Intact Red Blood Cells Research Articles

Hemolysis is a primary ATP-release mechanism in human erythrocytes

AbstractThe hypothesis that regulated ATP release from red blood cells (RBCs) contributes to nitric oxide-dependent control of local blood flow has sparked much interest in underlying release mechanisms. Several stimuli, including shear stress and hypoxia, have been found to induce significant RBC ATP release attributed to activation of ATP-conducting channels. In the present study, we first evaluated different experimental approaches investigating stimulated RBC ATP release and quantifying hemolysis. We then measured ATP and free hemoglobin in each and every RBC supernatant sample to directly assess the contribution of hemolysis to ATP release. Hypotonic shock, shear stress, and hypoxia, but not cyclic adenosine monophosphate agonists, significantly enhanced ATP release. It tightly correlated, however, with free hemoglobin in RBC supernatants, indicating that lysis was responsible for most, if not all, ATP release. Luminescence ATP imaging combined with simultaneous infrared cell imaging showed that ATP was released exclusively from lysing cells with no contribution from intact cells. In summary, with all stimuli tested, we found no evidence of regulated ATP release from intact RBCs other than by cell lysis. Such a release mechanism might be physiologically relevant in vivo, eg, during exercise and hypoxia where intravascular hemolysis, predominantly of senescent cells, is augmented.

The capacity of red blood cells to reduce nitrite determines nitric oxide generation under hypoxic conditions.

Nitric oxide (NO) is a key regulator of vascular tone. Endothelial nitric oxide synthase (eNOS) is responsible for NO generation under normoxic conditions. Under hypoxia however, eNOS is inactive and red blood cells (RBC) provide an alternative NO generation pathway from nitrite to regulate hypoxic vasodilation. While nitrite reductase activity of hemoglobin is well acknowledged, little is known about generation of NO by intact RBC with physiological hemoglobin concentrations. We aimed to develop and apply a new approach to provide insights in the ability of RBC to convert nitrite into NO under hypoxic conditions. We established a novel experimental setup to evaluate nitrite uptake and the release of NO from RBC into the gas-phase under different conditions. NO measurements were similar to well-established clinical measurements of exhaled NO. Nitrite uptake was rapid, and after an initial lag phase NO release from RBC was constant in time under hypoxic conditions. The presence of oxygen greatly reduced NO release, whereas inhibition of eNOS and xanthine oxidoreductase (XOR) did not affect NO release. A decreased pH increased NO release under hypoxic conditions. Hypothermia lowered NO release, while hyperthermia increased NO release. Whereas fetal hemoglobin did not alter NO release compared to adult hemoglobin, sickle RBC showed an increased ability to release NO. Under all conditions nitrite uptake by RBC was similar. This study shows that nitrite uptake into RBC is rapid and release of NO into the gas-phase continues for prolonged periods of time under hypoxic conditions. Changes in the RBC environment such as pH, temperature or hemoglobin type, affect NO release.

Tubulointerstitial nephritis and uveitis syndrome in an elderly woman.

Tubulointerstitial nephritis and uveitis syndrome in an elderly woman.

Flow cytometric analysis of red blood cell osmotic fragility.

This study investigated a new method of flow cytometry (FCM) for analysis of red blood cell (RBC) osmotic fragility. Venous peripheral blood collected in a sampling tube coated with EDTA 2Na was analyzed using FCM to determine RBC osmotic fragility. RBCs were represented as a double-peaked forward scatter (FSC) histogram in FCM. RBCs showed ballooning and spherical shape change in hypotonic solutions before hemolysis. The ballooning of RBCs was expressed as a disappearance of the minor peak and by narrowing and a shift to the right of the FSC histogram. The process of hemolysis was expressed as shrinking of the RBC cytogram in the right upper quadrant on the scatter plot of side scatter versus FSC and by emergence of a cell debris cytogram in the left lower quadrant. The ratio of intact RBCs in hypotonic solution was available as an indicator of osmotic fragility. Acidic solution made erythrocytes less tolerant to hypotonic solution by inducing spherical shape change. In conclusion, FCM can be used to assess RBC osmotic fragility.

Screening hypochromism (sieve effect) in red blood cells: a quantitative analysis.

Multiwavelength UV-visible spectroscopy, Kramers-Kronig analysis, and several other experimental and theoretical tools have been applied over the last several decades to fathom absorption and scattering of light by suspensions of micron-sized pigmented particles, including red blood cells, but a satisfactory quantitative analysis of the difference between the absorption spectra of suspension of intact and lysed red blood cells is still lacking. It is stressed that such a comparison is meaningful only if the pertinent spectra are free from, or have been corrected for, scattering losses, and it is shown that Duysens' theory can, whereas that of Vekshin cannot, account satisfactorily for the observed hypochromism of suspensions of red blood cells.

Information use and plasticity in the reproductive decisions of malaria parasites.

BackgroundInvestment in the production of transmissible stages (gametocytes) and their sex ratio are malaria parasite traits that underpin mosquito infectivity and are therefore central to epidemiology. Malaria parasites adjust their levels of investment into gametocytes and sex ratio in response to changes in the in-host environment (including red blood cell resource availability, host immune responses, competition from con-specific genotypes in mixed infections, and drug treatment). This plasticity appears to be adaptive (strategic) because parasites prioritize investment (in sexual versus asexual stages and male versus female stages) in manners predicted to maximize fitness. However, the information, or ‘cues’ that parasites use to detect environmental changes and make appropriate decisions about investment into gametocytes and their sex ratio are unknown.MethodsSingle genotype Plasmodium chabaudi infections were exposed to ‘cue’ treatments consisting of intact or lysed uninfected red blood cells, lysed parasitized RBCs of the same clone or an unrelated clone, and an unmanipulated control. Infection dynamics (proportion of reticulocytes, red blood cell and asexual stage parasite densities) were monitored, and changes in gametocyte investment and sex ratio in response to cue treatments, applied either pre- or post-peak of infection were examined.Results and conclusionsA significant reduction in gametocyte density was observed in response to the presence of lysed parasite material and a borderline significant increase in sex ratio (proportion of male gametocytes) upon exposure to lysed red blood cells (both uninfected and infected) was observed. Furthermore, the changes in gametocyte density and sex ratio in response to these cues depend on the age of infection. Demonstrating that variation in gametocyte investment and sex ratio observed during infections are a result of parasite strategies (rather than the footprint of host physiology), provides a foundation to investigate the fitness consequences of plasticity and explore whether drugs could be developed to trick parasites into making suboptimal decisions.

Single-Bacterial Profiling and Identification Based on Quantitative Phase Imaging

Rapid identification of bacterial species is crucial in medicine and food hygiene. For instance, bacterial invasion-related syndromes such as sepsis, pneumonia, abscess, meningitis, gastroenteritis and food poisoning require rapid cures which are appropriate for the invaded bacterial species. However, conventional culture-based or molecular-assay technologies do not match this time requirement in general, mainly due to their limited sensitivity. Here, we present the potential of quantitative phase imaging (QPI) techniques (1) for identification of bacterial species at single bacterium level. Employment of various QPI modalities provides the corresponding biophysical and biochemical properties of the bacterial cell in a rapid and label-free manner. First, we employed anisotropic Fourier transform light scattering (2) and demonstrated that light scattering from individual bacterium reflects structural information sufficient for identification of rod-shaped species. Second, the potential of spectroscopic QPI (3, 4) for extracting chemical composition of the bacterium with sub-genus sensitivity is suggested.1. Lee, K., K. Kim, J. Jung, J. H. Heo, S. Cho, S. Lee, G. Chang, Y. J. Jo, H. Park, and Y. K. Park. 2013. Quantitative phase imaging techniques for the study of cell pathophysiology: from principles to applications. Sensors 13:4170-4191.2. Kim, Y., J. M. Higgins, R. R. Dasari, S. Suresh, and Y. K. Park. 2012. Anisotropic light scattering of individual sickle red blood cells. J. Biomed. Opt. 17:040501.3. Park, Y., T. Yamauchi, W. Choi, R. Dasari, and M. S. Feld. 2009. Spectroscopic phase microscopy for quantifying hemoglobin concentrations in intact red blood cells. Optics Letters 34:3668-3670.4. Jang, Y., J. Jang, and Y. Park. 2012. Dynamic spectroscopic phase microscopy for quantifying hemoglobin concentration and dynamic membrane fluctuation in red blood cells. Optics Express 20.

A cancer cell-activatable aptamer-reporter system for one-step assay of circulating tumor cells.

The current antibody-mediated numeration assays of circulating tumor cells (CTCs) require multiple steps and are time-consuming. To overcome these technical limitations, a cancer cell-activatable aptamer-reporter was formulated by conjugating a biomarker-specific aptamer sequence with paired fluorochrome-quencher molecules. In contrast to the antibody probes, the intact aptamer-reporter was optically silent in the absence of cells of interest. However, when used in an assay, the aptamer selectively targeted cancer cells through interaction with a specific surface biomarker, which triggered internalization of the aptamer-reporter and, subsequently, into cell lysosomes. Rapid lysosomal degradation of the aptamer-reporter resulted in separation of the paired fluorochrome-quencher molecules. The released fluorochrome emitted bright fluorescent signals exclusively within the targeted cancer cells, with no background noise in the assay. Thus, the assays could be completed in a single step within minutes. By using this one-step assay, CTCs in whole blood and marrow aspirate samples of patients with lymphoma tumors were selectively highlighted and rapidly detected with no off-target signals from background blood cells. The development of the cancer cell-activatable aptamer-reporter system allows for the possibility of a simple and robust point-of-care test for CTC detection, which is currently unavailable.

T‐cell suppression by red blood cells is dependent on intact cells and is a consequence of blood bank processing

Red blood cells (RBCs) suppress T-cell responsiveness through a mechanism requiring cell-cell contact. Questions remain as to whether this effect is an allogeneic response, related to cell death, or dependent on particular components of the RBCs. Peripheral T cells were isolated from healthy donors and exposed to stored allogeneic RBCs or autologous RBCs after processing. RBCs were lysed by hypotonic solvent to produce cellular ghosts. Tritiated thymidine proliferation assays were utilized. Cultures were saturated with interleukin (IL)-2 to determine whether impaired IL-2 synthesis played a role. T-cell proliferation was suppressed by both autologous and allogeneic RBCs. RBC membrane integrity does enhance T-cell suppression. T-cell death is not responsible for the suppressive changes. IL-2 synthesis is suppressed in RBC-exposed T cells but addition of exogenous IL-2 does not rescue proliferative capabilities. Proliferation of T cells was inhibited with RBC exposure but mitigated with the addition of fresh RBCs. T-cell suppression is enhanced by intact RBCs but this effect is unrelated solely to alloantigens. Neither apoptosis nor necrosis of T cells contributes to this phenomenon. IL-2 synthesis is suppressed after RBC exposure as a consequence of T-cell inhibition, but is not the primary cause of suppression. Fresh RBCs do not mediate T-cell suppression, indicating that changes in the RBC and development of the storage lesion may occur during initial blood bank processing.

Stability and Stoichiometry of Bilayer Phospholipid–Cholesterol Complexes: Relationship to Cellular Sterol Distribution and Homeostasis

Is cholesterol distributed among intracellular compartments by passive equilibration down its chemical gradient? If so, its distribution should reflect the relative cholesterol affinity of the constituent membrane phospholipids as well as their capacity for association with the sterol. We examined this issue by analyzing the reactivity to cholesterol oxidase of large unilamellar vesicles (LUVs) containing phospholipids and varied levels of cholesterol. The rates of cholesterol oxidation differed among the various phospholipid environments by roughly 4 orders of magnitude. Furthermore, accessibility to the enzyme increased by orders of magnitude at cholesterol thresholds that suggested cholesterol:phospholipid association ratios of 1:1, 2:3, or 1:2 (moles:moles). The accessibility of cholesterol above these thresholds was still constrained by its particular phospholipid environment. One phospholipid, 1-stearoyl-2-oleoyl-sn-glycero-3-phosphatidylserine, exhibited no threshold. The analysis suggested values for the stoichiometries of the putative cholesterol-phospholipid complexes, their relative stabilities, and the fractions of bilayer cholesterol not in complexes at the threshold equivalence points. Predictably, the saturated phosphorylcholine species had the lowest apparent stoichiometric ratios and the strongest associations with cholesterol. These results are in general agreement with the equilibrium distribution of cholesterol between the various LUVs and methyl-β-cyclodextrin. In addition, the behavior of the cholesterol in intact human red blood cells matched predictions made from LUVs of the corresponding composition. These results support a passive mechanism for the intracellular distribution of cholesterol that can provide a signal for its homeostatic regulation.

<em>Ex Vivo</em> Red Blood Cell Hemolysis Assay for the Evaluation of pH-responsive Endosomolytic Agents for Cytosolic Delivery of Biomacromolecular Drugs

Phospholipid bilayers that constitute endo-lysosomal vesicles can pose a barrier to delivery of biologic drugs to intracellular targets. To overcome this barrier, a number of synthetic drug carriers have been engineered to actively disrupt the endosomal membrane and deliver cargo into the cytoplasm. Here, we describe the hemolysis assay, which can be used as rapid, high-throughput screen for the cytocompatibility and endosomolytic activity of intracellular drug delivery systems. In the hemolysis assay, human red blood cells and test materials are co-incubated in buffers at defined pHs that mimic extracellular, early endosomal, and late endo-lysosomal environments. Following a centrifugation step to pellet intact red blood cells, the amount of hemoglobin released into the medium is spectrophotometrically measured (405 nm for best dynamic range). The percent red blood cell disruption is then quantified relative to positive control samples lysed with a detergent. In this model system the erythrocyte membrane serves as a surrogate for the lipid bilayer membrane that enclose endo-lysosomal vesicles. The desired result is negligible hemolysis at physiologic pH (7.4) and robust hemolysis in the endo-lysosomal pH range from approximately pH 5-6.8.

Bacteria can promote calcium oxalate crystal growth and aggregation

Our previous report showed that uropathogenic bacteria, e.g., Escherichia coli, are commonly found inside the nidus of calcium oxalate (CaOx) kidney stones and may play pivotal roles in stone genesis. The present study aimed to prove this new hypothesis by direct examining CaOx lithogenic activities of both Gram-negative and Gram-positive bacteria. CaOx was crystallized in the absence (blank control) or presence of 10(5) CFU/ml E.coli, Klebsiella pneumoniae, Staphylococcus aureus, or Streptococcus pneumoniae. Fragmented red blood cell membranes and intact red blood cells were used as positive and negative controls, respectively. The crystal area and the number of aggregates were measured to initially screen for effects of bacteria on CaOx crystal growth and aggregation. The data revealed that all the bacteria tested dramatically increased the crystal area and number of crystal aggregates. Validation assays (spectrophotometric oxalate-depletion assay and an aggregation-sedimentation study) confirmed their promoting effects on both growth (20.17±3.42, 17.55±2.27, 16.37±1.38, and 21.87±0.85% increase, respectively) and aggregation (57.45±2.08, 51.06±5.51, 55.32±2.08, and 46.81±3.61% increase, respectively) of CaOx crystals. Also, these bacteria significantly enlarged CaOx aggregates, with the diameter greater than the luminal size of distal tubules, implying that tubular occlusion might occur. Moreover, these bacterial effects were dose-dependent and specific to intact viable bacteria, not intact dead or fragmented bacteria. In summary, intact viable E.coli, K.pneumoniae, S.aureus, and S.pneumoniae had significant promoting effects on CaOx crystal growth and aggregation. This functional evidence supported the hypothesis that various types of bacteria can induce or aggravate metabolic stone disease, particularly the CaOx type.

Pitfalls in the diagnosis of hepatic epithelioid hemangioendothelioma by FNA and needle core biopsy

Epithelioid hemangioendothelioma (EHE) of the liver is an extremely rare tumor that masquerades as an epithelial neoplasm and poses significant diagnostic pitfalls for the cytopathologist. This report describes a 64-year-old woman whom on computerized tomography was found to have multiple peripherally calcified hypodense lesions throughout the liver and an ipsilateral adrenal mass. Fine needle aspiration and needle cores biopsies of the largest liver lesion showed epithelioid cells and spindle cells in fibrous stroma. The epithelioid cells had round or oval, hyperchromatic nuclei with smooth nuclear contours, delicate cytoplasm and indistinct cytoplasmic borders. Rare cells had intracellular vascular lumen containing fragmented or intact red blood cells. The cells were individually dispersed or arranged in nests and vague tubular arrays. No mitotic figures or necrosis were seen. An immunohistochemical profile demonstrated diffuse CD31 and focal CD 34 and nuclear Fli-1 immunoexpression and low Ki-67 proliferative activity (1%) within lesional cells confirming the diagnosis of EHE. The differential diagnosis of EHE which includes cholangiocarcinoma, hepatocellular carcinoma, metastatic carcinoma and melanoma is discussed.

Red blood cell mechanical stability test

Red blood cells (RBC) are exposed to various levels of shear stress (SS) during their flow in the circulatory system, yet no significant damage occurs if their mechanical stability is not impaired. Alternatively, normal RBC may be damaged during flow in non-physiological environments and under extreme SS (e.g., extracorporeal circulation, ventricular assist devices). The shear-induced damage may result in hemolysis or in altered mechanical properties of RBC that, in turn, reduces the ability of RBC to withstand further damage by SS. An ektacytometer employing a Couette shearing system was used to apply SS at a constant level of 100 Pa for 300 seconds as a model of sub-hemolytic mechanical stress. The degree of cellular damage during and after the application was assessed by diffraction pattern analysis. The area of the diffraction pattern was found to correlate with the number of RBC in the sheared suspension. Monitoring the ellipse area during the application of gradually increasing SS provides the concentration of the remaining intact RBC and can therefore be used to estimate the hemolytic threshold as a measure of RBC mechanical stability. The hemolytic threshold determined after the mechanical stress application was found to be ~150 Pa, while it was ~250 Pa in the same samples before the SS application. Additionally, SS-elongation index curves recorded before and after the application of the sub-hemolytic SS significantly differed from each other, indicating the impairment in deformability following the mechanical stress. The Couette type ektacytometer can be used as a tool to assess the sub-hemolytic damage to RBC in testing the biomedical equipment.

Does the extracorporeal circulation worsen anemia in hemodialysis patients? Investigation with advanced microscopes of red blood cells drawn at the beginning and end of dialysis

BackgroundIn hemodialysis (HD) patients, anemia relates to three main factors: insufficient production of erythropoietin; impaired management of iron; and decreased lifespan of red blood cells (RBCs). The third factor can relate to structural deterioration of RBCs due to extrinsic (extracorporeal circuit; biochemical activation and/or mechanical stress during dialysis) and intrinsic (uremic milieu; biochemical interference of the RBC membrane constituents with toxins) mechanisms. Herein, we evaluate information accessed with advanced imaging techniques at the cellular level.MethodsAtomic force and scanning electron microscopes were employed to survey intact RBCs (iRBCs) of seven HD patients in comparison to seven healthy donors. The extrinsic factor was investigated by contrasting pre- and post-HD samples. The intrinsic environment was investigated by comparing the microscopy data with the clinical ones.ResultsThe iRBC membranes of the enrolled HD patients were overpopulated with orifice-like (high incidence; typical size within 100–1,000 nm) and crevice-like (low incidence; typical size within 500–4,000 nm) defects that exhibited a statistically significant (P < 0.05) relative increase (+55% and +350%, respectively) in respect to healthy donors. The relative variation of the orifice and crevice indices (mean population of orifices and crevices per top membrane surface) between pre- and post-HD was not statistically significant (−3.3% and +4.5%, respectively). The orifice index correlates with the concentrations of urea, calcium, and phosphorus, but not, however, with that of creatinine.ConclusionExtracorporeal circulation is not detrimental to the structural integrity of RBC membranes. Uremic milieu is a candidate cause of RBC membrane deterioration, which possibly worsens anemia.

Alkyl Substituted 2′-Benzoylpyridine Thiosemicarbazone Chelators with Potent and Selective Anti-Neoplastic Activity: Novel Ligands that Limit Methemoglobin Formation

Thiosemicarbazone chelators, including the 2'-benzoylpyridine thiosemicarbazones (BpT) class, show marked potential as anticancer agents. Importantly, 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP) has been investigated in >20 phase I and II clinical trials. However, side effects associated with 3-AP administration include methemoglobinemia. Considering this problem, novel BpT analogues were designed bearing hydrophobic, electron-donating substituents at the para position of the phenyl group (RBpT). Their Fe(III/II) redox potentials were all within the range accessible to cellular oxidants and reductants, suggesting they can redox cycle. These RBpT ligands exhibited potent and selective antiproliferative activity, which was comparable or exceeded their BpT counterparts. Major findings include that methemoglobin formation mediated by the lipophilic t-BuBpT series was significantly (p < 0.05-0.001) decreased in comparison to 3-AP in intact red blood cells and were generally comparable to the control. These data indicate the t-BuBpT ligands may minimize methemoglobinemia, which is a marked advantage over 3-AP and other potent thiosemicarbazones.

Treatment with a Novel Dinitroazetidine, Abdnaz, Improves Nitrite Reductase Activity of Sickle Red Blood Cells

AbstractAbstract 3246 IntroductionVaso-occlusion is a hallmark of sickle cell disease (SCD). Congestion of blood vessels by sickle red blood cells (RBC) is caused by hemoglobin polymerization (sickling), increased adhesion, and vasoconstriction due to a disregulated nitric oxide (NO) metabolism. NO generated from L-arginine by NO synthases plays a key role in vasodilation. Under hypoxia hemoglobin has been shown to be a potent alternative source of NO by reducing nitrite (NO2−). In SCD, modification of hemoglobin may reduce sickling and increase its ability to generate NO, and as such improve blood flow. In this study, we examined hemoglobin modification and consequent change in nitrite reductase capacity by 1-bromoacetyl-3,3-dinitroazetidine (ABDNAZ). This is a compound that has recently been described to bind to hemoglobin (Scicinski et al. Drug Met Disp. 2012) and has promising anticancer activity by altering blood flow in hypoxic tumors (Ning et al. Cancer Res. 2012). MethodBlood samples from SCD patients and control blood from healthy volunteers was collected with IRB approval. Red blood cells were isolated and resuspended to physiological hemactocrit in HEPES buffered saline (HBS) containing 2% bovine serum albumin (BSA). Samples were incubated with ABDNAZ, hydroxyurea (HU) or iodoacetamide at a final concentration of 3 mM for 30 minutes at room temperature. Intact RBC, hemolysates or purified hemoglobin samples were exposed to a constant nitrogen (N2) flow (200 mL/minute) at 37°C in a tonometer. At time zero, sodium nitrite was added to a final concentration of 5 mM. The influx of NO2− into the RBC was measured using Griess reagent on supernatant samples. The gas outflow from the tonometer was collected in Mylar balloons at various time points, and NO released in the gas phase was measured using a Nitric Oxide Analyzer. ResultsUpon NO2− addition under deoxygenated conditions, influx in RBC was rapid. An equilibrium between supernatant and RBC was reached within 10 minutes and indicated active recruitment of RBC rather than a simple diffusion equilibrium. Release of NO from SCD patient RBC was increased compared to normal control (HbAA) RBC. Under these conditions, hemolysates with the identical hemoglobin concentration as compared to intact RBC showed a pronounced increase in NO release. Using purified hemoglobin NO release was reduced to 50% as compared to hemolysates. Blood samples from both normal control and sickle cell patients treated with ABDNAZ, showed a significant increase in NO release as compared to untreated samples. Iodoacetamide and HU treatment, described to increase NO (Gladwin et al. Br. J. Hematol. 2002), also showed increased NO release but at a markedly lower level as compared to ABDNAZ treatment. ConclusionsEpisodes of vaso-occlusion result in pain and organ damage in SCD. Local generation of NO will improve vasodilation, and is potentially beneficial. The novel compound ABDNAZ, currently in human clinical trials, greatly increases nitrite reductase capacity of hemoglobin, in both normal and SCD blood samples. Our results indicate that this agent improves NO release from RBC and suggests that modification of sickle patient blood by ABDNAZ in the presence of plasma nitrite should be considered as a potential therapeutic approach. Disclosures:Scicinski:RadioRx, Inc: Employment. Oronsky:RadioRx, Inc: Employment.

69. All that’s needed is inside the cell: New method to measure permeability of red blood cell membrane using intrinsic fluorescence

To design effective cryopreservation procedures for cells and tissues, it is critical to know permeability of cell membrane to water and solutes. To determine the latter, one needs to measure the rate of cell volume changes when placed in anisotonic environment. Red blood cells (RBCs) respond very quickly to changes in extracellular solutes concentration, which makes it difficult to measure the rate of RBC volume change using traditional methods. Stopped-flow method captures rapid changes of RBC volume and is a common method to measure RBC osmotic permeability. There are two types of stopped-flow methods: the first measures RBC volume based on changes in the intensity of light scattered by RBCs (Sidel and Solomon, 1957), the second measures it based on the self-quenching of fluorescent dye entrapped inside RBCs (Chen et al., 1988). However, both techniques have flaws. A number of factors other than cell volume influence the intensity of scattered light (Chen et al., 1988). Absorbance of a significant portion of emitted fluorescent light by the broad spectrum of hemoglobin complicates the fluorescent quenching technique. Literature shows that RBC hemoglobin has autofluorescent properties (Hirstch, 2003). This work explored the possibility of using intrinsic fluorescence of intracellular hemoglobin to measure RBC permeability to water and solutes. Network Center for Applied Development in Vancouver provided adult RBCs for this study. We used SX20 stopped-flow reaction analyser (Applied Photophysics) to measure changes in RBC autofluorescence when mixed with sodium chloride or glycerol solutions of different concentration (excitation 280 nm, emission 314 nm). Equilibrium volumes of RBCs in sodium chloride solutions were measured on Coulter Counter Model ZBI (Coulter Electronics, Inc.). Lysed RBCs served as a negative control to verify that changes in autofluorescence intensity are caused by the changes in volume of intact RBCs. We also measured absolute values of autofluorescence as a function of hemoglobin concentration in the solution using SpectraMax GEMINI EM dual-scanning microplate spectrofluorometer (Molecular devices). Our results showed that the intensity of RBC autofluorescence and RBC volume measured on Coulter Counter were directly related. Specifically, mixing RBCs with hypotonic solution caused increase in cell volume and gradual increase in autofluorescence; cell shrinking in hypertonic solution resulted in autofluorescence decrease. On the contrary, tonicity of solution had no effect on autofluorescence of lysed RBCs. When RBCs were injected in glycerol solution, autofluorescence first dropped, then gradually increased, and finally reached a plateau. This response is similar to the well-known pattern of cell volume changes upon mixing with permeable cryoprotectant. We also found that at high (intracellular) hemoglobin concentrations fluorescence intensity and hemoglobin concentration are inversely related. Combined, our data shows that RBC autofluorescence is directly translated to RBC volume, and self-quenching of intracellular hemoglobin is responsible for the cell volume driven changes in RBC autofluorescence. Based on these findings, we are working on developing a new method to determine osmotic permeability of RBCs, which will significantly help with future design and optimization of cryopreservation protocols.

Defected red blood cell membranes and direct correlation with the uraemic milieu: the connection with the decreased red blood cell lifespan observed in haemodialysis patients

Together with impaired production of erythropoietin and iron deficiency, the decreased lifespan of red blood cells (RBCs) is a main factor contributing to the chronic anaemia observed in haemodialysis (HD) patients. Atomic force microscopy is employed in this work to thoroughly survey the membrane of intact RBCs (iRBCs) of HD patients in comparison to those of healthy donors, aiming to obtain direct information on the structural status of RBCs that can be related to their decreased lifespan. We observed that the iRBC membrane of the HD patients is overpopulated with extended circular defects, termed ‘orifices’, that have typical dimension ranging between 0.2 and 1.0 μm. The ‘orifice’ index—that is, the mean population of ‘orifices’ per top membrane surface—exhibits a pronounced relative increase of order 54 ± 12% for the HD patients as compared to healthy donors. Interestingly, for the HD patients, the ‘orifice’ index, which relates to the structural status of the RBC membrane, correlates strongly with urea concentration, which is a basic index of the uraemic milieu. Thus, these results indicate that the uraemic milieu downgrades the structural status of the RBC membrane, possibly triggering biochemical processes that result in their premature elimination from the circulation. This process could decrease the lifespan of RBCs, as observed in HD patients.

Fourier transform light scattering angular spectroscopy using digital inline holography

A simple and practical method for measuring the angle-resolved light scattering (ARLS) from individual objects is reported. Employing the principle of inline holography and a Fourier transform light scattering technique, both the static and dynamic scattering patterns from individual micrometer-sized objects can be effectively and quantitatively obtained. First, the light scattering measurements were performed on individual polystyrene beads, from which the refractive index and diameter of each bead were retrieved. Also, the measurements of the static and dynamic light scattering from intact human red blood cells are demonstrated. Using the present method, an existing microscope can be directly transformed into a precise instrument for ARLS measurements.