A single-view field filter device for rare tumor cell filtration and enumeration.

In this work, we demonstrate a single-view field filter (SVFF) device for the efficient filtration and enumeration of rare tumor cells in the blood. In our device, the track-etched membrane is integrated within a low-cost polymer-film microfluidic chip, and multiplex microfiltration chambers are designed. Our device permits the performing of multiple sample tests on a single membrane and the dynamical observation of the entire filtration process in a single field of view. To characterize the device performance, our device is first tested using tumor cells, and three different cell behaviors are observed during the filtration process. Finally, we successfully apply our device for the separation of rare tumor cells from the lysed blood samples at various flow rates. The recovery rates of 93.3, 87.6, and 84.1% can be respectively achieved at the throughputs of 50, 100, and 150 µL/min. Our single-view field filter (SVFF) device offers the advantages of label-free filtration, efficient enumeration, easy integration, and low cost, and holds the potential to be used as an efficient tool for the filtration and enumeration of rare cells.

Cost-effective microfabrication of sub-micron-depth channels by femto-laser anti-stiction texturing.

Micro Electro Mechanical Systems (MEMS) and microfluidic devices have found numerous applications in the industrial sector. However, they require a fast, cost-effective and reliable manufacturing process in order to compete with conventional methods. Particularly, at the sub-micron scale, the manufacturing of devices are limited by the dimensional complexity. A proper bonding and stiction prevention of these sub-micron channels are two of the main challenges faced during the fabrication process of low aspect ratio channels. Especially, in the case of using flexible materials such as polydimethylsiloxane (PDMS). This study presents a direct laser microfabrication method of sub-micron channels using an infrared (IR) ultrashort pulse (femtosecond), capable of manufacturing extremely low aspect ratio channels. These microchannels are manufactured and tested varying their depth from 0.5 µm to 2 µm and width of 15, 20, 25, and 30 µm. The roughness of each pattern was measured by an interferometric microscope. Additionally, the static contact angle of each depth was studied to evaluate the influence of femtosecond laser fabrication method on the wettability of the glass substrate. PDMS, which is a biocompatible polymer, was used to provide a watertight property to the sub-micron channels and also to assist the assembly of external microfluidic hose connections. A 750 nm depth watertight channel was built using this methodology and successfully used as a blood plasma separator (BPS). The device was able to achieve 100% pure plasma without stiction of the PDMS layer to the sub-micron channel within an adequate time. This method provides a novel manufacturing approach useful for various applications such as point-of-care devices.

Assessment and monitoring of agreement among professionals for morphological evaluation in compliance with International accreditation standard requirements.

OBJECTIVES: Standardized criteria guaranteeing harmonized interpretation among morphologists in the provision of morphology results represent an important tool to be adopted for risk management and patient safety. Aim of this work is to assess agreement among morphologists in the microscopic evaluation of the peripheral blood smear. METHODS: 17 morphologists participating in the external quality assessment (EQA) program individually evaluated the blood smear and recorded the results using a personal username and password. Agreement among operators was evaluated. RESULTS: The overall agreement rate in microscopic differential was 95% in 2016 and 97% in 2017 (acceptance limit 90%), with 6/120 and 4/120 incongruent results, respectively. The agreement for the diagnostic hypothesis was satisfactory with a full agreement being reached in 5 out of 16 cases. CONCLUSIONS: The creation of a tool to assess the agreement of readers providing morphological evaluations is a valuable step forward in ensuring patient safety and quality laboratory medicine.

A low-cost, accurate method for detecting reticulocytes at different maturation stages based on changes in the mitochondrial membrane potential.

In the clinical setting, reticulocytes are used as an index for the hematopoietic function of the bone marrow. Different maturation stages of reticulocytes are early markers for bone marrow hematopoietic stem cell transplantation and bone marrow regeneration after chemotherapy. Therefore, we aimed to establish a method for detecting the different reticulocyte maturation stages. Based on the decreases in mitochondrial membrane potential during reticulocyte maturation, we used MitoTracker Green (MTG)/tetramethylrhodamine, ethylester (TMRE) to identify the different reticulocyte maturation stages and used Hoechst33342 to exclude nucleated cells. The results show that this method was universal and could be applied to detect the proportions of reticulocytes in different samples. Their proportion in normal peripheral blood, a blood deficiency model, bone marrow, and spleen were (6 ± 2)%, (38 ± 4)%, (14 ± 4)%, and (3 ± 1)%, respectively. The results obtained using this method were similar to those obtained using the manual counting method (methylene blue); the correlation was good (R = 0.817; p < .01) and the coefficient of variation was lower for the method established. Moreover, reticulocytes in peripheral blood could be further divided into three distinct maturation stages: R1 (MTGneg/TMREhigh), R2 (MTGhigh/TMREhigh), and R3 (MTGhigh/TMREneg). Reticulocytes in the bone marrow and spleen could be further divided into four distinct maturation stages: R1 (MTGneg/TMREhigh), R2-1 (MTGhigh/TMREhigh/FSbig), R2-2 (MTGhigh/TMREhigh/FSsmall), and R3 (MTGhigh/TMREneg). Based on changes in mitochondrial membrane potential, MTG/TMRE/Hoechst33342 staining could be used to detect reticulocytes in different samples and at different maturation stages with low cost and high accuracy.

Comprehensive assessment of electrospun scaffolds hemocompatibility.

Biodegradable polyesters, namely polycaprolactone (PCL) and copolymer of polylactide and polycaprolactone (PLCL) were electrospun into various fibrous structures and their hemocompatibility was evaluated in vitro. Firstly, hemolytic effect was evaluated upon incubation with diluted whole blood. The results showed that the degree of hemolysis depended on chemical composition and fibrous morphology. Electrospun polycaprolactone induced slight degree of hemolysis depending on its molecular weight and fibrous morphology; copolymer PLCL did not cause detectable hemolysis. The influence of coagulation pathways was examined by measurement of coagulation times. It was showed that intrinsic coagulation pathway assessed by activated partial thromboplastin time (APTT) was moderately accelerated after incubation with PCL and prolonged after incubation with copolymer PLCL. Extrinsic activation of coagulation tested by prothrombin time (PT) was slightly accelerated after incubation with all tested electrospun samples. Thrombogenicity assessment of fibrous samples revealed high thrombogenic properties of fibrous materials that was comparable to high degree of collagen thrombogenicity. The level of platelet activation was dependent on chemical composition and surface morphology of tested materials.

Biocompatibility assessment of silk nanoparticles: hemocompatibility and internalization by human blood cells.

Many nanoparticles are designed for use as potential nanomedicines for parenteral administration. However, emerging evidence suggests that hemocompatibility is important, but is highly particle- and test-bed dependent. Thus, knowledge of bulk material properties does not predict the hemocompatibility of uncharacterized nanoparticles, including silk nanoparticles. This study compares the hemocompatibility of silk versus silica nanoparticles, using whole human blood under quasi-static and flow conditions. Substantial hemocompatibility differences are noted for some nanoparticles in quasi-static versus dynamic studies; i.e., the inflammatory response to silk nanoparticles is significantly lower under flow versus quasi-static conditions. Silk nanoparticles also have very low coagulant properties - an observation that scales from the macro- to the nano-level. These nanoparticle hemocompatibility studies are complemented by preliminary live cell measurements to evaluate the endocytosis and trafficking of nanoparticles in human blood cells. Overall, this study demonstrates that nanoparticle hemocompatibility is affected by several factors, including the test bed design.

Multivariate Assessment of the Effect of Pump Design and Pump Gap Design Parameters on Blood Trauma.

Pump gaps are the most critical regions in a rotary blood pump when it comes to blood trauma in the form of hemolysis, protein destruction, and platelet activation. This study investigated six pump design parameters affecting the flow in a radial pump gap. A multivariate approach was employed to determine individual and quantitative parameter effects on blood trauma as well as parameter interactions. To consider the effect of shear stress and blood cell residence time, a validated numerical Lagrangian particle tracking approach was used. Based on the results, small-diameter pumps can be as blood compatible, if not more blood compatible, as large-diameter pumps as long as identical circumferential velocities and clearance gaps are maintained. Furthermore, the results indicate that an eccentric rotor position in the casing is not harmful and that a pressure difference generating washout flow and thereby reducing the cell residence time is of significant importance.

Performance Assessment of Internal Quality Control (IQC) Products in Blood Transfusion Compatibility Testing in China.

Internal quality control (IQC) is a critical component of laboratory quality management, and IQC products can determine the reliability of testing results. In China, given the fact that most blood transfusion compatibility laboratories do not employ IQC products or do so minimally, there is a lack of uniform and standardized IQC methods. To explore the reliability of IQC products and methods, we studied 697 results from IQC samples in our laboratory from 2012 to 2014. The results showed that the sensitivity and specificity of the IQCs in anti-B testing were 100% and 99.7%, respectively. The sensitivity and specificity of the IQCs in forward blood typing, anti-A testing, irregular antibody screening, and cross-matching were all 100%. The reliability analysis indicated that 97% of anti-B testing results were at a 99% confidence level, and 99.9% of forward blood typing, anti-A testing, irregular antibody screening, and cross-matching results were at a 99% confidence level. Therefore, our IQC products and methods are highly sensitive, specific, and reliable. Our study paves the way for the establishment of a uniform and standardized IQC method for pre-transfusion compatibility testing in China and other parts of the world.

Hematological Assessment in Pet Rabbits: Blood Sample Collection and Blood Cell Identification.

Pet rabbits are presented to veterinary clinics for routine care and treatment of clinical diseases. In addition to obtaining clinical history, additional diagnostic testing may be required, including hematological assessments. This article describes common blood collection methods, including venipuncture sites, volume of blood that can be safely collected, and handling of the blood. Hematological parameters for normal rabbits are provided for comparison with in-house or commercial test results. A description of the morphology of rabbit leukocytes is provided to assist in performing a differential count. Differential diagnoses are provided for abnormal values identified in the hemogram.

Hematologic Assessment in Pet Rats, Mice, Hamsters, and Gerbils: Blood Sample Collection and Blood Cell Identification.

Hamsters, gerbils, rats, and mice are presented to veterinary clinics and hospitals for prophylactic care and treatment of clinical signs of disease. Physical examination, history, and husbandry practice information can be supplemented greatly by assessment of hematologic parameters. As a resource for veterinarians and their technicians, this article describes the methods for collection of blood, identification of blood cells, and interpretation of the hemogram in mice, rats, gerbils, and hamsters.

Hematological Assessment in Pet Guinea Pigs (Cavia porcellus): Blood Sample Collection and Blood Cell Identification.

Pet guinea pigs are presented to veterinary clinics for routine care and treatment of clinical diseases. In addition to obtaining clinical history and exam findings, diagnostic testing may be required, including hematological assessments. This article describes common blood collection methods, including venipuncture sites, the volume of blood that can be safely collected, and handling of the blood. Hematological parameters for normal guinea pigs are provided for comparison with in-house or commercial test results. A description of the morphology of guinea pig leukocytes is provided to assist in performing a differential count.

Assessing peripheral blood cell profile of Yorkshire pigs divergently selected for residual feed intake.

The cost of feed is a serious issue in the pork industry, contributing about 65 to 75% of the total production cost. To prevent economic losses and decreased productivity of the herd, it is important to select for animals that eat less for the same lean gain, or more efficient animals. Residual feed intake (RFI) is the difference between observed feed intake and expected feed intake based on estimated maintenance and production requirements. Selection for decreased RFI, or more efficient animals, is a potential solution to higher feed costs in pig production. However, animals that are highly selected for decreased RFI may have reduced energy input to the immune system and fail to withstand diseases and stressors after infection that negatively impact profitability. The objective of this study was to evaluate differences in circulating blood cell profiles at a young age between 2 lines of Yorkshire pigs that were divergently selected for RFI as well as the heritability of these traits, to investigate effects of selection for RFI on immune system parameters, and to identify potential biomarkers for feed efficiency. Previous work has shown that the 2 lines had diverged for IGF-1 in serum in young pigs and, therefore, this stage was investigated for other potential physiological differences. Blood samples were drawn for a complete blood count (CBC) analysis from 517 gilts and barrows, ages 35 to 42 d, across the 2 lines. In general, the low-RFI line had lower numbers of specific types of white blood cells but higher hemoglobin concentration and red blood cell volume compared to the high-RFI line. No significant correlations were found between CBC traits and RFI across and within the lines (0.05 < < 0.1). Of the 15 CBC traits that were measured, 3 were highly heritable (0.56 < < 0.62), 9 were moderately heritable (0.12 < < 0.47), and 3 were lowly heritable ( < 0.12), suggesting a substantial genetic component for CBC traits and that selection for CBC traits could be effective. Our results also show that selection for RFI has significantly impacted the number of circulating blood cells. In this experiment, we studied only healthy animals that were not under known pathogen challenge; therefore, our results cannot be directly applied to a disease challenge situation. Future work will be to challenge the animals and determine the effect of challenge on CBC levels.

Hematological assessment in pet rabbits: blood sample collection and blood cell identification.

Pet rabbits are presented to veterinary clinics for routine care and treatment of clinical diseases. In addition to obtaining clinical history, additional diagnostic testing may be required, including hematological assessments. This article describes common blood collection methods, including venipuncture sites, volume of blood that can be safely collected, and handling of the blood. Hematological parameters for normal rabbits are provided for comparison with in-house or commercial test results. A description of the morphology of rabbit leukocytes is provided to assist in performing a differential count. Differential diagnoses are provided for abnormal values identified in the hemogram.

Hematologic assessment in pet rats, mice, hamsters, and gerbils: blood sample collection and blood cell identification.

Hamsters, gerbils, rats, and mice are presented to veterinary clinics and hospitals for prophylactic care and treatment of clinical signs of disease. Physical examination, history, and husbandry practice information can be supplemented greatly by assessment of hematologic parameters. As a resource for veterinarians and their technicians, this article describes the methods for collection of blood, identification of blood cells, and interpretation of the hemogram in mice, rats, gerbils, and hamsters.

Hematological assessment in pet guinea pigs (Cavia porcellus): blood sample collection and blood cell identification.

Pet guinea pigs are presented to veterinary clinics for routine care and treatment of clinical diseases. In addition to obtaining clinical history and exam findings, diagnostic testing may be required, including hematological assessments. This article describes common blood collection methods, including venipuncture sites, the volume of blood that can be safely collected, and handling of the blood. Hematological parameters for normal guinea pigs are provided for comparison with in-house or commercial test results. A description of the morphology of guinea pig leukocytes is provided to assist in performing a differential count.

Low-cost mobile phone microscopy with a reversed mobile phone camera lens.

The increasing capabilities and ubiquity of mobile phones and their associated digital cameras offer the possibility of extending low-cost, portable diagnostic microscopy to underserved and low-resource areas. However, mobile phone microscopes created by adding magnifying optics to the phone's camera module have been unable to make use of the full image sensor due to the specialized design of the embedded camera lens, exacerbating the tradeoff between resolution and field of view inherent to optical systems. This tradeoff is acutely felt for diagnostic applications, where the speed and cost of image-based diagnosis is related to the area of the sample that can be viewed at sufficient resolution. Here we present a simple and low-cost approach to mobile phone microscopy that uses a reversed mobile phone camera lens added to an intact mobile phone to enable high quality imaging over a significantly larger field of view than standard microscopy. We demonstrate use of the reversed lens mobile phone microscope to identify red and white blood cells in blood smears and soil-transmitted helminth eggs in stool samples.

Kinetically limited differential centrifugation as an inexpensive and readily available alternative to centrifugal elutriation.

When separating two species with similar densities but differing sedimentation velocities (because of differences in size), centrifugal elutriation is generally the method of choice. However, a major drawback to this approach is the requirement for specialized equipment. Here, we present a new method that achieves similar separations using standard benchtop centrifuges by loading the seperands as a layer on top of a dense buffer of a specified length, and running the benchtop centrifugation process for a calculated amount of time, thereby ensuring that all faster moving species are collected at the bottom, while all slower moving species remain in the buffer. We demonstrate the use of our procedure to isolate bacteria from blood culture broth (a mixture of bacterial growth media, blood, and bacteria).

Systematic assessment in an animal model of the angiogenic potential of different human cell sources for therapeutic revascularization.

INTRODUCTION: Endothelial progenitor cells (EPC) capable of initiating or augmenting vascular growth were recently identified within the small population of CD34-expressing cells that circulate in human peripheral blood and which are considered hematopoietic progenitor cells (HPC). Soon thereafter human HPC began to be used in clinical trials as putative sources of EPC for therapeutic vascular regeneration, especially in myocardial and critical limb ischemias. However, unlike HPC where hematopoietic efficacy is related quantitatively to CD34+ cell numbers implanted, there has been no consensus on how to measure EPC or how to assess cellular graft potency for vascular regeneration. We employed an animal model of spontaneous neovascularization to simultaneously determine whether human cells incorporate into new vessels and to quantify the effect of different putative angiogenic cells on vascularization in terms of number of vessels generated. We systematically compared competence for therapeutic angiogenesis in different sources of human cells with putative angiogenic potential, to begin to provide some rationale for optimising cell procurement for this therapy. METHODS: Human cells employed were mononuclear cells from normal peripheral blood and HPC-rich cell sources (umbilical cord blood, mobilized peripheral blood, bone marrow), CD34+ enriched or depleted subsets of these, and outgrowth cell populations from these. An established sponge implant angiogenesis model was adapted to determine the effects of different human cells on vascularization of implants in immunodeficient mice. Angiogenesis was quantified by vessel density and species of origin by immunohistochemistry. RESULTS: CD34+ cells from mobilized peripheral blood or umbilical cord blood HPC were the only cells to promote new vessel growth, but did not incorporate into vessels. Only endothelial outgrowth cells (EOC) incorporated into vessels, but these did not promote vessel growth. CONCLUSIONS: These studies indicate that, since EPC are very rare, any benefit seen in clinical trials of HPC in therapeutic vascular regeneration is predominantly mediated by indirect proangiogenic effects rather than through direct incorporation of any rare EPC contained within these sources. It should be possible to produce autologous EOC for therapeutic use, and evaluate the effect of EPC distinct from, or in synergy with, the proangiogenic effects of HPC therapies.

A novel and simplified method of culture of human blood-derived early endothelial progenitor cells for the treatment of ischemic vascular disease.

Endothelial progenitor cells (EPCs) consist of two different subpopulations named early (eEPCs) and late EPCs (lEPCs) that are derived from CD14(+) and CD14(-) circulating cells, respectively. These cells are regularly cultured over fibronectin-coated surfaces in endothelial basal medium (EBM)-2 supplemented with insulin-like growth factor (IGF-1), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), and fibroblast growth factor (FGF). We have developed a new and simplified method for culturing human EPCs obtained from peripheral blood and tested their ability to preserve cardiac function following infarction. We first demonstrated that eEPCs derived from human peripheral blood mononuclear cells (PBMCs) and cultured in EBM-2 medium supplemented with autologous serum (10%) over fibronectin-coated surfaces (10 µg/ml) in the presence of IGF-1 (50 ng/ml) only, have a secretome similar to eEPCs cultured under regular conditions with IGF-1, VEGF, EGF, and FGF. Our data also indicate that IGF-1 modulates PBMC secretome in a dose-dependent manner. In another series of experiments, we showed that PBMCs cultured in suspension in bags (S-PBMCs) in basal medium supplemented with fibronectin and IGF-1 secrete significant amounts of stem cell factor (SCF, 31.3 ± 3.1 pg/ml)), hepatocyte growth factor (HGF, 438.6 ± 41.4 pg/ml), soluble tumor necrosis factor receptor 1 (sTNFR1, 127.1 ± 9.9 pg/ml), VEGF (139.3 ± 9.6 pg/ml), and IGF-1 (147.2 ± 46.1 pg/ml) but very low levels of TNF-α (13.4 ± 2.5 pg/ml). S-PBMCs injected intravenously into NOD SCID mice migrated to the injured myocardium, reduced cardiac fibrosis, enhanced angiogenesis, and preserved cardiac function after myocardial infarction (MI) in a manner similar to eEPCs cultured under standard conditions. In conclusion, we show in this study a refined and optimized method for culturing eEPCs. Our data indicate that S-PBMCs are composed of several cell populations including eEPCs and that they secrete high amounts of antiapoptotic, anti-inflammatory, and proangiogenic factors capable of preserving cardiac function following MI.

Cost-effective and compact wide-field fluorescent imaging on a cell-phone.

We demonstrate wide-field fluorescent and darkfield imaging on a cell-phone with compact, light-weight and cost-effective optical components that are mechanically attached to the existing camera unit of the cell-phone. For this purpose, we used battery powered light-emitting diodes (LEDs) to pump the sample of interest from the side using butt-coupling, where the pump light was guided within the sample cuvette to uniformly excite the specimen. The fluorescent emission from the sample was then imaged using an additional lens that was positioned right in front of the existing lens of the cell-phone camera. Because the excitation occurs through guided waves that propagate perpendicular to our detection path, an inexpensive plastic colour filter was sufficient to create the dark-field background required for fluorescent imaging, without the need for a thin-film interference filter. We validate the performance of this platform by imaging various fluorescent micro-objects in 2 colours (i.e., red and green) over a large field-of-view (FOV) of ∼81 mm(2) with a raw spatial resolution of ∼20 µm. With additional digital processing of the captured cell-phone images, through the use of compressive sampling theory, we demonstrate ∼2 fold improvement in our resolving power, achieving ∼10 µm resolution without a trade-off in our FOV. Further, we also demonstrate darkfield imaging of non-fluorescent specimen using the same interface, where this time the scattered light from the objects is detected without the use of any filters. The capability of imaging a wide FOV would be exceedingly important to probe large sample volumes (e.g., >0.1 mL) of e.g., blood, urine, sputum or water, and for this end we also demonstrate fluorescent imaging of labeled white-blood cells from whole blood samples, as well as water-borne pathogenic protozoan parasites such as Giardia Lamblia cysts. Weighing only ∼28 g (∼1 ounce), this compact and cost-effective fluorescent imaging platform attached to a cell-phone could be quite useful especially for resource-limited settings, and might provide an important tool for wide-field imaging and quantification of various lab-on-a-chip assays developed for global health applications, such as monitoring of HIV+ patients for CD4 counts or viral load measurements.