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Find video protocols related to scientific articles indexed in Pubmed.
Osmotic tolerance limits of red blood cells from umbilical cord blood.
Cryobiology
PUBLISHED: 04-07-2014
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Effective methods for long-term preservation of cord red blood cells (RBCs) are needed to ensure a readily available supply of RBCs to treat fetal and neonatal anemia. Cryopreservation is a potential long-term storage strategy for maintaining the quality of cord RBCs for the use in intrauterine and neonatal transfusion. However, during cryopreservation, cells are subjected to damaging osmotic stresses during cryoprotectant addition and removal and freezing and thawing that require knowledge of osmotic tolerance limits in order to optimize the preservation process. The objective of this study was to characterize the osmotic tolerance limits of cord RBCs in conditions relevant to cryopreservation, and compare the results to the osmotic tolerance limits of adult RBCs. Osmotic tolerance limits were determined by exposing RBCs to solutions of different concentrations to induce a range of osmotic volume changes. Three treatment groups of adult and cord RBCs were tested: (1) isotonic saline, (2) 40% w/v glycerol, and (3) frozen-thawed RBCs in 40% w/v glycerol. We show that cord RBCs are more sensitive to shrinkage and swelling than adult RBCs, indicating that osmotic tolerance limits should be considered when adding and removing cryoprotectants. In addition, freezing and thawing resulted in both cord and adult RBCs becoming more sensitive to post-thaw swelling requiring that glycerol removal procedures for both cell types ensure that cell volume excursions are maintained below 1.7 times the isotonic osmotically active volume to attain good post-wash cell recovery. Our results will help inform the development of optimized cryopreservation protocol for cord RBCs.
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Feasibility and transport of packed red blood cells into Special Forces operational conditions.
J Trauma Acute Care Surg
PUBLISHED: 03-26-2014
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Transfusing packed red blood cells (PRBCs) into Special Forces may provide a survival advantage from hemorrhage-induced battlefield injuries; however, the effect of the unique operational stressors on RBC integrity is not known.
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A lab-on-chip for malaria diagnosis and surveillance.
Malar. J.
PUBLISHED: 03-13-2014
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Access to timely and accurate diagnostic tests has a significant impact in the management of diseases of global concern such as malaria. While molecular diagnostics satisfy this need effectively in developed countries, barriers in technology, reagent storage, cost and expertise have hampered the introduction of these methods in developing countries. In this study a simple, lab-on-chip PCR diagnostic was created for malaria that overcomes these challenges.
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Osmotic parameters of red blood cells from umbilical cord blood.
Cryobiology
PUBLISHED: 02-14-2014
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The transfusion of red blood cells from umbilical cord blood (cord RBCs) is gathering significant interest for the treatment of fetal and neonatal anemia, due to its high content of fetal hemoglobin as well as numerous other potential benefits to fetuses and neonates. However, in order to establish a stable supply of cord RBCs for clinical use, a cryopreservation method must be developed. This, in turn, requires knowledge of the osmotic parameters of cord RBCs. Thus, the objective of this study was to characterize the osmotic parameters of cord RBCs: osmotically inactive fraction (b), hydraulic conductivity (Lp), permeability to cryoprotectant glycerol (Pglycerol), and corresponding Arrhenius activation energies (Ea). For Lp and Pglycerol determination, RBCs were analyzed using a stopped-flow system to monitor osmotically-induced RBC volume changes via intrinsic RBC hemoglobin fluorescence. Lp and Pglycerol were characterized at 4°C, 20°C, and 35°C using Jacobs and Stewart equations with the Ea calculated from the Arrhenius plot. Results indicate that cord RBCs have a larger osmotically inactive fraction compared to adult RBCs. Hydraulic conductivity and osmotic permeability to glycerol of cord RBCs differed compared to those of adult RBCs with the differences dependent on experimental conditions, such as temperature and osmolality. Compared to adult RBCs, cord RBCs had a higher Ea for Lp and a lower Ea for Pglycerol. This information regarding osmotic parameters will be used in future work to develop a protocol for cryopreserving cord RBCs.
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A quality monitoring program for red blood cell components: in vitro quality indicators before and after implementation of semiautomated processing.
Transfusion
PUBLISHED: 01-31-2014
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Canadian Blood Services has been conducting quality monitoring of red blood cell (RBC) components since 2005, a period spanning the implementation of semiautomated component production. The aim was to compare the quality of RBC components produced before and after this production method change.
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A method to measure permeability of red blood cell membrane to water and solutes using intrinsic fluorescence.
Clin. Chim. Acta
PUBLISHED: 01-21-2014
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Designing effective cryopreservation procedures for cells requires knowledge of permeability of cell membrane to water and solutes. To determine cell membrane permeability, one needs to measure the rate of cell volume changes in anisotonic environment. Red blood cells (RBCs) respond very quickly to changes in extracellular solutes concentration, which complicates the use of traditional methods. Preservation of RBCs from umbilical cord blood for neonatal transfusions is currently broadly discussed in the literature, but data on osmotic permeability of cord RBCs is controversial. Therefore, alternative methods to determine osmotic membrane permeability of these cells are warranted. We describe a technique to measure rapid changes in RBC volume through changes in the intensity of RBC autofluorescence.
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Coinfusion of dextrose-containing fluids and red blood cells does not adversely affect in vitro red blood cell quality.
Transfusion
PUBLISHED: 01-14-2014
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Transfusion guidelines advise against coinfusing red blood cells (RBCs) with solutions other than 0.9% saline. We evaluated the impact of coinfusion with dextrose-containing fluids (DW) on markers of RBC quality.
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Quality of red blood cells washed using an automated cell processor with and without irradiation.
Transfusion
PUBLISHED: 07-25-2013
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Sterile washing of red blood cells (RBCs) and use of an additive solution permits longer postwash storage. The effect of irradiation during this extended storage time is unclear.
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The effects of rejuvenation during hypothermic storage on red blood cell membrane remodeling.
Transfusion
PUBLISHED: 04-29-2013
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Our previous studies showed that hypothermic storage (HS) induces red blood cell (RBC) microparticle (RMP) generation and changes in phosphatidylserine (PS) and CD47 expression on RBCs and RMPs. The aim of this study was to evaluate the effect of cold rejuvenation treatment at multiple time points during storage on these prehemolytic indicators of RBC membrane storage lesion.
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Segments from red blood cell units should not be used for quality testing.
Transfusion
PUBLISHED: 04-01-2013
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Nondestructive testing of blood components could permit in-process quality control and reduce discards. Tubing segments, generated during red blood cell (RBC) component production, were tested to determine their suitability as a sample source for quality testing.
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Rapid removal of glycerol from frozen-thawed red blood cells.
Biotechnol. Prog.
PUBLISHED: 03-29-2013
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The storage of red blood cells (RBCs) in a refrigerated state allows a shelf life of a few weeks, whereas RBCs frozen in 40% glycerol have a shelf life of 10 years. Despite the clear logistical advantages of frozen blood, it is not widely used in transfusion medicine. One of the main reasons is that existing post-thaw washing methods to remove glycerol are prohibitively time consuming, requiring about an hour to remove glycerol from a single unit of blood. In this study, we have investigated the potential for more rapid removal of glycerol. Using published biophysical data for human RBCs, we mathematically optimized a three-step deglycerolization process, yielding a procedure that was less than 32 s long. This procedure was found to yield 70% hemolysis, a value that was much higher than expected. Consequently, we systematically evaluated three-step deglycerolization procedures, varying the solution composition and equilibration time in each step. Our best results consisted of less than 20% hemolysis for a deglycerolization time of 3 min, and it is expected that even further improvements could be made with a more thorough optimization and more reliable biophysical data. Our results demonstrate the potential for significantly reducing the deglycerolization time compared with existing methods. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:609-620, 2013.
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Quality of red blood cells washed using the ACP 215 cell processor: assessment of optimal pre- and postwash storage times and conditions.
Transfusion
PUBLISHED: 03-22-2013
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Washing of red blood cell concentrates (RCCs) is required for potassium-sensitive transfusion recipients, including neonates in need of large-volume transfusions. When open, nonsterile washing systems are used, postwash outdate time is limited to 24 hours, often leading to problems providing the component to the patient before expiry.
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An enclosed in-gel PCR amplification cassette with multi-target, multi-sample detection for platform molecular diagnostics.
Lab Chip
PUBLISHED: 03-07-2013
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This work describes a self-contained, simple, disposable, and inexpensive gel capillary cassette for DNA amplification in near point of care settings. The cassette avoids the need for pumps or valves during raw sample delivery or polymerase chain reaction (PCR) amplification steps. The cassette contains capillary reaction units that can be stored at room temperature for up to 3 months. The current cassette configuration format simultaneously tests up to 16 patients for two or more targets, accommodates different sample types on the same cassette, has integrated positive and negative controls and allows flexibility for multiple geometries. PCR reagents in the cassette are desiccated to allow storage at room temperature with rehydration by raw sample at the time of testing. The sample is introduced to the cassette via a transfer pipette simply by capillary force. DNA amplification was carried out in a portable prototype instrument for PCR thermal cycling with fluorescence detection of amplified products by melt curve analysis (MCA). To demonstrate performance, raw genital swabs and urine were introduced to the same cassette to simultaneously detect four sexually transmitted infections. Herpes Simplex Viruses (HSV-1 and HSV-2) were detected from raw genital swabs. Ureaplasma urealyticum (UU) and Mycoplasma homonis (MH) were detected from raw urine. Results for multiple patients were obtained in as little as 50 min. This platform allows multiparameter clinical testing with a pre-assembled cassette that requires only the introduction of raw sample. Modification of the prototype device to accommodate larger cassettes will ultimately provide high throughput simultaneous testing of even larger numbers of samples for many different targets, as is required for some clinical applications. Combinations of wax and/or polymer cassettes holding capillary reaction units are feasible. The components of the cassette are suited to mass production and robotic assembly to produce a readily manufactured disposable reaction cassette that can be configured for disease-specific testing panels. Rapid testing with a disposable reaction cassette on an inexpensive instrument will enable on the spot evaluation of patients in the clinic for faster medical decision-making and more informed therapeutic choices.
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Process improvement by eliminating mixing of whole blood units after an overnight hold prior to component production using the buffy coat method.
J Blood Transfus
PUBLISHED: 02-27-2013
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The elimination of a thorough manual mixing of whole blood (WB) which takes place following the overnight hold, but before the first centrifugation step, during buffy coat component production at Canadian Blood Services (CBS) was investigated. WB was pooled after donation and split. Pairs of platelet, red blood cell (RBC), and plasma components were produced, with half using the standard method and half using a method in which the mixing step was eliminated. Quality assessments included yield, pH, CD62P expression and morphology for platelets, hemoglobin, hematocrit, hemolysis, and supernatant K(+) for RBCs, and volume and factor VIII activity levels for plasma. All components, produced using either method, met CBS quality control criteria. There were no significant differences in platelet yield between components produced with and without mixing. A significant difference was seen for RBC hemolysis at expiry (P = 0.03), but for both groups, levels met quality control requirements. Noninferiority of components produced without mixing was confirmed for all parameters. Manual mixing is laborious and has a risk of repetitive strain for production staff and its significance is unclear. Elimination of this step will improve process efficiencies without compromising quality.
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Storage of red blood cells affects membrane composition, microvesiculation, and in vitro quality.
Transfusion
PUBLISHED: 01-16-2013
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During storage detrimental biochemical and biomechanical changes occur within a red blood cell (RBC). RBC microparticles (RMPs) produced during storage have been identified as biomarkers of RBC quality, being potentially immunogenic and inhibitory to nitric oxide regulation.
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Rejuvenation of ATP during storage does not reverse effects of the hypothermic storage lesion.
Transfusion
PUBLISHED: 01-04-2013
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Hypothermic storage (HS) of red blood cells (RBCs) leads to a progressive deterioration of cell quality. Mitigating these deleterious changes could allow maintenance or even an improvement of RBC in vitro quality. The aim was to determine the effect of a cold rejuvenation treatment of RBCs and in particular to assess the connection between ATP levels, RBC deformability, and morphology during RBC storage.
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Quality of red blood cells isolated from umbilical cord blood stored at room temperature.
J Blood Transfus
PUBLISHED: 10-16-2011
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Red blood cells (RBCs) from cord blood contain fetal hemoglobin that is predominant in newborns and, therefore, may be more appropriate for neonatal transfusions than currently transfused adult RBCs. Post-collection, cord blood can be stored at room temperature for several days before it is processed for stem cells isolation, with little known about how these conditions affect currently discarded RBCs. The present study examined the effect of the duration cord blood spent at room temperature and other cord blood characteristics on cord RBC quality. RBCs were tested immediately after their isolation from cord blood using a broad panel of quality assays. No significant decrease in cord RBC quality was observed during the first 65 hours of storage at room temperature. The ratio of cord blood to anticoagulant was associated with RBC quality and needs to be optimized in future. This knowledge will assist in future development of cord RBC transfusion product.
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Liposomes composed of unsaturated lipids for membrane modification of human erythrocytes.
Mol. Membr. Biol.
PUBLISHED: 09-28-2011
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Previous studies have shown that certain saturated lipids protect red blood cells (RBCs) during hypothermic storage but provide little protection during freezing or freeze-drying, whereas various unsaturated lipids destabilize RBCs during hypothermic storage but protect during freezing and freeze-drying. The protective effect of liposomes has been attributed to membrane modifications. We have previously shown that cholesterol exchange and lipid transfer between liposomes composed of saturated lipids and RBCs critically depends on the length of the lipid acyl chains. In this study the effect of unsaturated lipids with differences in their number of unsaturated bonds (18:0/18:1, 18:1/18:1, 18:2/18:2) on RBC membrane properties has been studied. RBCs were incubated in the presence of liposomes and both the liposomal and RBC fraction were analyzed by Fourier transform infrared spectroscopy (FTIR) after incubation. The liposomes caused an increase in RBC membrane conformational disorder at suprazero temperatures. The fluidizing effect of the liposomes on the RBC membranes, however, was found to be similar for the different lipids irrespective of their unsaturation level. The gel to liquid crystalline phase transition temperature of the liposomes increased after incubation with RBCs. RBC membrane fluidity increased linearly during the first 8 hours of incubation in the presence of liposomes. The increase in RBC membrane fluidity was found to be temperature dependent and displayed Arrhenius behaviour between 20 and 40°C, with an activation energy of 88 kJ mol?¹. Taken together, liposomes composed of unsaturated lipids increase RBC membrane conformational disorder, which could explain their cryoprotective action.
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Emerging Role for Use of Liposomes in the Biopreservation of Red Blood Cells.
Transfus Med Hemother
PUBLISHED: 01-12-2011
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SUMMARY: Biopreservation is the process of maintaining the integrity and functionality of cells held outside the native environment for extended storage times. The development of red blood cell (RBC) biopreservation techniques that maintain in vitro RBC viability and function represents the foundation of modern blood banking. The biopreservation of RBCs for clinical use can be categorized based on the techniques used to achieve biologic stability, including hypothermic storage and cryopreservation. This review will examine the emerging role of liposomes in the RBC biopreservation, including the incorporation of liposomes into RBC membranes as an effective approach for minimizing RBC hypothermic storage membrane lesion and use of liposomes as a permeabilization strategy for the intracellular accumulation of novel intracellular cryoprotectants. Integration of current biopreservation research with blood banking practices offers enormous potential for future improvements of safety and efficacy of RBC transfusion.
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In-gel technology for PCR genotyping and pathogen detection.
Anal. Chem.
PUBLISHED: 09-03-2010
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This work describes the use of polyacrylamide gel and PCR reagents photopolymerized in a mold to create an array of semisolid posts that serve as reaction vessels for parallel PCR amplification of an externally added template. DNA amplification occurred in a cylindrical, self-standing 9 × 9 array of gel posts each less than 1 ?L in volume. Photopolymerization of the gel with an intercalating dye added prior to polymerization permitted acquisition of real-time PCR data and melting curve analysis data without the need for any type of post-PCR staining procedures. PCR was equally efficient and reproducible when template DNA was polymerized within the gel or when exogenous template was added atop precast gel posts. PCR amplification occurred with template from purified DNA or from raw urine of patients with BK viruria. Multiple primer sets can be utilized per gel post array with no detectable cross contamination. As few as 34 BK virus templates were consistently detected by PCR in an individual gel post. Amplification of HPA1 and FGFR2 genes in human genomic DNA (gDNA) required as little as 2-5 ng of gDNA template/gel post. The device prototype includes a Peltier element for PCR thermal cycling and a CCD camera to capture fluorescence for product detection. Our technology is amenable to integration in point of care microdevices.
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Liposomes alter thermal phase behavior and composition of red blood cell membranes.
Biochim. Biophys. Acta
PUBLISHED: 08-09-2010
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Unilamellar liposomes composed of natural phospholipids provide a new promising class of protective agents for hypothermic storage, cryopreservation, or freeze-drying of red blood cells (RBCs). In this study, FTIR spectroscopy, MALDI-TOF MS, and colorimetric assays were used to investigate the effects of liposomes composed of a homologous series of linear saturated phosphatidylcholine phospholipids (18:0; 16:0; 14:0; 12:0) on RBC membranes. RBCs were incubated with liposomes at 37°C and both the liposomal and the RBC fraction were analyzed after incubation. FTIR studies showed that liposomes composed of short acyl chain length lipids cause an increase in RBC membrane conformational disorder at suprazero temperatures, whereas long acyl chain length lipids were found to have little effects. The increased lipid conformational disorder in the RBC membranes coincided with a decrease in the cholesterol-to-phospholipid ratio. The opposite effects were found in the liposomes after incubation with RBCs. MALDI-TOF MS analysis showed the presence of short acyl chain length lipids (14:0 and 12:0) in RBC membranes after incubation, which was not observed after incubation with liposomes containing long acyl chain length lipids (18:0 and 16:0). Liposomes alter RBC membrane properties by cholesterol depletion and lipid addition.
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Intracellular ice formation in confluent monolayers of human dental stem cells and membrane damage.
Cryobiology
PUBLISHED: 05-25-2010
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Dental pulp stem cells (DPSCs) are of interest to researchers and clinicians due to their ability to differentiate into various tissue types and potential uses in cell-mediated therapies and tissue engineering. Currently DPSCs are cryopreserved in suspension using Me(2)SO. However, preservation as two and three dimensional constructs, along with the elimination of toxic Me(2)SO, may be required. It was shown that intracellular ice formation (IIF), lethal to cells in suspensions, may be innocuous in cell monolayers due to ice propagation between cells through gap junctions that results in improved post-thaw recovery. We hypothesized that innocuous IIF protects confluent DPSC monolayers against injury during cryopreservation. The objective was to examine the effects of IIF on post-thaw viability of both confluent monolayers and suspensions of DPSCs. Confluent DPSC monolayers were assessed for the expression of gap junction protein Connexin-43. IIF was induced on the cryostage and in the methanol bath at different subzero temperatures. Membrane integrity and colony-forming ability were assessed post-thaw. Confluent DPSC monolayers expressed Connexin-43. In cell suspensions, 85.9+/-1.7% of cells were damaged after 100% IIF. In cell monolayers, after 100% IIF, only 25.5+/-5.5% and 14.8+/-3.3% of cells were damaged on the cryostage and in the methanol bath respectively. However, DPSC monolayers exposed to 100% IIF showed no colony-forming ability. We conclude that confluent monolayers of DPSCs express the gap junction-forming protein Connexin-43 and upon IIF retain membrane integrity, however lose the ability to proliferate.
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Biomonitoring of arsenic in urine and saliva of children playing on playgrounds constructed from chromated copper arsenate-treated wood.
Environ. Sci. Technol.
PUBLISHED: 04-10-2010
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Children may be exposed to arsenic during contact with structures treated with chromated copper arsenate (CCA). A high frequency of hand-to-mouth activity may increase their risk of ingesting arsenic. Previous work showed that arsenic concentrations in the hand-wash samples of children playing on CCA playgrounds were four times higher than those playing on non-CCA playgrounds. It is not clear whether playing on CCA playgrounds results in elevated overall exposure to arsenic. The objective of this study was to perform arsenic biomonitoring in children to determine whether playing on CCA-treated playgrounds substantially contributes to their overall exposure to arsenic. One hundred and twenty five saliva samples from 61 children and 101 urine samples from 45 children were collected after children played on 8 CCA and 8 non-CCA playgrounds. Arsenic speciation analysis was conducted using high performance liquid chromatography combined with inductively coupled plasma mass spectrometry. The arsenic species detected in the urine and saliva samples from children playing on CCA and non-CCA playgrounds were similar. Dimethylarsinic acid and arsenobetaine were the main arsenic species found in urine samples. The sum of inorganic trivalent and pentavalent arsenic, monomethylarsonic acid, and dimethylarsinic acid in urine was 15 +/- 28 microg/L in the CCA group and 12 +/- 23 microg/L in the non-CCA group (p = 0.60). The sum of these species in saliva was 1.1 +/- 2.1 microg/L in the CCA group and 1.4 +/- 1.1 microg/L in the non-CCA group (p = 0.32). These results show that there is no significant difference in the concentration or speciation of arsenic between the samples from children playing on CCA and non-CCA playgrounds. Contact with CCA playgrounds is not likely to significantly contribute to the overall arsenic exposure in children; other sources such as dietary arsenic may be a main contributor to their overall exposure.
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Mitochondria and membrane cryoinjury in micropatterned cells: effects of cell-cell interactions.
Cryobiology
PUBLISHED: 03-30-2010
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The maintenance of cell membrane integrity is an absolute minimum criterion for the selection of a successful cryopreservation process; however, it is often used as the sole determinant of cell "viability". Membrane stresses and strains that develop with cell volume fluctuations are only one component of the overall cellular response to freezing. Damage to organelles resulting from excessive concentration of intracellular solutes and/or the alternation of molecular signalling events may affect post-thaw outcomes. As the low temperature response of cells is affected by the presence of cell-cell interactions, the cryopreservation of tissues and tissue model systems would benefit from a more detailed understanding of the sites and mechanisms of cryoinjury. The purpose of this study was to examine the relationship between mitochondria and plasma membrane damage in frozen micropatterned cells and to identify the role of cell-cell interactions. Madin Darby Canine Kidney cells (MDCK) were micropatterned using a polydimethylsiloxane (PDMS) elastomeric stamp to create non-adhesive regions of agarose on untreated glass substrates. Five different cell arrangements were used to examine the effect of cell-cell contact: single cells, cell doublets, linear arrangement of cells, randomly arranged cells and confluent monolayers. Cells were cooled in a programmable alcohol bath at 1 degrees C/min to -40 degrees C after extracellular ice nucleation at -5 degrees C. Post-thaw plasma membrane integrity and mitochondria depolarization were determined using trypan blue and the lipophilic, cyanine derivative JC-1, respectively. alamarBlue was used to assess the post-thaw metabolic activity of the cell arrangements. We found that the incidence of plasma membrane damage and mitochondria integrity increased with decreasing temperature and was dependent on the degree of cell-cell interaction. Mitochondria damage was evident in cells that displayed intact plasma membranes, however this injury could be reversed in the micropatterned cells that are exposed to suprazero temperatures. The results from this study suggest that the exclusive use of membrane integrity as a measure of cell "viability" does not consider subcellular injury that may contribute to delayed recovery and/or cell death following low temperature exposures.
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Mechanism of hemoglobin-induced cellular injury in desiccated red blood cells.
Free Radic. Biol. Med.
PUBLISHED: 02-16-2010
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The current practice of red blood cell banking for transfusion medicine relies primarily on a 6-week liquid storage. A growing demand for red blood cell products has prompted the search for alternative preservation methods, including dry storage. We present a new mechanistic understanding of desiccation-induced cellular injury that is correlated with the oxidative state of the hemoglobin. We demonstrate that water loss induces a drastic increase in the rate of hemoglobin oxidation, formation of intracellular reactive oxygen species, and cellular death. Pharmacological treatment of the hemoglobin oxygen binding site reveals that hemoglobin-induced cellular injury is more prominent in red blood cells that are partially hydrated (about 5.5 to 3.5 g H(2)O/g dry wt) than in cells that are relatively dry (
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Red blood cell hemolysis during blood bank storage: using national quality management data to answer basic scientific questions.
Transfusion
PUBLISHED: 12-13-2009
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Hemolysis of red blood cells (RBCs) during blood bank storage is the most obvious manifestation of RBC storage system failure. However, its analysis is made difficult because the largest source of interunit difference is donor specific. Availability of data from national blood systems on large numbers of RBC units used for internal quality control (QC) purposes and stored and processed in uniform ways permits statistical analysis.
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Biopreservation of red blood cells--the struggle with hemoglobin oxidation.
FEBS J.
PUBLISHED: 11-26-2009
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One of the least recognized causes of cellular damage during ex vivo preservation of red blood cells is oxidative injury to the hemoglobin. The latter has been associated with hemolysis through the release of toxic substances and oxidation of vital cell components. This review delineates some of the major pathways that link hemoglobin oxidation and cellular damage, and summarizes the incidence of red blood cell oxidative injury during hypothermic storage, cryopreservation and desiccation stress. Red blood cell hypothermic storage, despite its success, is not exempt from oxidative injury. Growing evidence portrays a time-dependant oxidative assault including formation of reactive oxygen species, attachment of denatured hemoglobin to membrane phospholipids and the release of hemoglobin-containing membrane microvesicles throughout storage. Similar symptoms have been observed in attempts to stabilize red blood cells in the dried state, in which methemoglobin levels of reconstituted red blood cells reached 50%. Factors affecting the rate of hemoglobin oxidation during red blood cell ex vivo storage include compromised antioxidant activity, high concentrations of glucose in the storage media and the presence of molecular oxygen. Hemoglobin oxidation largely dictates our ability to effectively preserve red blood cells. Understanding its origins along with investigating methods to minimize it can significantly improve the quality of our future blood products.
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Blood preservation workshop: new and emerging trends in research and clinical practice.
Transfus Med Rev
PUBLISHED: 07-01-2009
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Preserving cell viability and function is an essential component in the translation and delivery of existing and emerging cell-based therapeutics from the research lab to the patient bedside. This workshop provided a summary of the advances and challenges that currently face the preservation sciences, together with a glimpse at the future applications and instrumentation that will enhance our ability to process, preserve, and store red blood cells (RBCs), platelets, and stem cells. It is clear from the presentations made during the workshop and the discussions that ensued after that, for us to overcome the challenges that face blood biopreservation, it will require a concerted effort from clinicians, scientists, and engineers from a variety of disciplines. Through this interdisciplinary research effort, significant progress will be made to improve the safety, quality, and potency of the blood products that are used in reparative medicine. As the need for effective preservation technologies will be the motivation for more concerted efforts in the biopreservation sciences, there are encouraging prospects for the future applications of biopreserved blood cells.
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Use of supernatant refractive index and supernatant hemoglobin concentration to assess residual glycerol concentration in cryopreserved red blood cells.
Clin. Chim. Acta
PUBLISHED: 03-26-2009
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Red blood cells (RBCs) cryopreserved in glycerol must be deglycerolized prior to transfusion. The adequacy of glycerol removal is commonly assessed by measurement of the refractive index (RI) of the supernatant fluid. However, the presence of free hemoglobin in the supernatant falsely increases the RI and may lead to discard of units that have an acceptable residual glycerol concentration.
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Effects of trehalose-loaded liposomes on red blood cell response to freezing and post-thaw membrane quality.
Cryobiology
PUBLISHED: 03-13-2009
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We are investigating the use of liposomes, which are synthetic, microscopic vesicles, for the intracellular delivery of trehalose into mammalian cells. This study focuses on the effects trehalose-containing liposomes improve the recovery and membrane quality of human RBCs following cryopreservation. Unilamellar liposomes consisting of a lipid bilayer composed of DPPC, PS and cholesterol (60:30:10 mol%) were synthesized using an extrusion method. Liposome-treated RBCs (l-RBCs) were resuspended in either physiological saline, 0.3M trehalose or liposome solution, then cooled with slow (0.95+/-0.02 degrees C/min), medium (73+/-3 degrees C/min) and fast (265+/-12 degrees C/min) cooling rates and storage in liquid nitrogen, followed by a 37 degrees C thawing step. RBC post-thaw quality was assessed using percent recovery, RBC morphology, PS and CD47 expression. Liposome treatment did not adversely affect the RBC membrane. Post-thaw recovery of l-RBCs was significantly higher (66%+/-5% vs 29%+/-4%) compared to control RBCs (c-RBC, p=0.003). Medium and high cooling rates resulted in significantly higher cell recovery compared to a slow cooling rate (p=0.039 and p=0.041, respectively). The recovery of l-RBCs frozen in liposome solution and trehalose solution was significantly higher than that of l-RBCs frozen in NaCl solution for all three cooling rates (p=0.021). Flow cytometry and morphology assessment showed that liposome treatment resulted in improved post-thaw membrane quality. There was no statistically significant difference in the post-thaw recovery between RBCs treated with liposomes containing trehalose in their aqueous core and RBCs treated with liposomes containing saline in their aqueous core (p=0.114). Liposome treatment significantly improves the recovery and membrane integrity of RBCs following low temperature exposure.
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Trehalose loading into red blood cells is accompanied with hemoglobin oxidation and membrane lipid peroxidation.
Cryobiology
PUBLISHED: 01-13-2009
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One of the recent approaches to enhance desiccation tolerance in red blood cells (RBCs) is by loading trehalose. This process has been shown to increase the recovery of lyophilized RBCs; conversely, it results in cellular damage including hemoglobin oxidation and loss of membrane integrity. The purpose of this study was to further investigate the extent of oxidative injury during the loading of trehalose into RBCs. RBCs were incubated in the absence (control) or presence of trehalose (0.8 mol/l) at 4 degrees C or 37 degrees C for different time scales. Oxidative damage was monitored by flow cytometry using dichlorofluorescin for reactive oxygen species formation, Annexin V-FITC for phosphatidylserine translocation and fluorescein-DHPE for lipid peroxidation. Percent methemoglobin, percent hemolysis and thiobarbituric acid reactive substances were measured by spectrophotometry. The extent of oxidative damage during trehalose loading is affected by the incubation temperature, incubation time and the presence of trehalose. Incubation at 4 degrees C was relatively innocuous; however, oxidative injury was evident at 37 degrees C in both RBC groups. The addition of trehalose is correlated with high osmotic pressure, which had minor effects during incubation at 4 degrees C, but seemed to have exacerbated the severity of cellular injury at 37 degrees C, as measured by higher levels of hemolysis, methemoglobin and lipid peroxidation. The process of trehalose-loading is problematic due to its requirement for prolonged incubations at 37 degrees C. These conditions are correlated with oxidative injury, even in the absence of trehalose. While trehalose is believed to be crucial for stabilizing biomembranes, the consequences of its introduction into the cells require further investigation.
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Phospholipidomics reveals differences in glycerophosphoserine profiles of hypothermically stored red blood cells and microvesicles.
Biochim. Biophys. Acta
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During their normal in vivo life cycle erythrocytes (red blood cells, RBCs) undergo biochemical changes leading to membrane microvesiculation and shedding. RBC microvesiculation also occurs in vitro under conditions of blood bank storage, so microvesicles (MVs) accumulate in the storage (preservation) medium over storage time. Considerable effort has been put into gaining a mechanistic understanding of the RBC microvesiculation process, as this is crucial to better understand RBC biology in disease and in health. Additionally, MVs accumulated in stored RBCs have been implicated in transfusion adverse inflammatory reactions, with chloroform extractable compounds, thus lipophilic, known to trigger the effect. However, because thin layer chromatography resolution of RBC and MV lipids has always enabled one to conclude high compositional similarities, in depth analysis of MV lipids has not been extensively pursued. Here we present an orbitrap mass spectrometry (MS) approach to compare the phospholipid composition of RBCs and MVs from leukoreduced, hypothermically (2-6°C) stored RBC units. We used shotgun MS analysis and electrospray ionization (ESI) intra-source separation, and demonstrated high similarity of compositional profiles, except for glycerophosphoserines (PS). Contrasting abundances of PS 38:4 and PS 38:1 characterized MV and RBC profiles and suggested that storage-associated microvesiculation possibly involves shedding of specific membrane rafts. This finding indicates that phospholipidomics could likely contribute to a better understanding of the RBC microvesiculation process.
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Evaluating the 4-hour and 30-minute rules: effects of room temperature exposure on red blood cell quality and bacterial growth.
Transfusion
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A 30-minute rule was established to limit red blood cell (RBC) exposure to uncontrolled temperatures during storage and transportation. Also, RBC units issued for transfusion should not remain at room temperature (RT) for more than 4 hours (4-hour rule). This study was aimed at determining if single or multiple RT exposures affect RBC quality and/or promote bacterial growth.
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Validation and implementation of a new hemoglobinometer for donor screening at Canadian Blood Services.
Transfusion
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Hemoglobin (Hgb) determination is an essential part of donor qualification. We assessed and implemented a new spectrophotometer for donor Hgb determination.
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An analysis of the bias in red blood cell hemolysis measurement using several analytical approaches.
Clin. Chim. Acta
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Percentage hemolysis in red cell concentrates (RCC) for transfusion is an indicator of RBC damage. As several factors need to be measured to determine hemolysis, and multiple assays are available for each, the choice of analytical methodology could critically influence results.
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Preserving and using germplasm and dissociated embryonic cells for conserving Caribbean and Pacific coral.
PLoS ONE
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Coral reefs are experiencing unprecedented degradation due to human activities, and protecting specific reef habitats may not stop this decline, because the most serious threats are global (i.e., climate change), not local. However, ex situ preservation practices can provide safeguards for coral reef conservation. Specifically, modern advances in cryobiology and genome banking could secure existing species and genetic diversity until genotypes can be introduced into rehabilitated habitats. We assessed the feasibility of recovering viable sperm and embryonic cells post-thaw from two coral species, Acropora palmata and Fungia scutaria that have diffferent evolutionary histories, ecological niches and reproductive strategies. In vitro fertilization (IVF) of conspecific eggs using fresh (control) spermatozoa revealed high levels of fertilization (>90% in A. palmata; >84% in F. scutaria; P>0.05) that were unaffected by tested sperm concentrations. A solution of 10% dimethyl sulfoxide (DMSO) at cooling rates of 20 to 30°C/min most successfully cryopreserved both A. palmata and F. scutaria spermatozoa and allowed producing developing larvae in vitro. IVF success under these conditions was 65% in A. palmata and 53% in F. scutaria on particular nights; however, on subsequent nights, the same process resulted in little or no IVF success. Thus, the window for optimal freezing of high quality spermatozoa was short (?5 h for one night each spawning cycle). Additionally, cryopreserved F. scutaria embryonic cells had?50% post-thaw viability as measured by intact membranes. Thus, despite some differences between species, coral spermatozoa and embryonic cells are viable after low temperature (-196°C) storage, preservation and thawing. Based on these results, we have begun systematically banking coral spermatozoa and embryonic cells on a large-scale as a support approach for preserving existing bio- and genetic diversity found in reef systems.
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Synergistic effects of liposomes, trehalose, and hydroxyethyl starch for cryopreservation of human erythrocytes.
Biotechnol. Prog.
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Red blood cells (RBCs) can be cryopreserved using glycerol as a cryoprotective agent, but one of the main disadvantages is the time-consuming deglycerolization step. Novel cryopreservation strategies for RBCs using nontoxic cryoprotective agents are urgently needed. The effect of DMPC, DOPC, and DPPC liposomes on survival of RBCs cryopreserved with trehalose and HES has been evaluated. DMPC caused hemolysis before freezing and affected RBC deformability parameters. DMPC treated RBCs displayed a strong increase in trehalose uptake compared to control cells, whereas DOPC treated liposomes only displayed a slight increase in trehalose uptake. High intracellular trehalose contents were observed after cryopreservation. The recovery of cells incubated with trehalose and liposomes, frozen in HES ranged between 92.6 and 97.4% immediately after freezing. Recovery values of RBCs frozen in HES, however, decreased to 66.5% after 96 h at 4°C compared to 77.5% for DOPC treated RBCs. The recovery of RBCs incubated and frozen in trehalose medium was 77.8%. After 96 hours post-thaw storage recovery of these cells was 81.6%. DOPC and DPPC treated RBCs displayed higher recovery rates (up to 89.7%) after cryopreservation in trehalose compared to control RBCs. Highest survival rates were obtained using a combination of trehalose and HES: 97.8% directly after thawing and 81.8% 96-h post-thaw. DOPC liposomes, trehalose and HES protect RBCs during cryopreservation in a synergistic manner. The advantage is that the protective compounds do not need to be removed before transfusion.
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In vitro platelet quality in storage containers used for pediatric transfusions.
Transfusion
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The in vitro quality of small-volume platelet (PLT) aliquots for pediatric transfusions was assessed to determine the best practice approach.
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What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

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In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.