Measurement and analysis of ionic leakage profiles in refrigerated human red blood cells using dielectrophoresis and inductively coupled mass spectroscopy.

Human red blood cells (RBCs) undergo ionic leakage through passive diffusion during refrigerated storage, affecting their quality and health. We investigated the dynamics of ionic leakage in human RBCs over a 20-day refrigerated storage period using extracellular ion quantification and dielectrophoresis (DEP). Four type O- human blood donors were examined to assess the relationship between extracellular ion concentrations (Na+, K+, Mg2+, Ca2+, and Fe2+), RBC cytoplasm conductivity, and membrane conductance. A consistent negative correlation between RBC cytoplasm conductivity and membrane conductance, termed the "ionic leakage profile" (ILP), was observed across the 20-day storage period. Specifically, we noted a gradual decline in DEP-measured RBC cytoplasm conductivity alongside an increase in membrane conductance. Further examination of the electrical origins of this ILP using inductively coupled plasma mass spectrometry revealed a relative decrease in extracellular Na+ concentration and an increase in K+ concentration over the storage period. Correlation of these extracellular ion concentrations with DEP-measured RBC electrical properties demonstrated a direct link between changes in the cytoplasmic and membrane domains and the leakage and transport of K+ and Na+ ions across the cell membrane. Our analysis suggests that the inverse correlation between RBC cytoplasm and membrane conductance is primarily driven by the passive diffusion of K+ from the cytoplasm and the concurrent diffusion of Na+ from the extracellular buffer into the membrane, resulting in a conductive reduction in the cytoplasmic domain and a subsequent increase in the membrane. The ILP's consistent negative trend across all donors suggests that it could serve as a metric for quantifying blood bank storage age, predicting the quality and health of refrigerated RBCs.

Functional asymmetry of the urea transporter UT-B in human red blood cells.

We studied urea, thiourea, and methylurea transport and interaction in human red blood cells (RBCs) under conditions of self-exchange (SE), net efflux (NE), and net influx (NI) at pH 7.2. We combined four methods, a four-centrifuge technique, the Millipore-Swinnex filtering technique, the continuous flow tube method, and a continuous pump method to measure the transport of the 14C-labeled compounds. Under SE conditions, both urea and thiourea show perfect Michaelis-Menten kinetics with half-saturation constants, K½,SE (mM), of ≈300 (urea) and ≈20 (thiourea). The solutes show no concentration-dependent saturation under NE conditions. Under NI conditions, transport displays saturation or self-inhibition kinetics with a K½,NI (mM) of ≈210 (urea) and ≈20 (thiourea). Urea, thiourea, and methylurea are competitive inhibitors of the transport of analog solutes. This study supports the hypothesis that the three compounds share the same urea transport system (UT-B). UT-B functions asymmetrically as it saturates from the outside only under SE and NI conditions, whereas it functions as a high-capacity channel-like transporter under NE conditions. When the red blood cell enters the urea-rich kidney tissue, self-inhibition reduces the urea uptake in the cell. When the cell leaves the kidney, the channel-like function of UT-B implies that intracellular urea rapidly equilibrates with external urea. The net result is that the cell during the passage in the kidney capillaries carries urea to the kidney to be excreted while the urea transfer from the kidney via the bloodstream is minimized.NEW & NOTEWORTHY The kinetics of urea transport in red blood cells was determined by means of a combination of four methods that ensures a high time resolution. In the present study, we disclose that the urea transporter UT-B functions highly asymmetric being channel-like with no saturation under conditions of net efflux and saturable under conditions of net influx and self-exchange in the concentration range 1-1,000 mM (pH 7.2 and 25-38 °C).

Core-Shell Microfiber Encapsulation Enables Glycerol-Free Cryopreservation of RBCs with High Hematocrit.

Cryopreservation of red blood cells (RBCs) provides great potential benefits for providing transfusion timely in emergencies. High concentrations of glycerol (20% or 40%) are used for RBC cryopreservation in current clinical practice, which results in cytotoxicity and osmotic injuries that must be carefully controlled. However, existing studies on the low-glycerol cryopreservation of RBCs still suffer from the bottleneck of low hematocrit levels, which require relatively large storage space and an extra concentration process before transfusion, making it inconvenient (time-consuming, and also may cause injury and sample lose) for clinical applications. To this end, we develop a novel method for the glycerol-free cryopreservation of human RBCs with a high final hematocrit by using trehalose as the sole cryoprotectant to dehydrate RBCs and using core-shell alginate hydrogel microfibers to enhance heat transfer during cryopreservation. Different from previous studies, we achieve the cryopreservation of human RBCs at high hematocrit (> 40%) with high recovery (up to 95%). Additionally, the washed RBCs post-cryopreserved are proved to maintain their morphology, mechanics, and functional properties. This may provide a nontoxic, high-efficiency, and glycerol-free approach for RBC cryopreservation, along with potential clinical transfusion benefits.

Course of Plasmodium infection studied using 2D-COS on human erythrocytes.

BACKGROUND: The threat of malaria is still present in the world. Recognizing the type of parasite is important in determining a treatment plan. The golden routine involves microscopic diagnostics of Giemsa-stained thin blood smears, however, alternative methods are also constantly being sought, in order to gain an additional insight into the course of the disease. Spectroscopic methods, e.g., Raman spectroscopy, are becoming increasingly popular, due to the non-destructive nature of these techniques. METHODS: The study included patients hospitalized for malaria caused by Plasmodium falciparum or Plasmodium vivax, in the Department of Infectious Diseases at the University Hospital in Krakow, Poland, as well as healthy volunteers. The aim of this study was to assess the possibility of using Raman spectroscopy and 2D correlation (2D-COS) spectroscopy in understanding the structural changes in erythrocytes depending on the type of attacking parasite. EPR spectroscopy and two-trace two-dimensional (2T2D) correlation was also used to examine the specificity of paramagnetic centres found in the infected human blood. RESULTS: Two-dimensional (2D) correlation spectroscopy facilitates the identification of the hidden relationship, allowing for the discrimination of Raman spectra obtained during the course of disease in human red blood cells, infected by P. falciparum or P. vivax. Synchronous cross-peaks indicate the processes taking place inside the erythrocyte during the export of the parasite protein towards the cell membrane. In contrast, moieties that generate asynchronous 2D cross-peaks are characteristic of the respective ligand-receptor domains. These changes observed during the course of the infection, have different dynamics for P. falciparum and P. vivax, as indicated by the asynchronous correlation cross-peaks. Two-trace two-dimensional (2T2D) spectroscopy, applied to EPR spectra of blood at the beginning of the infection, showed differences between P. falciparum and P. vivax. CONCLUSIONS: A unique feature of 2D-COS is the ability to discriminate the collected Raman and EPR spectra. The changes observed during the course of a malaria infection have different dynamics for P. falciparum and P. vivax, indicated by the reverse sequence of events. For each type of parasite, a specific recycling process for iron was observed in the infected blood.

Aging Injury Impairs Structural Properties and Cell Signaling in Human Red Blood Cells; Açaì Berry Is a Keystone.

Red blood cell (RBC) deformability is the ability of cells to modulate their shape to ensure transit through narrow capillaries of the microcirculation. A loss of deformability can occur in several pathological conditions, during natural RBC aging through an increase in membrane protein phosphorylation, and/or through the structural rearrangements of cytoskeletal proteins due to oxidative conditions, with a key role played by band 3. Due to the close relationship between aging and oxidative stress, flavonoid-rich foods are good candidates to counteract age-related alterations. This study aims to verify the beneficial role of Açaì extract in a d-Galactose (d-Gal)-induced model of aging in human RBCs. To this end, band 3 phosphorylation and structural rearrangements in membrane cytoskeleton-associated proteins, namely spectrin, ankyrin, and/or protein 4.1, are analyzed in RBCs treated with 100 mM d-Gal for 24 h, with or without pre-incubation with 10 µg/mL Açaì extract for 1 h. Furthermore, RBC deformability is also measured. Tyrosine phosphorylation of band 3, membrane cytoskeleton-associated proteins, and RBC deformability (elongation index) are analyzed using western blotting analysis, FACScan flow cytometry, and ektacytometry, respectively. The present data show that: (i) Açaì berry extract restores the increase in band 3 tyrosine phosphorylation and Syk kinase levels after exposure to 100 mM d-Gal treatment; and (ii) Açaì berry extract partially restores alterations in the distribution of spectrin, ankyrin, and protein 4.1. Interestingly, the significant decrease in membrane RBC deformability associated with d-Gal treatment is alleviated by pre-treatment with Açaì extract. These findings further contribute to clarify mechanisms of natural aging in human RBCs, and propose flavonoid substances as potential natural antioxidants for the treatment and/or prevention of oxidative-stress-related disease risk.

Integration of Trehalose Lipids with Dissociative Trehalose Enables Cryopreservation of Human RBCs.

Cryopreservation of red blood cells (RBCs) is imperative for transfusion therapy, while cryoprotectants are essential to protect RBCs from cryoinjury under freezing temperatures. Trehalose has been considered as a biocompatible cryoprotectant that naturally accumulates in organisms to tolerate anhydrobiosis and cryobiosis. Herein, we report a feasible protocol that enables glycerol-free cryopreservation of human RBCs by integration of the synthesized trehalose lipids and dissociative trehalose through ice tuning and membrane stabilization. Typically, in comparison with sucrose monolaurate or trehalose only, trehalose monolaurate was able to protect cell membranes against freeze stress, achieving 96.9 ± 2.0% cryosurvival after incubation and cryopreservation of human RBCs with 0.8 M trehalose. Moreover, there were slight changes in cell morphology and cell functions. It was further confirmed by isothermal titration calorimetry and osmotic fragility tests that the moderate membrane-binding activity of trehalose lipids exerted cell stabilization for high cryosurvival. The aforementioned study is likely to provide an alternative way for glycerol-free cryopreservation of human RBCs and other types of cells.

A Dynamic Membrane-Active Glycopeptide for Enhanced Protection of Human Red Blood Cells against Freeze-Stress.

Intracellular delivery of freezing-tolerant trehalose is crucial for cryopreservation of red blood cells (RBCs) and previous strategies based on membrane-disruptive activity usually generate severe hemolysis. Herein, a dynamic membrane-active glycopeptide is developed by grafting with 25% maltotriose and 50% p-benzyl alcohol for the first time to effectively facilitate entry of membrane-impermeable trehalose in human RBCs with low hemolysis. Results of the mechanism acting on cell membranes suggest that reversible adsorption of such benzyl alcohol-grafted glycopeptide on cell surfaces upon weak perturbation with phospholipids and dynamic transition toward membrane stabilization are essential for keeping cellular biofunctions. Furthermore, the functionalized glycopeptide is indicative of typical α-helical/ß-sheet structure-driven regulations of ice crystals during freeze-thaw, thereby strongly promoting efficient cryopreservation. Such all-in-one glycopeptide enables achieving both high cell recovery post-thaw >85% and exceptional cryosurvival >95% in direct freezing protocols. The rationally designed benzyl alcohol-modified glycopeptide permits the development of a competent platform with high generality for protection of blood cells against freeze-stress.

Achieving high intracellular trehalose in hRBCs by reversible membrane perturbation of maltopyranosides with synergistic membrane protection of macromolecular protectants.

Trehalose is considered as a biocompatible cryoprotectant for solvent-free cryopreservation of cells, but the difficulty of the current trehalose delivery platforms to human red blood cells (hRBCs) limits its wide applications. Due to cell injuries caused by incubation at 37 °C and low intracellular loading efficiency, development of novel methods to facilitate trehalose entry in hRBCs is essential. Herein, a reversible membrane perturbation and synergistic membrane stabilization system based on maltopyranosides and macromolecular protectants was constructed, demonstrating the ability of efficient trehalose loading in hRBCs at 4 °C. Results of confocal laser scanning microscopy exhibited that the intracellular loading with the assistance of maltopyranosides was a reversible process, while the membrane protective effect of macromolecular protectants on trehalose loading in hRBCs was necessary. It was suggested that introduction of 30 mM poly(vinyl pyrrolidone) 8000 combined with 1 mM dodecyl-ß-D-maltopyranoside and 0.8 M trehalose could increase the intracellular trehalose to 84.0 ± 11.3 mM in hRBCs, whereas poly(ethylene glycol), dextran, human serum albumin or hydroxyethyl starch had a weak effect. All the macromolecular protectants could promote the cryosurvival of hRBCs, exhibiting membrane stabilization, and incubation and followed by cryopreservation did not change the basic functions and normal morphology of hRBCs substantially. This study provided an alternative strategy for glycerol-free cryopreservation of cells and the delivery of membrane-impermeable cargos.

The Relation Between Extracellular Vesicles Released From Red Blood Cells, Their Cargo, and the Clearance by Macrophages.

Extracellular vesicles (EVs) are cell-derived membrane particles that include exosomes, ectosomes, microvesicles, microparticles, apoptotic bodies, and other EV subsets. EVs are involved in intercellular communication and the transport of macromolecules between cells. Here, we propose and test the ability of red blood cell (RBC)-derived EVs (RBC-EVs) as putative drug carriers. EVs were produced by treating RBCs with Phorbol-12-myristate-13-acetate (PMA) and separating from the cells by differential centrifugation steps. RBC-EVs were characterized by size determination, flow cytometry, and scanning electron microscopy (SEM). EVs were loaded with DNA plasmids coding for the green fluorescent protein (GFP) by electroporation. The DNA-loaded EVs (DNA-EVs) were used to transfect THP-1-derived macrophages and analyzed by fluorescence microscopy and flow cytometry. The results showed that RBC-EVs had an almost spherical shape and a polydispersity in their size with an average of 197 ± 44 nm and with a zeta potential of -36 ± 8 mV. RBC-EVs were successfully loaded with DNA but associated with an increase of the polydispersity index (PdI) and showed a positive signal with Picogreen. DNA-EVs were almost completely taken up by macrophages within 24 h, however, resulting in the expression of the GFP in a subpopulation of macrophages. As the way, we designed that RBC-EVs could be potential nucleic acid carriers when the immune system was addressed. This study may contribute to the understanding of the role of EVs in the development of microvesicle-based vehicles.

Dielectrophoretic detection of electrical property changes of stored human red blood cells.

The ability to transport and store a large human blood inventory for transfusions is an essential requirement for medical institutions. Thus, there is an important need for rapid and low-cost characterization tools for analyzing the properties of human red blood cells (RBCs) while in storage. In this study, we investigate the ability to use dielectrophoresis (DEP) for measuring the storage-induced changes in RBC electrical properties. Fresh human blood was collected, suspended in K2-EDTA anticoagulant, and stored in a blood bank refrigerator for a period of 20 days. Cells were removed from storage at 5-day intervals and subjected to a glutaraldehyde crosslinking reaction to "freeze" cells at their ionic equilibrium at that point in time and prevent ion leakage during DEP analysis. The DEP behavior of RBCs was analyzed in a high permittivity DEP buffer using a three-dimensional DEP chip (3DEP) and also compared to measurements taken with a 2D quadrupole electrode array. The DEP analysis confirms that RBC electrical property changes occur during storage and are only discernable with the use of the cell crosslinking reaction above a glutaraldehyde fixation concentration of 1.0 w/v%. In particular, cytoplasm conductivity was observed to decrease by more than 75% while the RBC membrane conductance was observed to increase by more than 1000% over a period of 20 days. These results show that the presented combination of chemical crosslinking and DEP can be used as rapid characterization tool for monitoring electrical properties changes of human RBCs while subjected to refrigeration in blood bank storage.

Lateral Deformation of Human Red Blood Cells by Optical Tweezers.

In this paper, we studied the lateral deformation of human red blood cells (RBCs) during lateral indentation by an optically trapped silica bead with a diameter of 4.5 µm (Bangs Laboratories, Inc. Fishers, IN, USA). The images were captured using a CCD camera and the Boltzmann statistics method was used for force calibration. Using the Hertz model, we calculated and compared the elastic stiffness resulting from the lateral force, showing that the differences are important and that the force should be considered. Besides the lateral component, this setup also allowed us to examine the lateral cell-bead interaction. The mean values of the cell shear stiffness measured during indentation were 3.37 ± 0.40 µN/m for biconcave RBCs, 3.48 ± 0.23 µN/m for spherical RBCs, and 3.80 ± 0.22 µN/m for crenelated RBCs, respectively. These results show that this approach can be used as a routine method for RBC study, because it enabled us to manipulate the cell without contact with the wall.

Inter-study and time-dependent variability of metabolite abundance in cultured red blood cells.

BACKGROUND: Cultured human red blood cells (RBCs) provide a powerful ex vivo assay platform to study blood-stage malaria infection and propagation. In recent years, high-resolution metabolomic methods have quantified hundreds of metabolites from parasite-infected RBC cultures under a variety of perturbations. In this context, the corresponding control samples of the uninfected culture systems can also be used to examine the effects of these perturbations on RBC metabolism itself and their dependence on blood donors (inter-study variations). METHODS: Time-course datasets from five independent studies were generated and analysed, maintaining uninfected RBCs (uRBC) at 2% haematocrit for 48 h under conditions originally designed for parasite cultures. Using identical experimental protocols, quadruplicate samples were collected at six time points, and global metabolomics were employed on the pellet fraction of the uRBC cultures. In total, ~ 500 metabolites were examined across each dataset to quantify inter-study variability in RBC metabolism, and metabolic network modelling augmented the analyses to characterize the metabolic state and fluxes of the RBCs. RESULTS: To minimize inter-study variations unrelated to RBC metabolism, an internal standard metabolite (phosphatidylethanolamine C18:0/20:4) was identified with minimal variation in abundance over time and across all the samples of each dataset to normalize the data. Although the bulk of the normalized data showed a high degree of inter-study consistency, changes and variations in metabolite levels from individual donors were noted. Thus, a total of 24 metabolites were associated with significant variation in the 48-h culture time window, with the largest variations involving metabolites in glycolysis and synthesis of glutathione. Metabolic network analysis was used to identify the production of superoxide radicals in cultured RBCs as countered by the activity of glutathione oxidoreductase and synthesis of reducing equivalents via the pentose phosphate pathway. Peptide degradation occurred at a rate that is comparable with central carbon fluxes, consistent with active degradation of methaemoglobin, processes also commonly associated with storage lesions in RBCs. CONCLUSIONS: The bulk of the data showed high inter-study consistency. The collected data, quantification of an expected abundance variation of RBC metabolites, and characterization of a subset of highly variable metabolites in the RBCs will help in identifying non-specific changes in metabolic abundances that may obscure accurate metabolomic profiling of Plasmodium falciparum and other blood-borne pathogens.

Energy absorption of human red blood cells and conductivity of the cytoplasm influenced by temperature.

The energy absorbed into tissues is known as the specific energy absorption (SAR) which is dependent on conductivity of the tissue. We calculated cytoplasmic conductivity of human red blood cell (HRBC) using the intracellular ionic concentrations and the Debye-Hückel-Onsager relation. The overall concentration is determined by cell volume and cell water content. The calculated HRBC conductivity at 25 o C was σc,25 = 0.5566 ± 0.0146 S m-1, ±SE). It is exponentially related to temperature: Q10 ≈ 1.866. At 37 o C, the calculated SAR value is 1.6 W kg-1 using a linear temperature compensation of conductivity. However, if using a biologically realistic non-linear temperature compensated conductivity, the SAR is ≈ 2.62 ± 0.05 W kg-1. The relationship between SAR and temperature increase is not straightforward. Since there is a wide variance in cellular ionic and water perfusion rates more tissue-specific SAR limits which consider temperature-related factors would be valuable.

Helix pomatia albumen gland water soluble protein extract as powerful antiaging agent.

Accounting for increasingly developed population aging and dramatic elevation of aging-related severe disorders worldwide, search of the efficient antiaging agents is becoming one of the urgent problems of contemporary biomedical science. The aim of current study was to reveal the potential protective effects of water-soluble proteins extracted from albumen gland of snails against aging processes. We evaluated the antioxidant effect of the extract in 20 older adult rats in vivo and on 60 human blood samples ex vivo at the cellular level under physiological and oxidative stress conditions using the methods of spectrophotometric analysis, two-photon imaging and cell viability assay. The in vivo animal experiments showed significant increase in the levels of catalase and superoxide dismutase in treated older adult rats, compared to non-treated group. The ex vivo studies involving three human groups (young, middle aged and older adult), demonstrated that the extract has no effect on the cell viability, moreover significantly increases the number of erythrocytes, decreases age-related oxidative stress and the percentage of hemolysis of erythrocytes by aging. Thus, the snails albumen gland protein extract can be considered as effective natural antioxidative antiaging agent in prevention of aging-related pathological processes associated with oxidative stress.

Poly(oxanorbornene)-Coated CdTe Quantum Dots as Antibacterial Agents.

In this study, synthetic mimics of antimicrobial peptides based on poly(oxanorbornene) molecules (or PONs) were used to coat CdTe quantum dots (QDs). These PONs-CdTe QDs were investigated for their activity against Escherichia coli, a bacterium with antibiotic resistant strains. At the same time, the antibacterial activity of the PONs-CdTe QDs was compared to the antibacterial activity of free PONs and free CdTe QDs. The observed antibacterial activity of the PONs-CdTe QDs was additive and concentration dependent. The conjugates had a significantly lower minimum inhibitory concentration (MIC) than the free PONs and QDs, particularly for PONs-CdTe QDs which contained PONs of high amine density. The maximum activity of PONs-CdTe QDs was not realized by conjugating PONs with the highest intrinsic antibacterial activity (i.e., the lowest MIC in solution as free PONs), indicating that the mechanism of action for free PONs and PONs-CdTe QDs is different. Equally important, conjugating PONs to CdTe QDs decreased their hemolytic activity against red blood cells compared to free PONs, lending to higher therapeutic indices against E. coli. This could potentially enable the use of higher, and therefore more effective, PONs-QDs concentrations when addressing bacterial contamination, without concerns of adverse impacts on mammalian cells and organisms.

Nonclinical safety, tolerance and pharmacodynamics evaluation for meplazumab treating chloroquine-resistant Plasmodium falciparum.

Meplazumab is an anti-CD147 humanized IgG2 antibody. The purpose of this study was to characterize the nonclinical safety, tolerance and efficacy evaluation of meplazumab treating chloroquine resistant Plasmodium falciparum. Meplazumab was well tolerated in repeat-dose toxicology studies in cynomolgus monkeys. No observed adverse effect level was 12 mg/kg. No difference between genders in the primary toxicokinetic parameters after repeat intravenous injection of meplazumab. No increased levels of drug exposure and drug accumulation were observed in different gender and dose groups. Meplazumab had a low cross-reactivity rate in various tissues and did not cause hemolysis or aggregation of red blood cells. The biodistribution and excretion results indicated that meplazumab was mainly distributed in the plasma, whole blood, and hemocytes, and excreted in the urine. Moreover, meplazumab effectively inhibited the parasites from invading erythrocytes in humanized mice in a time-dependent manner and the efficacy is superior to that of chloroquine. All these studies suggested that meplazumab is safe and well tolerated in cynomolgus monkeys, and effectively inhibits P. falciparum from invading into human red blood cells. These nonclinical data facilitated the initiation of an ongoing clinical trial of meplazumab for antimalarial therapy.

Human red blood cell uptake and sequestration of arsenite and selenite: Evidence of seleno-bis(S-glutathionyl) arsinium ion formation in human cells.

Over 200 million people worldwide are exposed to the human carcinogen, arsenic, in contaminated drinking water. In laboratory animals, arsenic and the essential trace element, selenium, can undergo mutual detoxification through the formation of the seleno-bis(S-glutathionyl) arsinium ion [(GS)2AsSe]-, which undergoes biliary and fecal elimination. [(GS)2AsSe]-, formed in animal red blood cells (RBCs), sequesters arsenic and selenium, and slows the distribution of both compounds to peripheral tissues susceptible to toxic effects. In human RBCs, the influence of arsenic on selenium accumulation, and vice versa, is largely unknown. The study aims were to characterize arsenite (AsIII) and selenite (SeIV) uptake by human RBCs, to determine if SeIV and AsIII increase the respective accumulation of the other in human RBCs, and ultimately to determine if this occurs through the formation and sequestration of [(GS)2AsSe]-. 75SeIV accumulation was temperature and Cl--dependent, inhibited by 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonic acid (H2DIDS) (IC50 1 ± 0.2 µM), and approached saturation at 30 µM, suggesting uptake is mediated by the erythrocyte anion-exchanger 1 (AE1 or Band 3, gene SLC4A1). HEK293 cells overexpressing AE1 showed concentration-dependent 75SeIV uptake. 73AsIII uptake by human RBCs was temperature-dependent, partly reduced by aquaglyceroporin 3 inhibitors, and not saturated. AsIII increased 75SeIV accumulation (in the presence of albumin) and SeIV increased 73AsIII accumulation in human RBCs. Near-edge X-ray absorption spectroscopy revealed the formation of [(GS)2AsSe]- in human RBCs exposed to both AsIII and SeIV. The sequestration of [(GS)2AsSe]- in human RBCs potentially slows arsenic distribution to susceptible tissues and could reduce arsenic-induced disease.

Human Red Blood Cells as Oxygen Carriers to Improve Ex-Situ Liver Perfusion in a Rat Model.

Ex-situ machine perfusion (MP) has been increasingly used to enhance liver quality in different settings. Small animal models can help to implement this procedure. As most normothermic MP (NMP) models employ sub-physiological levels of oxygen delivery (DO2), the aim of this study was to investigate the effectiveness and safety of different DO2, using human red blood cells (RBCs) as oxygen carriers on metabolic recovery in a rat model of NMP. Four experimental groups (n = 5 each) consisted of (1) native (untreated/control), (2) liver static cold storage (SCS) 30 min without NMP, (3) SCS followed by 120 min of NMP with Dulbecco-Modified-Eagle-Medium as perfusate (DMEM), and (4) similar to group 3, but perfusion fluid was added with human RBCs (hematocrit 15%) (BLOOD). Compared to DMEM, the BLOOD group showed increased liver DO2 (p = 0.008) and oxygen consumption ( V O ˙ 2) (p < 0.001); lactate clearance (p < 0.001), potassium (p < 0.001), and glucose (p = 0.029) uptake were enhanced. ATP levels were likewise higher in BLOOD relative to DMEM (p = 0.031). V O ˙ 2 and DO2 were highly correlated (p < 0.001). Consistently, the main metabolic parameters were directly correlated with DO2 and V O ˙ 2. No human RBC related damage was detected. In conclusion, an optimized DO2 significantly reduces hypoxic damage-related effects occurring during NMP. Human RBCs can be safely used as oxygen carriers.

A Continuous Flow-through Microfluidic Device for Electrical Lysis of Cells.

In contrast to the delicate 3D electrodes in the literature, a simple flow-through device is proposed here for continuous and massive lysis of cells using electricity. The device is essentially a rectangular microchannel with a planar electrode array built on its bottom wall, actuated by alternating current (AC) voltages between neighboring electrodes, and can be incorporated easily into other biomedical systems. Human whole blood diluted 10 times with phosphate-buffered saline (about 6 108 cells per mL) was pumped through the device, and the cells were completely lysed within 7 s after the application of a 20 V peak-to-peak voltage at 1 MHz, up to 400 µL/hr. Electric field and Maxwell stress were calculated for assessing electrical lysis. Only the lower half-channel was exposed to an electric field exceeding the irreversible threshold value of cell electroporation (Eth2), suggesting that a cross flow, proposed here primarily as the electro-thermally induced flow, was responsible for bringing the cells in the upper half-channel downward to the lower half-channel. The Maxwell shear stress associated with Eth2 was one order of magnitude less than the threshold mechanical stresses for lysis, implying that an applied moderate mechanical stress could aid electrical lysis.

Utilidad de la microscopia holográfica digital para el estudio histomorfométrico de eritrocitos humanos / Usefulness of the digital holographic microscopy for the histomorphometric study of human red blood cells

Se realizó una investigación descriptiva y transversal para determinar la utilidad de la microscopia holográfica digital en el estudio histomorfométrico de eritrocitos humanos, mediante el análisis comparativo con la microscopia óptica, en el Departamento de Holografía Digital de la Universidad de Oriente de Santiago de Cuba, de abril del 2014 a igual mes del 2015. A tal efecto se seleccionaron muestras de eritrocitos diluidos en las soluciones amortiguadoras Hepes y Hepa, se emplearon el programa Holodig® y el sistema Matlab® para las mediciones en la técnica holográfica, y en el procesamiento estadístico se aplicó la prueba de la t de Student para la comparación de medias entre ambas técnicas microscópicas. Se obtuvo que la microscopia holográfica digital permitiera observar los límites y contornos de los eritrocitos estudiados y las mínimas variaciones en su forma, además de realizar estudios morfométricos, cuyos resultados mostraron similitud a los obtenidos con la microscopia óptica, y de otros parámetros, como el volumen

A descriptive and cross-sectional investigation to determine the usefulness of the digital holographic microscopy in the histomorphometric study of human red blood cells, by means of the comparative analysis with the optic microscopy, was carried out in the Digital Holography Department of Oriente University in Santiago de Cuba, from April, 2014 to the same month in 2015. To such an effect, red blood cells samples diluted in the Hepes and Hepa shock-absorbing solutions were selected, Holodig program and Matlab® system were used for the measurement in the holographic technique, and in the statistical processing the Student t test was applied for the comparison of means between both microscopic techniques. It was concluded that the digital holographic microscopy allowed to observe the limits and contours of the studied red blood cells and the minimum variations in their form, besides carrying out morphometric studies which results showed similarity to those obtained with the optic microscopy, and of other parameters, as volume