Preservation Of Erythrocytes Research Articles

A novel mini-invasive finger-prick protocol for preparing blood micro-samples for morphological analysis of human erythrocytes by scanning electron microscopy

Here we describe the development and optimization of a new protocol for the preparation and surface imaging by scanning electron microscope of human erythrocytes from blood micro-samples obtained by finger prick. By testing several key pre-analytical conditions for blood sampling, erythrocyte preservation, storage and imaging, we designed a rapid new minimally-invasive reproducible method for obtaining uniform deposition of an adequate number of erythrocytes with well-preserved morphology on a substrate. The possibility of obtaining reliable reproducible high resolution morphometric data on peripheral erythrocytes makes this protocol valuable for diagnostic and basic research purposes.

Cryopreservation of Canine Erythrocytes Using Dimethyl Sulfoxide, Polyethylene Glycol and Sucrose

Cryoprotective properties of combined media of permeable (dimethyl sulfoxide) and impermeable (polyethylene glycol with m. w. 1500) cryoprotective agents during rapid cooling in liquid nitrogen of canine erythrocytes using saline and sucrose-saline media have been investigated. It was found that the use of combined solutions of cryoprotective agents based on polyethylene glycol with m.w. 1500 (15%) and dimethyl sulfoxide (2.5–10%) in saline was not quite effective for cryopreservation of canine erythrocytes. Reducing the salt concentration and adding cell-impermeable sucrose to the cryopreservation medium increase the preservation of erythrocytes after warming. The best cryoprotective properties for canine erythrocytes were demonstrated by 10% dimethyl sulfoxide based on sucrose-saline medium, with high preservation of cells after freeze-warming, mechanical and osmotic stability of warmed erythrocytes. This indicates the possibility of a long-term storage and use of cryopreserved canine erythrocytes for transfusions.

Effect of ozonation on resistance of ovine and human erythrocytes to hypothermic storage

Long-term hypothermic storage of animal blood can lead to the loss of its quality and can cause complications in recipient animals after transfusion, so the search for new methods of increasing the preservation of erythrocytes after hypothermic storage continues. The article presents the data of the ozonation effect on the preservation rate of ovine and human erythrocytes during hypothermic storage with Alsever’s solution and mannitol medium. Hemolysis, osmotic fragility and distribution density of ovine and human erythrocytes by the sphericity index were determined at different stages of hypothermic storage. The ovine erythrocytes in the control had a lower osmotic resistance compared to human erythrocytes; however, their osmotic fragility did not change significantly after hypothermic storage for 8 weeks, and for human erythrocytes, it significantly increased. Storage of ovine and human erythrocytes longer than 8 weeks led to a sharp hemolysis, while the level of hemolysis of ovine erythrocytes was lower than that of human erythrocytes. Preservation of ozonated erythrocytes is higher than non-ozonated ones during prolonged hypothermic storage. The effect of ozonation on the preservation rate of red blood cells depended on the composition of the preservation media. Hypothermal storage of human erythrocytes in Alsever’s solution for up to 8 weeks led to a shift in the density of distribution according to the sphericity index towards spheroidization of cells; in a medium with mannitol, the number of flattened cell forms increased. After 8 weeks of hypothermic storage of ovine erythrocytes, most of the cells had high sphericity indices. Pretreatment of human and sheep erythrocytes with ozone allows the distribution of cells to be kept closer to the control during long-term storage, which is especially pronounced in mannitol medium. Ovine erythrocytes retained the ability to form rosettes with human T-lymphocytes after hypothermic storage for up to 12 weeks. Thus, ozone pretreatment and the use of mannitol as part of hypothermic storage medium could be an approach to improve the quality of preserved ovine erythrocytes.

Ultrasound-induced molecular delivery to erythrocytes using a microfluidic system.

Preservation of erythrocytes in a desiccated state for storage at ambient temperature could simplify blood transfusions in austere environments, such as rural clinics, far-forward military operations, and during space travel. Currently, storage of erythrocytes is limited by a short shelf-life of 42 days at 4 °C, and long-term preservation requires a complex process that involves the addition and removal of glycerol from erythrocytes before and after storage at -80 °C, respectively. Natural compounds, such as trehalose, can protect cells in a desiccated state if they are present at sufficient levels inside the cell, but mammalian cell membranes lack transporters for this compound. To facilitate compound loading across the plasma membrane via ultrasound and microbubbles (sonoporation), a polydimethylsiloxane-based microfluidic device was developed. Delivery of fluorescein into erythrocytes was tested at various conditions to assess the effects of parameters such as ultrasound pressure, ultrasound pulse interval, microbubble dose, and flow rate. Changes in ultrasound pressure and mean flow rate caused statistically significant increases in fluorescein delivery of up to 73 ± 37% (p < 0.05) and 44 ± 33% (p < 0.01), respectively, compared to control groups, but no statistically significant differences were detected with changes in ultrasound pulse intervals. Following freeze-drying and rehydration, recovery of viable erythrocytes increased by up to 128 ± 32% after ultrasound-mediated loading of trehalose compared to control groups (p < 0.05). These results suggest that ultrasound-mediated molecular delivery in microfluidic channels may be a viable approach to process erythrocytes for long-term storage in a desiccated state at ambient temperatures.

Correlation of cavitation activity with ultrasound-enhanced delivery of compounds to erythrocytes ex vivo

Blood transfusions are one of the most common medical procedures in hospitals, but shortages of erythrocytes often occur due to their limited shelf life (6 weeks) when refrigerated. Preservation of erythrocytes in a dried state offers a potential solution to challenges faced with blood storage, and preservative compounds such as trehalose have been identified in organisms that survive desiccation in nature. However, these compounds do not readily cross mammalian cell membranes. Therefore, we are exploring the use of sonoporation to facilitate their delivery into erythrocytes ex vivo. In this study, we assessed the effect of cavitation activity on delivery of a fluorescent compound similar in size to trehalose (fluorescein) to human erythrocytes. Microbubbles were added to erythrocyte solutions and sonicated (2.5 MHz, 4 cycles) at various pressures and durations. Fluorescence was quantified with flow cytometry. The amplitude of broadband emissions in the first 8 seconds of sonication did not correlate with delivery (r2 = 0.23), whereas after 8 seconds the broadband emissions amplitude was associated with increased delivery to erythrocytes (r2 = 0.97). These results suggest that the timing of cavitation activity, rather than the amplitude alone, may be an important factor in ultrasound-mediated delivery of compounds into erythrocytes.

СУЧАСНІ УЯВЛЕННЯ ПРО МЕТОДИ КРІОКОНСЕРВУВАННЯ ЕРИТРОЦИТІВ ТВАРИН

Nowadays, the method of blood transfusion is often used in veterinary practice. Hypothermic storage allows to save blood cells for a limited time, while morphofunctional parameters are getting worse. Cryopreservation allows to save and receive high-quality cells for the use in veterinary practice. Therefore, the development of reserves of donor blood is possible with long-term preservation in the frozen state. The use of cryopreservation makes it possible to avoid a number of problems: finding a donor at the right time for transfusion, the cost of maintaining the donor, etc. These days high therapeutic efficacy of using cryopreserved red blood cells was confirmed in intensive therapy and hematological diseases. The survival of biological objects under cryopreservation conditions is due to the ability of cells to withstand a complex of negative factors, including: the formation of crystals, an increase in the concentration of salts and osmotic pressure, dehydration of macromolecules and phase transitions of membrane lipids. Fundamental studies of physicochemical processes in cell suspensions under cooling and freezing conditions revealed important patterns that determine the basic principles of damage and protection of biological objects. The safety of cells under the influence of factors of low-temperature exposure is ensured by the use of special agents&nbsp; -&nbsp; cryoprotectants. Cryoprotective agents belong to different classes of organic compounds and are able to protect cells according to endo-and exocellular principle. However, the high efficiency of cryopreservation of red blood cells is achieved mainly by using endocellular compounds which must be removed after thawing from cell suspensions in order to maintain osmotic stability of cells under physiological conditions. Alternative to this may be cell-free cell cryopreservation methods developed on the basis of exocellular cryoprotectants. It should be noted that the cryoprotective properties of different compounds selectively manifest themselves depending on the type of cells. Biological preservation of erythrocytes for the use in veterinary practice is based on technologies for achieving biological stability and, accordingly, preserving a viable state after prolonged storage. This article reviews the mechanisms of cryodamage of erythrocytes, the cryoprotectants used in cryopreservation, as well as the existing low temperature storage methods.

An Unrecognized Function of Cholesterol: Regulating the Mechanism Controlling Membrane Phospholipid Asymmetry.

An asymmetric distribution of phospholipids in the membrane bilayer is inseparable from physiological functions, including shape preservation and survival of erythrocytes, and by implication other cells. Aminophospholipids, notably phosphatidylserine (PS), are confined to the inner leaflet of the erythrocyte membrane lipid bilayer by the ATP-dependent flippase enzyme, ATP11C, counteracting the activity of an ATP-independent scramblase. Phospholipid scramblase 1 (PLSCR1), a single-transmembrane protein, was previously reported to possess scrambling activity in erythrocytes. However, its function was cast in doubt by the retention of scramblase activity in erythrocytes of knockout mice lacking this protein. We show that in the human erythrocyte PLSCR1 is the predominant scramblase and by reconstitution into liposomes that its activity resides in the transmembrane domain. At or below physiological intracellular calcium concentrations, total suppression of flippase activity nevertheless leaves the membrane asymmetry undisturbed. When liposomes or erythrocytes are depleted of cholesterol (a reversible process in the case of erythrocytes), PS quickly appears at the outer surface, implying that cholesterol acts in the cell as a powerful scramblase inhibitor. Thus, our results bring to light a previously unsuspected function of cholesterol in regulating phospholipid scrambling.

Construction of d-α-tocopheryl polyethylene glycol succinate/PEO core-shell nanofibers on a blood-contacting surface to reduce the hemolysis of preserved erythrocytes.

The hemolysis of erythrocytes is a big obstacle to the development of new non-plasticizer polymer containers for erythrocyte preservation. To construct a long-term anti-hemolytic surface of a plasticizer-free polymer, we coaxially electrospin core-shell structured d-α-tocopheryl polyethylene glycol 1000 succinate (TPGS)/poly(ethylene oxide) nanofibers on the surface of a styrene-b-(ethylene-co-butylene)-b-styrene (SEBS) elastomer that is covered with grafted poly(ethylene glycol) (PEG) chains. Our strategy is based on the fact that the grafted layers of PEG reduce mechanical damage to red blood cells (RBCs) while the TPGS released from the nanofibers on a blood-contacting surface can act as an antioxidant to protect RBCs from oxidative damage. We demonstrate that TPGS/PEO core-shell structured nanofibers have been well prepared on the surface of PEG modified SEBS; the controlled release of TPGS in distilled water is obtained and the release can last for almost 4 days at 4 °C; during RBC preservation, TPGS acts as the antioxidant to decrease the membrane oxidation and hemolysis of RBCs. Our work paves a new way for the development of non-plasticizer polymers for RBC preservation, which may be helpful for the fabrication of long-term anti-hemolytic biomaterials in vivo.

148 Defining the operating space for spray drying of red blood cells

The ability to store red blood cells (RBCs) in the dry state at room temperature would be extremely beneficial, particularly in areas where reliable access to electricity is unavailable. In this study, we explore the potential for using spray drying for preservation of RBCs in the dried state. Spray drying involves atomization of a liquid feed into small droplets and drying of the droplets using a heated gas stream, resulting in a powder composed of the solid components of the liquid feed. Previously, we demonstrated the ability to atomize a suspension of RBCs using a two-fluid nozzle with humidified nitrogen gas without significant shear-induced hemolysis [M. McLean, X.Y. Han, A.Z. Higgins, Spray Drying for Preservation of Erythrocytes: Effect of Atomization on Hemolysis, Biopreservation and Biobanking 11 (2013) 122–123]. Here, we explore the potential spray drying process space using bench-scale tests in order to understand the balance between producing sufficiently dry RBC powder and maintaining the integrity of thermally labile RBCs. We investigated the temperature sensitivity of RBCs in order to determine a maximum tolerated drying gas temperature, recognizing that evaporative cooling of drying droplets results in droplet temperatures at wet-bulb. Suspensions of RBCs were exposed to temperatures up to 65 °C for a period of 10 s. At 50 °C and below the RBCs remained intact, exhibiting hemolysis levels of less than 10%. However, exposure to higher temperatures resulted in substantial increases in hemolysis. A wet bulb temperature of 50 °C is consistent with a drying gas inlet temperature of 115 °C at 5% relative humidity, indicating that a process inlet temperature of 115 °C would result in less than 10% hemolysis due to process temperatures. We also investigated the equilibrium relationship between relative humidity and RBC moisture content by drying RBC suspensions in the presence of various saturated salt solutions. The results of these experiments indicate that an equilibrium moisture content of 5% corresponds with a relative humidity between 15% and 20%. Assuming equilibrium at the drying chamber outlet, these results bracket the liquid feed to drying gas feed ratio necessary to achieve a target moisture content of 5%. In addition, our current results together with our previous measurements of droplet size after atomization allow estimation of the droplet drying time. The predicted drying time for a 70 μ diameter aqueous droplet in isolation with a drying gas at 115 °C is on the order of 1 s. Although in actual practice increased mass transfer resistance would be expected as a result of droplet–droplet interactions, previous experimental observations of industrial spray drying confirm the drying time estimate to within an order of magnitude. This short drying time underscores a major advantage of spray drying as a method for achieving the dry state. Source of funding: None declared. Conflict of interest: None declared. adam.higgins@oregonstate.edu

Technical note: A histological technique for detecting the cryptic preservation of erythrocytes and soft tissue in ancient human skeletonized remains

Bone samples from a Middle Bronze Age (ca., 1600-1300 BC) site were prepared for histological analysis. Preliminary results suggested that components of bone marrow remained preserved. To verify these findings and optimize the sample preparation procedure, we conducted experiments varying the type of acid used to decalcify the bones for histology preparation, as well as the exposure time to the demineralizing agents and thickness of sections taken from the samples for slide preparation. Subsequent examination of the slides revealed the presence of well-preserved erythrocytes and other cellular structures consistent with bone marrow. Our results demonstrate that the traditional methods used to prepare bone samples for histology may be adjusted to improve the quality of the soft tissue architecture and cellular morphology for histological observation.

Spray drying for preservation of erythrocytes: effect of atomization on hemolysis.

Spray drying has the potential to enable storage of erythrocytes at room temperature in the dry state. The spray drying process involves atomization of a liquid into small droplets and drying of the droplets in a gas stream. In this short report, we focus on the atomization process. To decouple atomization from drying, erythrocyte suspensions were sprayed with a two-fluid atomizer nozzle using humid nitrogen as the atomizing gas. The median droplet size was less than 100 μm for all of the spray conditions investigated, indicating that the suspensions were successfully atomized. Hemolysis was significantly affected by the hematocrit of the erythrocyte suspension, the suspension flow rate, and the atomizing gas flow rate (p<0.01 in all cases). Under appropriate conditions, it was possible to achieve less than 2% hemolysis, suggesting that spray drying may be a feasible option for erythrocyte biopreservation.

A Proteomics-Based Translational Approach Reveals an Antifolate Resistance Inherent in Human Plasma Derived from Blood Donation

The inhibition of dihydrofolate reductase (DHFR) by antifolates is a common practice both in cell culture and in chemotherapy. Surprisingly, antifolate resistance was also observed in cultured murine myeloma cells (SP2/0) in the presence of human plasma (HP); thus, we used a proteomic approach to identify novel plasma biomarker(s) for this condition. In contrast to the in vitro antifolate response, metabolic enzymes and translation machinery proteins were found to be up-regulated in the presence of HP. The antifolate resistance inherent in HP may be explained by a simultaneous promotion of cell proliferation and the maintenance of DNA integrity. Furthermore, the factor(s) was found to be extrinsic, heat stable and very small in size. Adenine, a supplemented additive in erythrocyte preservation, was subsequently identified as the contributing factor and exogenous addition in cultures reversed the cytotoxicity induced by antifolates. Importantly, adenine-containing blood components, which may provide enhanced survival to otherwise sensitive antifolate-targeted cells, showed a dose-dependent adverse effect in transfusion recipients receiving antifolate (methotrexate) medications. These findings not only highlight a previously unnoticed role of adenine, but also emphasize a novel mechanistic link between transfusion and subsequently reduced survival in patients taking methotrexate.

Effect of Vitamin E on Human Erythrocyte Preservation Solutions

Objective To explore a protocol for prolonging the in vitro preservation of red blood cells (RBCs)for biological tests.Methods The fresh blood of healthy adult was preservated in the well-established blood eell preservation solutions(CPDA and the Alserver's solution).After the preservation solutions were added by Vitamin E as an additive,the RBC-SOD,RBC-ATP,free hemoglobin(Free Hb)were examined every 7 days in 56-day experimental period.Results The average lifespan of RBCs had risen from 28 days to 42 days in CPDA+Vitamin E and from 7 days to 21 days in Alserve'S solution+Vitamin E respectively at CC.However,the preservation effects of two kinds of solutions are the same in initial 7 days.Conclusion Vitamin E can significantly enhance the protection effects of well-established erythroeyte prescrvation solutions.It is recommended that valuable RBCs sample can be preserved for short period of time in the simpled-component Alserve's with the additive Vitamin E.and be long-term saved with the balanced CPDA and Vitamin E. Key words: red cell preservation; CPDA; Alserve'S; Vitamin EEffect of Vitamin E on Human Erythrocyte Preservation Solutions,LIU Jian,LJ Xiao-mian

Effect of the haematocrit layer geometry on Plasmodium falciparum static thin-layer in vitro cultures

BackgroundIn vitro cultivation of Plasmodium falciparum is usually carried out through the continuous preservation of infected erythrocytes deposited in static thin layers of settled haematocrit. This technique, called the candle-jar method, was first achieved by Trager and Jensen in 1976 and has undergone slight modifications since then. However, no systematic studies concerning the geometry of the haematocrit layer have been carried out. In this work, a thorough investigation of the effects of the geometric culturing conditions on the parasite's development is presented.MethodsSeveral experimental trials exploring different settings have been carried out, covering haematocrit layer depths that ranged from 6 mm to 3 mm and separation between the walls of the culturing device that ranged from 7.5 mm to 9 mm. The obtained results have been analysed and compared to different system-level models and to an Individual-Based Model.ConclusionIn line with the results, a mechanism governing the propagation of the infection which limits it to the vicinity of the interface between the haematocrit layer and the culture medium is deduced, and the most appropriate configurations are proposed for further experimental assays.

Mammalian erythrocyte preservation in a 3500-year-old Egyptian pot

A ca. 3500-year-old Egyptian pot from the Fayoum site of Gurob contains ethmoid bones characteristic of the sinuses and morphological remains of erythrocytes. The pot contents are interpreted as the remnants of an embalmers studio, and substantiate records of mummification.

Red Blood Cells (RBC) and High Fluorescence Reticulocytes (HFR) production increased by induction chemotherapy and GM-CSF plus G-CSF in peripheral blood of breast cancer patients

The preservation of erythrocytes is important during cytostatic chemotherapy. To study the synergistic effect of GM-CSF plus G-CSF on the red blood recovery, we looked at the kinetics of red blood cells, total reticulocytes and High Fluorescence Reticulocytes (HFR) from peripheral blood of 26 breast cancer patients, between 2 cycles of chemotherapy. After four days of TNCF (Thp-doxorubicin, Vinorelbine, Cyclophosphamide, Fluorouracil) treatment, 12 patients received either GM-CSF or G-CSF (D5-D16) and 14 received both of them (GM-CSF, D5-D14 and G-CSF, D10-D14). Our results showed a significantly lesser RBC decrease for patients receiving both CSFs versus only one CSF (p < 0.05). This greater RBC preservation was explained by the increase of HFR and total reticulocytes from D12 to D17 in the peripheral blood of patients receiving the two CSFs, with significantly values greater than that of patients receiving one CSF (p < 0.01) it showed that the synergistic GM-CSFplus G-CSF effect observed previously on PBPC results in a positive effect on erythroid lineage. GM-CSF plus G-CSF association exhibit in vivo multilineage (myeloid and erythroid) activity showing the interest to use different cytokines in combination to obtain a better hematological recovery after induction chemotherapy as well as a better quality of PBPC mobilization for transplantation after high-dose chemotherapy.

Stability of blood coagulation factors and inhibitors in blood drawn into half-strength citrate anticoagulant.

Drawing of blood into a citrate-phosphate-dextrose (CPD) solution with a reduced citrate concentration has been shown to improve the maintenance of coagulation factor VIII (F VIII) in plasma and to give possibilities to improve erythrocyte preservation. We studied the quality of plasma obtained from whole blood drawn under continuous mixing into CPD in which the citrate concentration was reduced by 50% (0.5CPD). The blood was stored at room temperature for 8 h before component preparation. We confirmed improved stability of F VIII by 0.5CPD. We found no clinically significant changes in inhibitors to the coagulation and kallikrein systems, and no signs of activation of these systems, during the 8-hour holding time. In control blood drawn into CPD, F VIII and coagulation factor IX decreased by 0.09 IU/ml (8%) and 0.07 U/ml (7%), respectively, otherwise we found no significant differences between 0.5CPD plasma and CPD plasma.

Stability of Blood Coagulation Factors and Inhibitors in Blood Drawn into Half-Strength Citrate Anticoagulant

Drawing of blood into a citrate-phosphate-dextrose (CPD) solution with a reduced citrate concentration has been shown to improve the maintenance of coagulation factor VIII (F VIII) in plasma and to give possibilities to improve erythrocyte preservation. We studied the quality of plasma obtained from whole blood drawn under continuous mixing into CPD in which the citrate concentration was reduced by 50% (0.5CPD). The blood was stored at room temperature for 8 h before component preparation. We confirmed improved stability of F Vili by 0.5CPD. We found no clinically significant changes in inhibitors to the coagulation and kallikrein systems, and no signs of activation of these systems, during the 8-hour holding time. In control blood drawn into CPD, F Vili and coagulation factor IX decreased by 0.09 IU/ml (8%) and 0.07 U/ml (7%), respectively, otherwise we found no significant differences between 0.5CPD plasma and CPD plasma.

Sem analysis of red blood cells in aged human bloodstains

Mammal red blood cells (RBC) in bloodstains have been previously detected by light microscopy on stone tools from as early as 100 000 ± 25 000 years ago. In order to evaluate the degree of morphological preservation of erythrocytes in bloodstains, an accidental human blood smear on white chert and several experimental bloodstains on hard substrates (the same stone - white chert; another type of stone - graywacke; a non-stone support - stainless steel), were stored in a room, in non-sterile and fluctuating conditions, for lengths of time ranging from 3 to 18 months. Afterwards, the specimens were coated with gold and examined by a Cambridge Stereoscan 120 scanning electron microscope. Results revealed a high preservation of RBC integrity, with the maintenance of several discocytary shapes, a low tendency to echinocytosys and a frequent appearance of a moon-like erythrocytary shape in the thinner areas of the bloodstains.

169 PURINE METABOLISM IN NORMAL AND ITP PYROPHOSPHOHYDRO-LASE-DEFICIENT ERYTHROCYTES

The activity of ITP pyrophosphohydrolase (ITP-ase) was measured in erythrocyte lysates of 48 healthy volunteers. This group could clearly be divided into two different polulations. Mean activity was 48.3 ± 13.1 nkat/g Hb (x ±SD, n=38) in one population, and only one-fourth thereof in the other population (11.4 ± 4.3 nkat/g Hb, n=10). In two subjects, previously selected because of high levels of ITP in fresh or stored erythrocytes, the ITP-ase activity was 0.2 and 2.4 nkat/g Hb, respectively.In normal erythrocytes incubated in phosphate-rich buffer (20 mM), the rate of conversion of allopurinol, hypoxanthine and guanine to their respective nucleotides followed a biphasic pattern. Initial “salvage rate” was 130 nmol/l/s, which leveled of after 15 min to about 50 nmol/l/s. Exactly the same pattern was observed in erythrocytes from ITP-ase deficient subjects. Routine liquid preservation of erythrocytes from both groups in CPD-SAGMAN (containing citrate, phosphate, dextrose, sodium chloride, adenine, glucose and mannitol) resulted in a slow decline in salvage rate (0,7 % /day). The salvage rate of AICA (aminoimidazolecarboxamide) and AICA-riboside was 8 and 40 nmol/l/s, respectively.We conclude that, despite the fact that the energy consumption in the IMP-ITP cycle can amount to up to 10 % erythrocyte energy turnover in vitro, ITP-ase deficiency and lack of activity in the IMP-ITP cycle, does not influence purine salvage.