Diffusional water permeability of mammalian red blood cells

An extensive programme of comparative nuclear magnetic resonance measurements of the membrane diffusional permeability for water (P_d) and of the activation energy (E_a,d) of this process in red blood cells (RBCs) from 21 mammalian species was carried out. On the basis of P_d, these species could be divided into three groups. First, the RBC's from humans, cow, sheep and “large” kangaroos (Macropus giganteus and Macropus rufus) had P_d values 5 × 10⁻³ cm/s at 25°C and 7 × 10⁻³ cm/s at 37°C. The RBCs from other marsupial species, mouse, rat, guinea pig and rabbit, had P_d values roughly twice higher, whereas echidna RBCs were twice lower than human RBCs. The value of E_a,d was in most cases correlated with the values of P_d. A value of E_a,d -26 kJ/mol was found for the RBCs from humans and the species having similar P_d values. Low values of E_a,d (ranging from 15 to 22 kJ/mol) appeared to be associated with relatively high values of P_d. The highest value of E_a,d (33 kJ/mol) was found in echidna RBCs. This points to specialized channels for water diffusion incorporated in membrane proteins; a relatively high water permeability of the RBC membrane could be due to a greater number of channel proteins. There are, however, situations where a very high water permeability of RBCs is associated with a high value of E_a,d (above 25 kJ/mol) as in the case of RBCs from mouse, rat and tree kangaroo. Moreover, it was found that P_d in different species was positively correlated to the RBC membrane phosphatidylcholine and negatively correlated to the sphingomyelin content. This suggests that in addition to the number of channel proteins, other factors are involved in the water permeability of the RBC membrane.     

Reducing the immediate availability of red blood cells in cardiac surgery,a single-centre experience

Background

In our institution, we have redefined our criteria for direct availability of red blood cell (RBC) units in the operation room. In this study, we sought to evaluate the safety of applying this new logistical policy of blood transfusion in the first preliminary group of patients.

Methods

In March 2010, we started a new policy concerning the elective availability of RBC units in the operation room. This policy was called: No Elective Red Cells (NERC) program. The program was applied for patients undergoing primary isolated coronary artery bypass grafting (CABG) or single valve surgery. No elective RBC units were preoperatively ordered for these patients. In case of urgent need, blood was delivered to the operating room within 20 min. The present study includes the first 500 patients who were managed according to this policy. Logistic regression analyses were performed to investigate the impact of biomedical variables on fulfilling this NERC program.

Results

The majority of patients (n = 409, 81 %) did not receive any RBCs during the hospital stay. In patients who did receive RBCs (n = 91, 19 %), 11 patients (2.2 %) received RBCs after 24 h postoperatively. Female gender, left ventricular ejection fraction (LVEF) and EuroSCORE were significant predictors for the need of blood transfusion (OR = 3.12; 2.79; 1.17 respectively).

Conclusion

In a selected group of patients, it is safe to perform cardiac surgery without the immediate availability of RBCs in the operating room. Transfusion was avoided in 81 % of these patients. Female gender, LVEF and EuroSCORE were associated with blood transfusion.     

Changes of phosphatidylserine distribution in human red blood cells during the process of loading sugars

The plasma membrane of red blood cells permits sugars to be loaded into the cytoplasm simply by incubation in a suitable buffer solution containing the sugar. This may provide some hope for the freeze-drying of human red blood cells. However, the effect of the loading process on red blood cells has not been fully investigated. The exposure of phosphatidylserine (PS) on the surface of the cell can be recognized by macrophages and result in shortened circulation in vivo. This study evaluates the effects of the concentration, the incubation time, and the temperature of exposure of human red blood cells to extracellular trehalose or glucose. Exposure of PS was demonstrated by annexin V labeling. It was shown that the efficiency of loading of glucose was significantly greater than that of trehalose. The loading efficiency of both sugars increased with increase in extracellular sugar concentration, prolongation of incubation time, and increase of incubation temperature. The percentages of cells with exposed PS and of damaged cells were dependent on the extracellular sugar concentration, the incubation time, and the temperature. With an extracellular glucose concentration of 0.8M, the percentage of cells with exposed PS was more than 80% and significantly higher than that of red blood cells loaded with trehalose (approximate 20%, P<0.01). As the incubation time was prolonged, the percentage of PS exposure and of damaged cells also increased. After incubation for 5h, the percentage of red cells with exposed PS following loading with glucose was more than 80% and significantly higher than that of cells loaded with trehalose (40%, P<0.01). In addition, the incubation temperature had a major effect on PS exposure. The percentage of cells with PS exposure and the proportion of damaged cells increased with increase of incubation temperature. At 37 degrees C, the percentage of cells with exposed PS and of damaged cells after loading with glucose was more than 80% and significantly higher than that of cells loaded with trehalose (P<0.01). However, when the temperature was below 25 degrees C, the percentage of cells with exposed PS and of damaged cells after loading with glucose or trehalose were both less than 10%. In conclusion, the loading efficiency for glucose was higher than that for trehalose, but the lesser effect of trehalose on exposure of PS suggests that it can maintain the asymmetrical distribution of membrane phospholipids and the intracellular trehalose can increase the osmotic tolerance of cells.     

Comparative study of human red blood cell analysis with three different field-flow fractionation systems

An extensive multi-laboratory study was conducted to compare three different field-flow fractionation (FFF) systems for use in the analysis of human erythrocytes. The object of this study was to determine the relationship between the FFF elution properties for each system and the traditional hematological blood cell parameters. One centrifugal system (Utah) and two gravitational systems (Paris and Abbott) were compared. In order to analyze erythrocyte populations with a broad range of hematological indices, blood samples were collected from individuals heterozygous for sickle cell anemia (A/S) and also from normal controls (A/A), and these were analyzed at each site. Identical samples were analyzed by the Abbott and Utah sites. With all three systems, blood samples from each category produced narrow, overlapping distributions of FFF retention ratios, with the Abbott and Utah systems showing slight elevations in the mean retention ratios for the sickle cell samples. Blood cell elution peak characteristics were compared with standard hematological parameters for each of the FFF systems, and negative correlations were consistently found between mean corpuscular volume (MCV) and retention ratios. Positive correlations were found between red cell distribution width (RDW) and retention ratios. Elevated FFF retention ratios were frequently found with blood samples having abnormal hematological profiles. These results demonstrate that the three differently configured systems all produce similar analysis profiles for erythrocytes from the classes studied here. The relationships between FFF parameters and hematological indices were consistent for all systems.     

Effects of pre-freeze incubation of human red blood cells with various sugars on postthaw recovery when using a dextran-rapid cooling protocol

Polymer has been used as substitute to replace glycerol for cryopreservation of red blood cells (RBCs). But polymer can not penetrate cell membrane, it can not efficiently protect the inner membrane. In this study, RBCs were incubated with glucose, fructose, galactose or trehalose and frozen in liquid nitrogen for 24 h using dextran as the extracellular protectant. The postthaw quality was assessed by RBC hemolysis, RBC morphology, PS distribution, osmotic fragility, and the 4 °C stability. The results indicated the loading efficiency of monosaccharide was significantly higher than that of trehalose. Adding trehalose and 40% dextran caused more serious hemolysis before freezing. The percent hemolysis of RBCs loaded with high concentration of trehalose was approximately 16% and significantly more than that of RBCs loaded with glucose (approximately 5%, P < 0.05). Intracellular trehalose can not increase the postthaw recovery of RBCs compared with cells frozen without sugar. However, low concentration of intracellular glucose or galactose can reduce the percent hemolysis to less than 5% and significantly less than that of RBCs frozen without sugar (P < 0.05). Finally, the ability of galactose or fructose to maintain the 4 °C stability was significantly more than that of glucose. In conclusion, the injuries caused by trehalose loading may directly lead to postthaw hemolysis and poor quality of RBCs. However, monosaccharide can enhance the recovery of frozen RBCs. The cryoprotective effect of galactose may be better than that of glucose or fructose. In the future, we will continue to look for a safe and efficient trehalose loading process and try to decrease the osmotic fragility of RBCs frozen with polymers and sugars.     

Evaluation of trehalose and sucrose as cryoprotectants for hematopoietic stem cells of umbilical cord blood

Bone marrow transplantation (BMT) is a therapeutic procedure that involves transplantation of hematopoietic stem cells (HSC). To date, there are three sources of HSC for clinical use: bone marrow; mobilized peripheral blood; and umbilical cord blood (UCB). Depending on the stem cell source or type of transplantation, these cells are cryopreserved. The most widely used cryoprotectant is dimethylsulfoxide (Me₂SO) 10% (v/v), but infusion of Me₂SO-cryopreserved cells is frequently associated with serious side effects in patients. In this study, we assessed the use of trehalose and sucrose for cryopreservation of UCB cells in combination with reduced amounts of Me₂SO. The post-thawed cells were counted and tested for viability with Trypan blue, the proportion of HSC was determined by flow cytometry, and the proportion of hematopoeitic progenitor cells was measured by a colony-forming unit (CFU) assay. A solution of 30 mmol/L trehalose with 2.5% Me₂SO (v/v) or 60 mmol/L sucrose with 5% Me₂SO (v/v) produced results similar to those for 10% (v/v) Me₂SO in terms of the clonogenic potential of progenitor cells, cell viability, and numbers of CD45⁺/34⁺ cells in post-thawed cord blood cryopreserved for a minimum of 2 weeks. Thus, cord blood, as other HSC, can be cryopreserved with 1/4 the standard Me₂SO concentration with the addition of disaccharides. The use of Me₂SO at low concentrations in the cryopreservation solution may improve the safety of hematopoietic cell transplantation by reducing the side effects on the patient.     

Development of a high-performance liquid chromatography-based assay for carotenoids in human red blood cells: application to clinical studies

Peroxidized phospholipid-mediated cytotoxicity is involved in the pathophysiology of many diseases; for example, there is an abnormal increase of phospholipid hydroperoxides in red blood cells (RBCs) of dementia patients. Dietary carotenoids have gained attention as potent inhibitors of RBC phospholipid hydroperoxidation, thereby making them plausible candidates for preventing disease. However, the occurrence of carotenoids in human RBCs is still unclear. This is in contradistinction to plasma carotenoids, which have been investigated thoroughly for analytical methods as well as biological significance. In this study, we developed a method to analyze RBC carotenoids using high-performance liquid chromatography (HPLC) coupled with ultraviolet (UV) diode array detection (DAD) and atmospheric pressure chemical ionization (APCI) mass spectrometry (MS). Under optimized conditions that included extraction, separation, and detection procedures, six carotenoids (lutein, zeaxanthin, β-cryptoxanthin, α-carotene, β-carotene, and lycopene) were separated, detected by DAD, and concurrently identified based on APCI/MS and UV spectra profiles when an extract from human RBCs was subjected to HPLC-DAD-APCI/MS. The amounts of carotenoids varied markedly (1.3-70.2 nmol/L packed cells), and polar oxygenated carotenoids (xanthophylls) were predominant in RBCs. The HPLC-DAD-APCI/MS method would be a useful tool for clinical studies for evaluating the bioavailability of RBC carotenoids.     

Influence of absorption on polarization effects in light scattering from human red blood cells

Polarization effects in light scattering are sensitive indicators of cell structure and structural changes in time. In the spectral regions where the optical properties of the scatterers are relatively constant, the scattering pattern scales, it contracts or expands in a predictable manner as a function of the wavelength. In the spectral regions where the optical properties are strongly wavelength dependent (near absorption bands, etc.) the scattering curves do not scale, but change drastically in phase and amplitude as the wavelength is varied. Reported here is an empirical study of the magnitude of the influence of absorption on the polarization effects in light scattering. Scattering curves have been obtained for human red blood cells in the absorption band (blue light) and far from the absorption band (red light). The scattering at these wavelengths shows very strong nonscaling differences. These observations suggest the use of polarization effects in light scattering and their wavelength dependence for the studies of structural changes in cell nuclei. Nucleoproteins have strong absorption, optical rotatory dispersion and circular dichroism bands in the ultraviolet region of the spectrum, whereas there is little ψ-dependence in the visible range. There is also the possibility of binding specific chromophoric dyes to cell components, thus introducing absorption bands in the visible range, where scattering instrumentation and laser light sources are more readily available.     

Glycine uptake by trout (Salmo trutta) red blood cells

The present study demonstrates the presence of different amino acid carriers in the membrane of trout red cells. Most glycine is taken up through the Na⁺-dependent system ASC, although the nearly specific Gly system is also active. Besides these carriers, glycine is taken up by means of Na⁺-independent transporters, system l being the most important. A system asc of high affinity and low capacity has been found, and band 3 is unable to transport glycine under physiological conditions. These results suggest that although all these carriers are already present in primitive vertebrates, several differences exist in their properties with respect to those found in mammalian cells.We would like to express our sincere thanks to Mr. Antonino Clemente (Piscifactoria de Bagà, Medi Natural, Generalitat de Catalunya) for his help and logistical assistance and to Mr. Robin Rycroft for his editorial help.This work was supported by a grant of Comisió Interdepartamental de Recerca i Technologia (AR90-3.3394). M.A.G. is recipient of a fellowship from the Generalitat de Catalunya.