) pattern. Modified spatial filtering is employed in a cylindrical lens system to generate the MLS pattern. The transmission window [the difference (α - β) angle] of the spatial filter determines the characteristics of the pattern and the fabricated microfluidic channel array. After exposing to a negative photoresist (DPHPA monomer with rose bengal as the photoinitiator), this gives rise to an array of micro-fluidic channels (post development process). We studied the effect of micro-channel geometry (channel width, inter-channel separation, and aspect ratio) for varying exposure times that show near-linear dependence. The results show that the fabricated array has 7 prominent channels with an individual channel width and inter-channel separation of approximately 5 μm and 12 μm, respectively. The proposed technique enables selective plane patterning and reduces the overall cost for large-scale production.

Free-standing hydrogel-particle composite membrane with dynamically controlled permeability.

The preparation and investigation of a free-standing membrane made from a composite of thermoresponsive poly(N-isopropylacrylamide) (pNIPAAm) and polystyrene nanoparticles (PS NP) with temperature-controlled permeability is reported. The method exploits the light-induced crosslinking of the photo-reactive pNIPAAm-based polymer and mechanical reinforcement of the membrane structure by the polystyrene nanoparticles. About micrometer thick layers were either directly attached to a gold surface or prepared as free-standing layers spanning over arrays of microfluidic channels with a width of about hundred microns by using template stripping. Diffusion of liquid medium, low molecular weight molecules, and large molecular weight proteins contained in blood through the composite membrane was observed with combined surface plasmon resonance (SPR) and optical waveguide spectroscopy (OWS). The swelling ratio, permeability, and nonspecific sorption to these composite membranes were investigated by SPR and OWS as a function of molecular weight of analyte, loading of PS NP in the composite film, and temperature. The authors show successful preparation of a defect-free membrane structure that acts as a thermoresponsive filter with nanoscale pores spanning over an area of several square millimeters. This membrane can be reversibly switched to block or allow the diffusion of low mass molecules to the sensor surface by temperature-triggered swelling and collapsing of the hydrogel component. Blocking of diffusion and low unspecific sorption of proteins contained in blood serum is observed. These features make this platform interesting for potential future applications in continuous monitoring biosensors for the analysis of low molecular weight drug analytes or for advanced cell-on-chip microfluidic studies.

Micro-Viscometer for Measuring Shear-Varying Blood Viscosity over a Wide-Ranging Shear Rate.

In this study, a micro-viscometer is developed for measuring shear-varying blood viscosity over a wide-ranging shear rate. The micro-viscometer consists of 10 microfluidic channel arrays, each of which has a different micro-channel width. The proposed design enables the retrieval of 10 different shear rates from a single flow rate, thereby enabling the measurement of shear-varying blood viscosity with a fixed flow rate condition. For this purpose, an optimal design that guarantees accurate viscosity measurement is selected from a parametric study. The functionality of the micro-viscometer is verified by both numerical and experimental studies. The proposed micro-viscometer shows 6.8% (numerical) and 5.3% (experimental) in relative error when compared to the result from a standard rotational viscometer. Moreover, a reliability test is performed by repeated measurement (N = 7), and the result shows 2.69 ± 2.19% for the mean relative error. Accurate viscosity measurements are performed on blood samples with variations in the hematocrit (35%, 45%, and 55%), which significantly influences blood viscosity. Since the blood viscosity correlated with various physical parameters of the blood, the micro-viscometer is anticipated to be a significant advancement for realization of blood on a chip.

Light‐sheet based lithography technique for patterning an array of microfluidic channels

We propose a Light-sheet laser interference lithography technique for fabricating periodic microfluidic channels. This technique uses multiple light-sheet illumination pattern that is generated using a spatial filter at the back-aperture of the cylindrical lens. Specially designed spatial filter is used that give rise to a periodic pattern at the focal plane which is essentially a 1D Fourier transform of the spatial filter transfer function. One-dimensional focusing property of the cylindrical lens result in the generation of line shaped channel geometry. To design microfluidic channels, the illumination pattern is exposed to the glass substrate coated with a photopolymer sensitized to 532 nm and subsequently developed using standard chemical protocols. Experimentally, the 1D periodic channel structure has an approximate width and periodicity of approximately 11.25 microns. Light-sheets based lithography technique offer a fast and single-shot process to generate microfluidic channels.

Soft capacitive tactile sensing arrays fabricated via direct filament casting

Advances in soft electronics are enabling the development of mechanical sensors that can conform to curved surfaces or soft objects, allowing them to interface seamlessly with the human body. In this paper, we report on intrinsically deformable tactile sensing arrays that achieve a unique combination of high spatial resolution, sensitivity, and mechanical stretchability. The devices are fabricated via a casting process that yields arrays of microfluidic channels in low modulus polymer membranes with thickness as small as one millimeter. Using liquid metal alloy as a conductor, we apply matrix-addressed capacitive sensing in order to resolve spatially distributed strain with millimeter precision over areas of several square centimeters. Due to the use of low-modulus polymers, the devices readily achieve stretchability greater than 500%, making them well suited for novel applications in wearable tactile sensing for biomedical applications.

Development of a Label-Free Electrochemical Immunosensor for the Detection of Prostate Cancer

Prostate cancer or carcinoma of the prostate is one of the most frequently diagnosed malignancies in the world. Prostate cancer is the second leading cause of cancer death in men worldwide and the fourth most common cancer overall, with more than 1.1 million new cases diagnosed in 2012, and more than 307,000 deaths from the disease1. The prevalence of the condition and mortality will clearly vary from one country to another but tends to be higher in developed countries2. Prostate cancer incidence is strongly linked to age with the lowest incidence rates being in younger men. In the UK between 2009 and 2011, only 1% of cases were diagnosed in men under 50 and an average of 36% of cases was diagnosed in men aged 75 years and over1. In a population with increasing longevity, it is likely that prostate cancer will become even more clinically prevalent in the future. This projected increase is a major concern for the public health sector, especially when there are both problems associated with the detection and the treatment of prostate cancer. Specifically, current practice in prostate cancer and staging leads to inaccurate assessments often resulting in unwanted or even unnecessary treatments that adversely affect the patient’s quality of life with little gain. It will also place a considerable burden on the healthcare provider. The presentation will detail a new approach to the development of microfluidic paper-based electrochemical devices that will be capable of label-free multiparametric detection. Rather than examining single parameter detection, the strategy adopted here involves the laser machining of cellulose based substrates to create an array of microfluidic channels and detection wells as detailed in Figure 1. Screen printed electrodes functionalised with gold nanoparticles onto which a variety of molecular recognition elements (enzyme or antibody) are immobilised, serve as the principal detection elements. These can be independently addressed thereby providing a system which is capable of screening a panel of biomarkers relevant to the diagnosis of prostate cancer. An innovative approach to the detection methodology is taken where the use of a nitro functionalised biotin – streptavidin conjugate is employed as a redox probe through which to assess the degree of antibody-antigen binding events at the electrode array. The use of Label-free detection over traditional methods such as enzyme-linked immunosorbent assay (ELISA) provides several advantages including speed, simplicity, low cost and sensitivity, and also lays the foundations for the development of a point of care device. The design, development and bioanalytical characterisation of the prototype sensing systems will be presented and the applicability to point of care diagnosis critically assessed. 1.UK, C. R. Prostate cancer mortality statistics.(2014); 2.Ferlay J et al. GLOBOCAN 2012 v1.0, Cancer Incidence and Mortality Worldwide: IARC CancerBase No.11. Lyon, France: International Agency for Research on Cancer; 2013. Figure 1

Deformability measurement of red blood cells using a microfluidic channel array and an air cavity in a driving syringe with high throughput and precise detection of subpopulations.

Red blood cell (RBC) deformability has been considered a potential biomarker for monitoring pathological disorders. High throughput and detection of subpopulations in RBCs are essential in the measurement of RBC deformability. In this paper, we propose a new method to measure RBC deformability by evaluating temporal variations in the average velocity of blood flow and image intensity of successively clogged RBCs in the microfluidic channel array for specific time durations. In addition, to effectively detect differences in subpopulations of RBCs, an air compliance effect is employed by adding an air cavity into a disposable syringe. The syringe was equally filled with a blood sample (V(blood) = 0.3 mL, hematocrit = 50%) and air (V(air) = 0.3 mL). Owing to the air compliance effect, blood flow in the microfluidic device behaved transiently depending on the fluidic resistance in the microfluidic device. Based on the transient behaviors of blood flows, the deformability of RBCs is quantified by evaluating three representative parameters, namely, minimum value of the average velocity of blood flow, clogging index, and delivered blood volume. The proposed method was applied to measure the deformability of blood samples consisting of homogeneous RBCs fixed with four different concentrations of glutaraldehyde solution (0%-0.23%). The proposed method was also employed to evaluate the deformability of blood samples partially mixed with normal RBCs and hardened RBCs. Thereafter, the deformability of RBCs infected by human malaria parasite Plasmodium falciparum was measured. As a result, the three parameters significantly varied, depending on the degree of deformability. In addition, the deformability measurement of blood samples was successfully completed in a short time (∼10 min). Therefore, the proposed method has significant potential in deformability measurement of blood samples containing hematological diseases with high throughput and precise detection of subpopulations in RBCs.

Fluorescence enhancement and multiple protein detection in ZnO nanostructure microfluidic devices.

In this study, different morphological ZnO nanostructures, those of sharp nanowires (NWs), rod NWs, and hexahedral-puncheon nanostructures, were grown in microfluidic channels on the same glass substrate. Characterizations of correspondent biomolecule binding properties were simulated and demonstrated. The surface was modified using 3-ammineopropyl-triethoxysilane (3-APTES) and biotin-N-hydroxysuccinimide ester (NHS-biotin). Different concentrations (4.17pM to 41.7nM) of dye-conjugated streptavidin were simultaneously infused through the second microfluidic channels, which lie 90° from the first microfluidic channels. The florescent intensity at the crossover areas showed good agreement with simulations, with sharp ZnO NWs exhibiting the largest dynamic range and the highest fluorescent intensity. We further characterize correspondent protein detection using sharp ZnO NWs. The surfaces of these ZnO NWs were modified with mouse immunoglobulin G (IgG), infused through the second microfluidic channels with dye-conjugated (Alexa 546) anti-mouse IgG in different concentrations. Concentrations ranging from 417fM to 41.7nM can be resolved using sharp ZnO NWs. Finally, multiple protein detection was demonstrated using a five-by-eight microfluidic channel array. Fluorescence images present clear multiple detections at the crossover areas when using the sharp ZnO NWs for simultaneous dye-conjugated anti-mouse IgG and dye-conjugated anti-rabbit IgG (Alexa 647) detection.

Rapid homogeneous endothelialization of high aspect ratio microvascular networks.

Microvascularization of an engineered tissue construct is necessary to ensure the nourishment and viability of the hosted cells. Microvascular constructs can be created by seeding the luminal surfaces of microfluidic channel arrays with endothelial cells. However, in a conventional flow-based system, the uniformity of endothelialization of such an engineered microvascular network is constrained by mass transfer of the cells through high length-to-diameter (L/D) aspect ratio microchannels. Moreover, given the inherent limitations of the initial seeding process to generate a uniform cell coating, the large surface-area-to-volume ratio of microfluidic systems demands long culture periods for the formation of confluent cellular microconduits. In this report, we describe the design of polydimethylsiloxane (PDMS) and poly(glycerol sebacate) (PGS) microvascular constructs with reentrant microchannels that facilitates rapid, spatially homogeneous endothelial cell seeding of a high L/D (2 cm/35 μm; > 550:1) aspect ratio microchannels. MEMS technology was employed for the fabrication of a monolithic, elastomeric, reentrant microvascular construct. Isotropic etching and PDMS micromolding yielded a near-cylindrical microvascular channel array. A 'stretch - seed - seal' operation was implemented for uniform incorporation of endothelial cells along the entire microvascular area of the construct yielding endothelialized microvascular networks in less than 24 h. The feasibility of this endothelialization strategy and the uniformity of cellularization were established using confocal microscope imaging.

Manufacturing a nanowire-based sensing system via flow-guided assembly in a microchannel array template

A novel flow-guided assembly approach is presented to accurately align and position nanowire arrays in pre-defined locations with high throughput and large-scale manufacturing capability. In this approach, a polymer solution is first filled in an array of microfluidic channels. Then a gas flow is introduced to blow out most of the solution while pushing a little leftover against the channel wall for assembly into polymer nanowires. In this way, highly ordered nanowires are conveniently aligned in the flow direction and patterned along both sides of the microchannels. In this study, we demonstrated this flow-guided assembly process by producing millimetre-long nanowires across a 5 × 12 mm area in the same orientation and with basic ‘I-shape’, ‘T-shape’, and ‘cross’ patterns. The assembled polymer nanowires were further converted to conductive carbon nanowires through a standard carbonization process. After being integrated into electronic sensors, high sensitivity was found in model protein sensing tests. This new nanowire manufacturing approach is anticipated to open new doors to the fabrication of nanowire-based sensing systems and serve as good manufacturing practice for its simplicity, low cost, alignment reliability, and high throughput.

Multiplex serum cytokine immunoassay using nanoplasmonic biosensor microarrays.

Precise monitoring of the rapidly changing immune status during the course of a disease requires multiplex analysis of cytokines from frequently sampled human blood. However, the current lack of rapid, multiplex, and low volume assays makes immune monitoring for clinical decision-making (e.g., critically ill patients) impractical. Without such assays, immune monitoring is even virtually impossible for infants and neonates with infectious diseases and/or immune mediated disorders as access to their blood in large quantities is prohibited. Localized surface plasmon resonance (LSPR)-based microfluidic optical biosensing is a promising approach to fill this technical gap as it could potentially permit real-time refractometric detection of biomolecular binding on a metallic nanoparticle surface and sensor miniaturization, both leading to rapid and sample-sparing analyte analysis. Despite this promise, practical implementation of such a microfluidic assay for cytokine biomarker detection in serum samples has not been established primarily due to the limited sensitivity of LSPR biosensing. Here, we developed a high-throughput, label-free, multiarrayed LSPR optical biosensor device with 480 nanoplasmonic sensing spots in microfluidic channel arrays and demonstrated parallel multiplex immunoassays of six cytokines in a complex serum matrix on a single device chip while overcoming technical limitations. The device was fabricated using easy-to-implement, one-step microfluidic patterning and antibody conjugation of gold nanorods (AuNRs). When scanning the scattering light intensity across the microarrays of AuNR ensembles with dark-field imaging optics, our LSPR biosensing technique allowed for high-sensitivity quantitative cytokine measurements at concentrations down to 5-20 pg/mL from a 1 μL serum sample. Using the nanoplasmonic biosensor microarray device, we demonstrated the ability to monitor the inflammatory responses of infants following cardiopulmonary bypass (CPB) surgery through tracking the time-course variations of their serum cytokines. The whole parallel on-chip assays, which involved the loading, incubation, and washing of samples and reagents, and 10-fold replicated multianalyte detection for each sample using the entire biosensor arrays, were completed within 40 min.

Integrating nanopore sensors within microfluidic channel arrays using controlled breakdown.

Nanopore arrays are fabricated by controlled dielectric breakdown (CBD) in solid-state membranes integrated within polydimethylsiloxane (PDMS) microfluidic devices. This technique enables the scalable production of independently addressable nanopores. By confining the electric field within the microfluidic architecture, nanopore fabrication is precisely localized and electrical noise is significantly reduced. Both DNA and protein molecules are detected to validate the performance of this sensing platform.

A combined microfluidic-microstencil method for patterning biomolecules and cells.

Despite the myriad of soft lithography based micropatterning methods available to researchers, it is still challenging to define small features (10-100 μm) that are spaced far apart (1-10 mm). In this report, we describe a combined microfluidic-microstencil patterning method that can produce multifunctional substrates of small features, O(10 μm), with a large pitch, O(1 mm). In that, we fabricate microstencils using an UV curable polyurethane (Norland Optical Adhesive 81) with dense arrays of 10-100 μm holes. Overlaying arrays of microfluidic channels over these microstencils allow for the control of the spacing between features and the ability to pattern multiple substrates. We show that this method is capable of patterning soluble proteins, fibrillar insoluble collagen, liposomes, cells, and nanoparticles. We demonstrate the utility of the method by measuring platelet adhesion under flow to three adhesive proteins (insoluble fibrillar collagen, laminin, and reconstituted acid solubilized collagen fibers) in a single assay.

Microfluidic artificial “vessels” for dynamic mechanical stimulation of mesenchymal stem cells

Cells in the cardiovascular system are constantly exposed to complex mechanical stimulation due to the pulsatile nature of blood flow and the haemodynamic forces that are key to the regulation of vascular development, remodeling and pathophysiology. Mechanical stretch can also modulate the differentiation of stem cells toward vascular cell lineages (i.e., vascular smooth muscle cells), and represent a critical factor in vascular tissue engineering. Here we report on the development of a microchip platform that can emulate several key aspects of the vascular mechanical environment, such as cyclic stimulation and circumferential strain. This chip consists of an array of microfluidic channels with widths ranging from 20 to 500 micrometers. These channels are covered by suspended deformable membranes, on which cells are cultured and stimulated by cyclic circumferential strain of up to 20% via hydrodynamic actuation of the fluid in the microfluidic channels, thereby mimicking the biomechanical conditions of small blood vessels. We show that human mesenchymal stem cells (MSCs) can be cultured and continuously stimulated by cyclic stretch over a period of 7 days with no evidence of device fatigue or performance degradation. We observed localization and alignment of MSCs when mechanical stretch is larger than 10%, indicating the importance of mechanical stimulation in modulating cellular behavior. We further demonstrated simultaneous detection of proteins in multiple signaling pathways, including SMAD1/SMAD2 and canonical Wnt/β-catenin. This microchip represents a generic and versatile platform for high-throughput and rapid screening of cellular responses, including signal transduction cascades, in response to mechanical cues. The system emulates the physiological conditions of blood vessels and other tissues that are subject to cyclic strain, and may have a wide range of applications in the fields of stem cell mechanobiology, vascular tissue engineering, and other areas of regenerative medicine.

Formation of lipid bilayers inside microfluidic channel array for monitoring membrane-embedded nanopores of phi29 DNA packaging nanomotor

An efficient method to form lipid bilayers inside an array of microfluidic channels has been developed and applied to monitor the membrane-embedded phi29 DNA packaging motor with an electrochemical characterization on a lab-on-a-chip (LOC) platform. A push-pull junction capturing approach was applied to confine a small amount of the lipid solution inside a microchannel. The selective permeability between solvents and water in PDMS was utilized to extract the solvent from the lipid solution, resulting in a self-formation of the lipid bilayer in the microchannel array. Each microchannel was independently connected to a silver/silver chloride (Ag/AgCl) electrode array, leading to a high-throughput monitoring of the nanopore insertion in the formed lipid bilayers. The formation of multiple lipid bilayers inside an array of microchannels and the simultaneous electrical and optical monitoring of multiple bilayer provides an efficient LOC platform for the further development of single phi29 motor pore sensing and high throughput single pore dsDNA sequencing.

Microfabricated multiple field of view imaging flow cytometry

The combination of microscopy and flow cytometry enables image based screening of large collections of cells. Despite the proposition more than thirty years ago, adding high resolution wide-field imaging to flow cytometers remains challenging. The velocity of cells in flow cytometry can surpass a meter per second, requiring either sub-microsecond exposure times or other sophisticated photodetection techniques. Instead of faster detectors and brighter sources, we demonstrate that by imaging multiple channels simultaneously, a high throughput can be maintained with a flow velocity reduced in proportion to the degree of parallelization. The multi-field of view imaging flow cytometer (MIFC) is implemented with parallel arrays of microfluidic channels and diffractive lenses that produce sixteen wide field images with a magnification of 45 and submicron resolution. Using this device, we have imaged latex beads, red blood cells, and acute myeloid leukemia cells at rates of 2,000-20,000 per second.

Well plate microfluidic system for investigation of dynamic platelet behavior under variable shear loads

The study of platelet behavior in real-time under controlled shear stress offers insights into the underlying mechanisms of many vascular diseases and enables evaluation of platelet-focused therapeutics. The two most common methods used to study platelet behavior at the vessel wall under uniform shear flow are parallel plate flow chambers and cone-plate viscometers. Typically, these methods are difficult to use, lack experimental flexibility, provide low data content, are low in throughput, and require large reagent volumes. Here, we report a well plate microfluidic (WPM)-based system that offers high throughput, low reagent consumption, and high experimental flexibility in an easy to use well plate format. The system consists of well plates with an integrated array of microfluidic channels, a pneumatic interface, an automated microscope, and software. This WPM system was used to investigate dynamic platelet behavior under shear stress. Multiple channel designs are presented and tested for shear loads with whole blood to determine their applicability to study thrombus formation. Normal physiological shear (0.1-20 dyn/cm(2) ) and pathological shear (20-200 dyn/cm(2) ) devices were used to study platelet behavior in vitro under various shear, matrix coating, and monolayer conditions. The high physiological relevance, low blood consumption, and increased throughput create a valuable technique available to vascular biology researchers. The approach also has extensibility to other research areas including inflammation, cancer biology, and developmental/stem cell research.

A Highly Accurate and Consistent Microfluidic Viscometer for Continuous Blood Viscosity Measurement

A high-precision microfluidic viscometer with a microfluidic channel array composed of 100 indicating channels is demonstrated in this study. The relative viscosity of the sample fluid could be measured by simply counting the number of the indicating channels occupied by the sample and the reference fluids. Using lumped parameter modeling, an analytical solution of the relative viscosity is derived. In order to evaluate the performance of the developed microfluidic viscometer, the viscosity values obtained by the microfluidic viscometer are compared with the ones obtained by a conventional viscometer. In Newtonian fluid (sodium dodecyl sulfate [SDS] solution) tests, the normalized differences in the viscosities measured by two methods are less than 2.5%. In non-Newtonian fluid (whole blood, 45% hematocrit) tests at various shear rates, the viscosities measured by two methods are evaluated by a regression analysis via power law (). The k values for both the microfluidic viscometer and the conventional viscometer are 12.953 and 13.175, respectively; the n values are 0.797 and 0.807, respectively. The normalized differences in two parameters measured by two methods are less than 2%. Thus, it could be concluded that the microfluidic viscometer developed in this study is capable of measuring viscosity of both Newtonian fluid (SDS solution) and non-Newtonian fluid (whole blood) with a relatively high accuracy in a continuous and near real-time fashion. Furthermore, the viscometer could be potentially employed in cardiopulmonary bypass procedures by continuously monitoring viscosity changes due to blood damages and hemodilution.