Red Blood Cell Samples Research Articles

Towards Mechanical Clinical Markers in Sickle Cell Disease: Dynamics of Red Blood Cells in Low Shear Flow

Several regimes of motion are evident in red blood cells (RBCs) under shear flow and are known to be controlled by the shear flow rate and by the deformability of the cell. Above a certain shear flow rate, healthy RBCs present a fluidized regime analogous to that of a droplet: the membrane adopts a tank-treading motion, i.e. rotates around the centre of mass of the cell, and its orientation oscillates around a mean value (Fig. 1). This motion is not observed in rigid RBCs which keep the same regime of motion as with low shear flow rate. The transition from one motion to the other depends on several intrinsic parameters including membrane viscosity, elasticity and cytoplasmic viscosity.Sickle cell disease (SCD) is a hereditary hemolytic anemia due to the presence of mutant hemoglobin, Hb S, which tends to polymerize in RBCs, reducing its deformability and resulting ultimately in RBC sickling. We postulate that SCD may affect RBCs capacity to adopt the tank-treading motion.Thanks to microfluidic chips coupled with high speed video, we studied the fraction of RBCs displaying a tank-treading motion (% of TT) at moderate shear stresses (0.4-0.6 Pa), in homozygous SCD patients (n=30) and in normal subjects (n=12). At the time of sampling, SCD patients were in steady state (no vaso-occlusive crisis or other acute complications) and not transfused for at least 3 months. Around 3 microL of total blood were necessary for each measurement. RBCs were studied under atmospheric conditions and exhibited the normal discoid shape.We showed first that the % of TT is significantly different in SCD patients compared to controls, with no overlap between the values of each groups (respectively 70.4±12.9% versus 98.6±0.9%). Second, measurements performed on different cells fractions isolated by cell density or in the presence of different NaCl concentrations, showed that the % of TT cells is sensitive to cell density and hydration status, both in normal subjects and in SCD patients. Finally, we evaluate the % of TT in RBC samples drawn just before or during 10 episodes of vaso-occlusive crisis from 8 different patients. The definition of a vaso-occlusive crisis, for this specific study, was set as an episode with pain in at least 2 different locations requiring hospitalization at least for 24h. The results revealed that, for a given patient, the % of TT cells varies significantly for 12h before as well as during vaso-occlusive crises with kinetics comparable from one patient to another.In conclusion, we described here a new inexpensive biological test able to discriminate healthy RBCs from sickle RBCs, with preliminary results suggesting potential interest for monitoring the clinical status of SCD patients. Further analysis will be necessary to determine if this parameter is suitable for predicting the occurrence of vaso-occlusive crises early enough to allow preventive actions.Fig1: Schematic view of the regime of motion corresponding to tank-treading. Flow direction is from left to right and RBCs are observed from the direction of the shear gradient. The black dot on the RBCs displays the motion of a membrane element on the RBC surface. The axis of symmetry remains in the shear plane, the body of the RBC oscillates slightly as the membrane rotates around it DisclosuresThuret:Addmedica: Research Funding; bluebird bio: Research Funding; Novartis: Research Funding.

Integrated Microfluidic Automated Particle Tracking Enables High-Throughput Cell Deformability Cytometry for Red Cell Disorders

Background: Studying individual red blood cells (RBCs) is critical to understanding hematologic diseases, as pathology often originates at the single-cell level. Measuring biophysical properties, such as deformability, of RBCs is also clinically important as RBC deformability is pathologically altered in numerous disease states including sickle cell disease (SCD), beta-thalassemia major (β-thal), and the “storage lesion” that occurs over time in stored samples of otherwise normal RBCs1-3. While ektacytometry-based systems measure RBC deformability, these assays do not have single-cell resolution4. As evidenced by flow cytometry, single cell measurements are important to detect pathologic cellular subpopulations in hematologic diseases. Recently, microfluidic devices that model the microvascular environment have enabled single RBC deformability measurements, but truly high-throughput measurements have remained elusive due to technical issues such as image processing inaccuracy or aberrant signaling when reconstructing RBC velocity profiles.Methods: To that end, here we introduce a novel integration of a microfluidic device coupled with our UMUTracker, an innovative MATLAB-based automated particle tracking program that is run on a standard desktop computer5. This innovative pairing of the two technologies results in high-throughput velocity tracking of single RBCs through a microfluidic model of a capillary bed that translates to a single deformability index (sDI) for each RBC (Fig 1). To demonstrate the sDI heterogeneity of RBCs across different conditions, data was obtained from healthy volunteers, SCD and β-thal patients, and stored packed RBCs.Results: sDI distribution curves were obtained for healthy RBCs and glutaraldehyde-stiffened RBCs (as positive controls) in which the latter resulted in an expected “left shift” and decrease in the mean sDI (Fig 2). Interestingly, while the mean sDI for SCD RBCs was also expectedly decreased, the sDI distribution was clearly non-normal, indicating the existence of heterogeneous RBC subpopulations with different deformabilities. These heterogeneous sDI distributions were observed in multiple SCD patients, including those on hydroxyurea (HU) and not, although patients on HU exhibited RBCs subpopulations with relatively higher sDIs (Fig 3). While stored RBC samples showed an expected drop in mean sDI over time, our system detected marked shifts in peak sDI with high temporal resolution over only 4 day intervals (Fig 4). Finally, each time a β-thal patient was transfused, the peak sDI shifted to the right and then gradually decreased over the course of the transfusion cycle, while the mean sDI exhibited minimal change over time (Fig 5).Conclusions: Our novel combined microfluidic/portable image analysis system demonstrates the high-throughput capability to detect distinct RBC subpopulations, at the single cell level, of different deformabilities in SCD, β-thal, and aging stored RBCs. This heterogeneity indicates that, in these disease states, RBC deformability cannot be fully characterized with mean or bulk biophysical measurements such as those obtained with ektacytometry. Ongoing studies will determine how changes in sDI profiles are associated with clinical events and different therapies as well as the biological significance of these RBC subpopulations with varied deformabilities and the underlying mechanisms for these differences.

Intradonor reproducibility and changes in hemolytic variables during red blood cell storage: results of recall phase of the REDS-III RBC-Omics study.

Genetic determinants may underlie the susceptibility of red blood cells (RBCs) to hemolyze in vivo and during routine storage. This study characterized the reproducibility and dynamics of in vitro hemolysis variables from a subset of the 13,403 blood donors enrolled in the RBC-Omics study. RBC-Omics donors with either low or high hemolysis results on 4°C-stored leukoreduced (LR)-RBC samples from enrollment donations stored for 39 to 42 days were recalled 2 to 12 months later to donate LR-RBCs. Samples of stored LR-RBCs from the unit and from transfer bags were evaluated for spontaneous and stress-induced hemolysis at selected storage time points. Intradonor reproducibility of hemolysis variables was evaluated in transfer bags over two donations. Hemolysis data at serial storage time points were generated on LR-RBCs from parent bags and analyzed by site, sex, race/ethnicity, and donation frequency. A total of 664 donors were successfully recalled. Analysis of intradonor reproducibility revealed that osmotic and oxidative hemolysis demonstrated good and moderate reproducibility (Pearson's r = 0.85 and r = 0.53, respectively), while spontaneous hemolysis reproducibility was poor (r = 0.40). Longitudinal hemolysis in parent bags showed large increases over time in spontaneous (508.6%) and oxidative hemolysis (399.8%) and smaller increases in osmotic (9.4%) and mechanical fragility (3.4%; all p < 0.0001). Spontaneous hemolysis is poorly reproducible in donors over time and may depend on site processing methods, while oxidative and osmotic hemolysis were reproducible in donors and hence could reflect consistent heritable phenotypes attributable to genetic traits. Spontaneous and oxidative hemolysis increased over time of storage, whereas osmotic and mechanical hemolysis remained relatively stable.

Blood, sweat, and tears: Red Blood Cell-Omics study objectives, design, and recruitment activities.

The Red Blood Cell (RBC)-Omics study was initiated to build a large data set containing behavioral, genetic, and biochemical characteristics of blood donors with linkage to outcomes of the patients transfused with their donated RBCs. The cohort was recruited from four US blood centers. Demographic and donation data were obtained from center records. A questionnaire to assess pica, restless leg syndrome, iron supplementation, hormone use, and menstrual and pregnancy history was completed at enrollment. Blood was obtained for a complete blood count, DNA, and ferritin testing. A leukocyte-reduced RBC sample was transferred to a custom storage bag for hemolysis testing at Storage Days 39 to 42. A subset was recalled to evaluate the kinetics and stability of hemolysis measures. A total of 13,403 racially/ethnically diverse (12% African American, 12% Asian, 8% Hispanic, 64% white, and 5% multiracial/other) donors of both sexes were enrolled and ranged from 18 to 90 years of age; 15% were high-intensity donors (nine or more donations in the prior 24 mo without low hemoglobin deferral). Data elements are available for 97% to 99% of the cohort. The cohort provides demographic, behavioral, biochemical, and genetic data for a broad range of blood donor studies related to iron metabolism, adverse consequences of iron deficiency, and differential hemolysis (including oxidative and osmotic stress perturbations) during RBC storage. Linkage to recipient outcomes may permit analysis of how donor characteristics affect transfusion efficacy. Repository DNA, plasma, and RBC samples should expand the usefulness of the current data set.

Assessment of the Deformability and Velocity of Healthy and Artificially Impaired Red Blood Cells in Narrow Polydimethylsiloxane (PDMS) Microchannels.

Malaria is one of the leading causes of death in underdeveloped regions. Thus, the development of rapid, efficient, and competitive diagnostic techniques is essential. This work reports a study of the deformability and velocity assessment of healthy and artificially impaired red blood cells (RBCs), with the purpose of potentially mimicking malaria effects, in narrow polydimethylsiloxane microchannels. To obtain impaired RBCs, their properties were modified by adding, to the RBCs, different concentrations of glucose, glutaraldehyde, or diamide, in order to increase the cells’ rigidity. The effects of the RBCs’ artificial stiffening were evaluated by combining image analysis techniques with microchannels with a contraction width of 8 µm, making it possible to measure the cells’ deformability and velocity of both healthy and modified RBCs. The results showed that healthy RBCs naturally deform when they cross the contractions and rapidly recover their original shape. In contrast, for the modified samples with high concentration of chemicals, the same did not occur. Additionally, for all the tested modification methods, the results have shown a decrease in the RBCs’ deformability and velocity as the cells’ rigidity increases, when compared to the behavior of healthy RBCs samples. These results show the ability of the image analysis tools combined with microchannel contractions to obtain crucial information on the pathological blood phenomena in microcirculation. Particularly, it was possible to measure the deformability of the RBCs and their velocity, resulting in a velocity/deformability relation in the microchannel. This correlation shows great potential to relate the RBCs’ behavior with the various stages of malaria, helping to establish the development of new diagnostic systems towards point-of-care devices.

Intra-individual variability of long-chain fatty acids (C12–C24) in plasma and red blood cells

Long-chain fatty acids (LCFA) play key roles in mammalian cells as sources of energy, structural components and signaling molecules. Given their importance in numerous physiological processes, the roles of LCFAs in health and disease have been extensively investigated. In the majority of studies, correlations are established using a single measurement in plasma or red blood cells (RBCs). Although a few studies have reported on reproducibility of individual fatty acid measurements, the comprehensive analysis of intra-individual LCFA variability has not been performed. Therefore, our goal was to determine intra-individual variability for the 22 most abundant LCFAs in both plasma and RBC samples collected from healthy individuals on a regular diet after overnight fasting. The measurements of LCFAs in RBCs were consistent throughout the course of study reflecting long-term nutritional status. In contrast, the results in plasma showed considerable LCFA intra-individual variability, even between fatty acids of the same type. Plasma intra-individual variability for omega-3 alpha-linolenic and eicosapentaenoic acids in some participants were >40% whereas the variability of docosahexaenoic acid was consistently <12.8%. Omega-6 linoleic and arachidonic acids also showed low variability in plasma. The results suggest that some LCFAs have less variability and would be more reliable as biomarkers. Reliability of biomarkers can have a profound impact on the research outcomes. Intra-individual variability of LCFAs should be taken into consideration in designing, conducting and interpreting results of clinical studies.

NLRP3 Inflammasome Activation Regulates Aged RBC Clearance

The NLR family pyrin domain-containing protein 3 (NLRP3) inflammasome is triggered by various stimuli. Whether the NLRP3 inflammasome is activated during the monocyte clearing of aged or damaged erythrocytes is unknown. This work aimed to determine whether the NLRP3 inflammasome is activated during the THP-1 cell engulfing of aged erythrocytes. In the study, THP-1 cells were treated with PMA and then coincubated with untreated red blood cells (RBCs), 42 °C-treated RBCs, immunoglobulin G (IgG) anti-D-sensitized RBCs, Rhnull/Rhmod RBC sample, hemoglobin, and RBC ghost. The activation of the NLRP3 inflammasome and production of some proinflammatory cytokines were determined using immunoblotting, cytometric bead array, and digital PCR. An NLRP3 inflammasome inhibitor was also used to evaluate the alteration of the NLRP3 activation and RBC clearance rate. The untreated RBCs, 42 °C-incubated RBCs, IgG-opsonized RBCs, Rhnull/Rhmod RBCs, RBC ghosts, and hemoglobin induced the THP-1-cell-mediated activation of the NLRP3 inflammasome and the production of inflammatory cytokines. The RBC clearance rate exhibited a positive correlation with the expression of proinflammatory cytokines. The NLRP3 inflammasome inhibitor reduced the NLRP3 activation and RBC phagocytosis rate. The NLRP3 inflammasome was activated during the clearance of the aged erythrocytes through unopsonized and opsonized pathways. However, the mechanism of such phenomenon needs to be further elucidated. Such mechanism may provide new insight into the assessment of the safety of transfusing long-storage RBC based on cytokine levels.

Mossbauer study of different factors influence on donated blood quality

Packed red blood cells (RBC) are main blood components used in transfusiology. During storage, preserved blood cells undergo progressive structural and functional changes that may reduce red-cell function and viability after transfusion. As the hemoglobin is the main and active component of erythrocytes, we consider that oxidized hemoglobin derivatives formation cause those changes. With the aim of developing the new criteria for donated blood suitability for transfusion, we studied storage-related changes in red blood cells hemoglobin by means of analytical fitting of Mossbauer spectra. We analyzed Mossbauer spectra of set RBC’s samples from volunteer donors every few days during their storage. Using those data, we built kinetic curves of various hemoglobin derivatives changes during RBC storage for different hemoglobin forms concentration in RBC samples. We found that kinetic curves are specific for each donor. They also depend on the donor’s age and health condition at the time of donation. Based on our spectroscopy results we suggested an objective criteria for duration of storage of regular donors RBC.

Recovery of autologous sickle cells by hypotonic wash

Abstract It is important to isolate autologous red blood cells (RBCs) from transfused RBCs in samples from recently transfused patients to ensure that accurate serologic results are obtained. Typically, this isolation can be performed using methods that separate patient reticulocytes from transfused, older donor RBCs. Patients with sickle cell disease (SCD), however, characteristically have RBCs with altered membrane and morphological features, causing their RBCs to take on a sickle-shape appearance different from the biconcave disc-shape appearance of “normal” RBCs. These characteristics enable the use of hypotonic saline solution to lyse normal RBCs while allowing “sickle cells” to remain intact. Because many patients with SCD undergo frequent transfusions to treat their condition, the use of hypotonic saline solution provides a rapid method to obtain autologous RBCs for serologic testing from this patient population using standard laboratory equipment and supplies. Immunohematology 2018;34: 16–18.

Validity and reliability of serologic immunophenotyping of multiple blood group systems by ORTHO Sera with fully automated procedure

The increase of immunization against blood group antigens has reinforced the need for automated extensive blood typing. The aim of this study was to assess both the validity and reliability of red blood cell (RBC) automated agglutination technology in testing for antigens of Kidd (Jk), Duffy (Fy), and MNS (Ss) blood systems. ORTHO Sera (Ortho Clinical Diagnostics, Raritan, NJ) anti-Jka, anti-Jkb, Anti-Fya, anti-Fyb, anti-S, and anti-s reagents were each tested on RBC samples previously typed. Replicates were performed on three separate testing sessions with three consecutive repetitions within each session, thus obtaining 486 test results. Accuracy was assessed by aggregate analysis of sensitivity, specificity, and area under the receiver operating characteristics curve (AUC). Reliability was estimated by a cross-classified mixed-effect logistic model. All reagents tested yielded optimal accuracy (100% for sensitivity and specificity, and 1.00 for AUC), except for anti-S, for which performance was slightly lower (98%, 100%, and 0.99, respectively), owing to misclassification of one sample in a single replicate. Anomalous automated measurements were recorded in 38 of 486 tests (7.8%), which then needed additional manual interpretation. Different sessions and samples were the major contributors to measurement failures (38% and 18%, separately). Order of repetitions and antigen specificity across replicates did not contribute to the risk of failures, although weak evidence of enhanced risk was observed with Jk testing. Automated RBC typing with ORTHO Sera reagents against antigens in the Kidd, Duffy, and MNS blood group systems displayed nearly 100 percent accuracy. However, a sizable number of replicates needed additional ad hoc interpretation, thus suggesting that the reliability could still be improved. Automated agglutination technology represents a viable option for phenotyping large volumes of samples.

Occurrence and Partitioning of Bisphenol Analogues in Adults’ Blood from China

Widespread human exposure and associated adverse health effects led to regulations on the usage of bisphenol A (BPA). Several bisphenol analogues (BPs) have been introduced as BPA alternatives in various applications. However, these BPs have been shown to exhibit similar or even stronger endocrine-disrupting activities compared with that of BPA. Currently, information on the human exposure to BPA alternatives remains limited. In this study, nine BPs were quantified in 81 pairs of plasma and red blood cell (RBC) samples from Chinese participants. In human plasma, the predominant BPs was BPA, bisphenol S (BPS), and bisphenol AF (BPAF), with the mean concentrations of 0.40, 0.15, and 0.073 ng/mL, respectively. BPA (accounting for 63% of total BPs) and BPS (18%) were the major BPs in the RBC fraction. Mass fractions in plasma (Fp) were found to be highest for BPS (mean, 0.78), followed by BPAF (0.71) and BPA (0.67), indicating strong partitioning to the plasma fraction. However, bisphenol AP was more frequently detected in the RBC fraction. Estimated total daily intake (EDI) of BPA was in the range of 0.0048-0.75 μg/kg bw/day for the participants, and adults aged >50 years had comparatively lower EDI. To our knowledge, this is the first study to assess the occurrence and partitioning of BPA alternatives in paired human plasma and RBCs from the Chinese general population.

The changes of red blood cell viscoelasticity and sports anemia in male 24-hr ultra-marathoners

BackgroundIn endurance sports, stress, dehydration and release of chemical factors have been associated with red blood cell (RBC) alterations of structure and function, which may contribute to sports anemia, a well-observed phenomenon during long-distance running. Until now, the investigation of the changes of viscoelastic properties of RBC membrane, a decisive factor of RBC deformability to avoid hemolysis, is lacking, especially in an Oriental population. Methodsnineteen runners were prospectively recruited into our study. Hematological parameters were analyzed before and immediately after the 2015 Taipei 24H Ultra-Marathon Festival, Taiwan. Video particle tracking microrheology was used to determine viscoelastic properties of each RBC sample by calculating the dynamic elastic modulus G′(f) and the viscous modulus G″(f) at frequency f = 20 Hz. ResultsHaptoglobin, RBC count, hemoglobin, hematocrit, mean cell hemoglobin, plasma free hemoglobin and unsaturated iron-binding capacity values of the recruited runners showed a statistically significant drop in the post-race values. Blood concentration of reticulocyte and ferritin were significantly higher at post-race compared with pre-race. 15 out of the 19 runners had a concurrent change in the elastic and the viscous moduli of their RBCs. Changes in the elastic and the viscous moduli were correlated with changes in the RBC count, hemoglobin and hematocrit. ConclusionViscoelasticity properties, the elastic modulus G′(f) and the viscous modulus G″(f) of RBCs are associated with endurance exercise-induced anemia.

High prevalence of very-low Plasmodium falciparum and Plasmodium vivax parasitaemia carriers in the Peruvian Amazon: insights into local and occupational mobility-related transmission

BackgroundThe incidence of malaria due both to Plasmodium falciparum and Plasmodium vivax in the Peruvian Amazon has risen in the past 5 years. This study tested the hypothesis that the maintenance and emergence of malaria in hypoendemic regions such as Amazonia is determined by submicroscopic and asymptomatic Plasmodium parasitaemia carriers. The present study aimed to precisely quantify the rate of very-low parasitaemia carriers in two sites of the Peruvian Amazon in relation to transmission patterns of P. vivax and P. falciparum in this area.MethodsThis study was carried out within the Amazonian-ICEMR longitudinal cohort. Blood samples were collected for light microscopy diagnosis and packed red blood cell (PRBC) samples were analysed by qPCR. Plasma samples were tested for total IgG reactivity against recombinant PvMSP-10 and PfMSP-10 antigens by ELISA. Occupation and age 10 years and greater were considered surrogates of occupation-related mobility. Risk factors for P. falciparum and P. vivax infections detected by PRBC-qPCR were assessed by multilevel logistic regression models.ResultsAmong 450 subjects, the prevalence of P. vivax by PRBC-PCR (25.1%) was sixfold higher than that determined by microscopy (3.6%). The prevalence of P. falciparum infection was 4.9% by PRBC-PCR and 0.2% by microscopy. More than 40% of infections had parasitaemia under 5 parasites/μL. Multivariate analysis for infections detected by PRBC-PCR showed that participants with recent settlement in the study area (AOR 2.1; 95% CI 1.03:4.2), age ≥ 30 years (AOR 3.3; 95% CI 1.6:6.9) and seropositivity to P. vivax (AOR 1.8; 95% CI 1.0:3.2) had significantly higher likelihood of P. vivax infection, while the odds of P. falciparum infection was higher for participants between 10 and 29 years (AOR 10.7; 95% CI 1.3:91.1) and with a previous P. falciparum infection (AOR 10.4; 95% CI 1.5:71.1).ConclusionsThis study confirms the contrasting transmission patterns of P. vivax and P. falciparum in the Peruvian Amazon, with stable local transmission for P. vivax and the source of P. falciparum to the study villages dominated by very low parasitaemia carriers, age 10 years and older, who had travelled away from home for work and brought P. falciparum infection with them.

Laser-induced changes of in vitro erythrocyte sedimentation rate.

The study of the effects of low-level laser (LLL) radiation on blood is important for elucidating the mechanisms behind the interaction of LLL radiation and biologic tissues. Different therapy methods that involve blood irradiation have been developed and used for clinical purposes with beneficial effects. The aim of this study was to compare the effects of different irradiation protocols using a diode-pumped solid-state LLL (λ=405nm) on samples of human blood by measuring the erythrocyte sedimentation rate (ESR). Human blood samples were obtained through venipuncture into tubes containing EDTA as an anticoagulant. Every sample was divided into two equal aliquots to be used as an irradiated sample and a non-irradiated control sample. The irradiated aliquot was subjected to a laser beam with a wavelength of 405nm and an energy density of 72J/cm2. The radiation source had a fixed irradiance of 30mW/cm2. The ESR change was observed for three different experimental protocols: irradiated whole blood, irradiated red blood cells (RBCs) samples re-suspended in non-irradiated blood plasma, and non-irradiated RBCs re-suspended in irradiated blood plasma. The ESR values were measured after laser irradiation and compared with the non-irradiated control samples. Irradiated blood plasma in which non-radiated RBCs were re-suspended was found to result in the largest ESR decrease for healthy human RBCs, 51%, when compared with RBCs re-suspended in non-irradiated blood plasma. The decrease in ESR induced by LLL irradiation of the plasma alone was likely related to changes in the plasma composition and an increase in the erythrocyte zeta potential upon re-suspension of the RBCs in the irradiated blood plasma.

Pathogen inactivation of Dengue virus in red blood cells using amustaline and glutathione.

Dengue virus (DENV) is an arbovirus primarily transmitted through mosquito bite; however, DENV transfusion-transmitted infections (TTIs) have been reported and asymptomatic DENV RNA-positive blood donors have been identified in endemic countries. DENV is considered a high-risk pathogen for blood safety. One of the mitigation strategies to prevent arbovirus TTIs is pathogen inactivation. In this study we demonstrate that the amustaline and glutathione (S-303/GSH) treatment previously found effective against Zika virus in red blood cells (RBCs) is also effective in inactivating DENV. Red blood cells were spiked with high levels of DENV. Viral RNA loads and infectious titers were measured in the untreated control and before and after pathogen inactivation treatment of RBC samples. DENV infectivity was also assessed over five successive cell culture passages to detect any potential residual replicative virus. The mean ± SD DENV titer in RBCs before inactivation was 6.61 ± 0.19 log 50% tissue culture infectious dose (TCID50 )/mL and the mean viral RNA load was 8.42 log genome equivalents/mL. No replicative DENV was detected either immediately after completion of treatment using S-303/GSH or after cell culture passages. Treatment using S-303/GSH inactivated high levels of DENV in RBCs to the limit of detection. In combination with previous studies showing the effective inactivation of DENV in plasma and platelets using the licensed amotosalen/UVA system, this study demonstrates that high levels of DENV can be inactivated in all blood components.

Adherence to 6-Mercaptopurine in children and adolescents with Acute Lymphoblastic Leukemia.

ObjectiveStudies on children with Acute Lymphoblastic Leukemia (ALL) reported non-adherence in 2–54% of cases. The primary objective of this study was to assess rates of adherence to 6-MP using two different methods in children and adolescents with ALL. Secondary aim was to identify factors that influence adherence to 6-MP in children with ALL.MethodsAll eligible children with ALL who are (≤ 19) years old and receive 6-MP therapy for at least 1 month were approached to participate in the study. A total of 52 children with ALL and their primary caregivers were recruited. Adherence measures included an objective method (measuring 6-MP metabolites in packed Red Blood Cells (RBCs)) and a subjective method (using parent and child self-report via the Medication Adherence Report Scale; MARS; Adherence was defined as 90% or greater).ResultsRates of adherence varied across the measurement methods. Packed RBCs sample analysis indicated forty-four patients (84.6%) to be adherent. Using the MARS questionnaires, a total of 49 children (94.2%) were classified as being adherent according to the parental MARS questionnaire scores, while all the 15 children (100%) who answered the MARS (child) questionnaire were classified as adherent. Overall adherence rate was 80.8% within the studied population.ConclusionMARS scale was shown to overestimate adherence compared to measurement of 6-MP metabolites in the blood. A combination of both methods led to increased detection of non-adherence to thiopurine in children with ALL.

Spectrum analysis for assessing red blood cell aggregation using high-frequency ultrasound array transducer.

The purpose of this study is to investigate a spectrum analysis technique for detecting and monitoring red blood cell (RBC) aggregation using a high-frequency array transducer. To assess the feasibility of this approach, the backscattered radio-frequency signal from non-aggregated and aggregated RBC samples with two hematocrit levels were acquired by using a 30-MHz linear array transducer and analyzed in frequency domain. Three parameters such as spectral slope, midband fit and Y intercept were extracted in a static condition. Fresh porcine blood was used and degrees of aggregation were changed by diluting plasma concentration. From the experiments, it was demonstrated that the spectral slope related to a size of scatterer progressively declined as the level of aggregation increased; its mean values at hematocrit of 40% were 1.10 and -0.22dB/MHz for RBCs suspended in isotonic phosphate buffered saline and solution with 70% plasma concentrations, respectively. For the midband fit and Y intercept, the mean values were increased by 9.1 and 46.4dB, respectively. These results indicated that the spectrum analysis technique is useful for monitoring RBC aggregation and can be potentially developed for assessing aggregation in clinical applications.

Platelet Detection in Unaltered Whole Blood

Abstract Platelets are non-nucleated cellular fragments circulating in the peripheral blood, derived from megakaryocytes. They are critical in the maintenance of hemostasis. Alterations in their function are associated with a range of clinical conditions. Using flow cytometry, light scatter signals from platelets typically overlap signals from red blood cells (RBC). Since RBC outnumber platelets by more than an order of magnitude in whole blood, platelets are typically studied by depleting samples of RBCs by centrifugation methods or selective RBC cell lysis. These sample preparation procedures require manipulations that may affect platelet health and function, and result in loss of cells. Additionally, lysed samples require labeling of the platelets with a fluorescent marker to adequately resolve the platelets from debris/noise. We present a novel technique for resolving leukocytes and platelets from RBC in whole blood using orthogonal light scatter from 2 lasers. This method exploits the differences in light-scattering properties between RBCs and platelets and white blood cells (WBC). Hemoglobin readily absorbs 405 nm laser light, reducing the scatter signal from RBC relative to WBCs and platelets, resulting in a reproducible scatter pattern when analyzing human whole blood with both blue (488 nm) and violet (405 nm) side scatter. Using this approach, a distinct platelet population can be discerned in unaltered whole blood, requiring only dilution of whole blood in buffer. Combining this approach with acoustic cytometry allows great flexibility in determining optimal dilution and sample collection rates for minimal coincidence and rapid analysis. For research use only. Not for use in diagnostic procedures.

Description of 15 DNA-positive and antibody-negative "window-period" blood donations identified during prospective screening for Babesia microti.

Blood donation screening detecting only antibodies fails to identify donors in the earliest stage of infection, before a detectable immunologic response, that is, the "window period" (WP). We present data on WP donations identified during prospective screening for Babesia microti, a transfusion-transmissible parasite of increasing concern in the United States. Blood donations collected in Connecticut, Massachusetts, Minnesota, and Wisconsin were screened using polymerase chain reaction (PCR) and arrayed fluorescence immunoassay (AFIA) to detect B. microti DNA and antibodies, respectively. Parasite loads were estimated using quantitative PCR. Red blood cell (RBC) samples were inoculated into hamsters to assess infectivity. Donors screening reactive were indefinitely deferred, tested by supplemental methods, and followed to assess DNA and antibody clearance. Demographic data from WP donors (i.e., those screening PCR positive and AFIA negative) were compared to data from other positive donors. Of 220,479 donations screened from June 2012 to August 2016, a total of 700 were positive, of which 15 (2% of positive donations or 1 per 14,699 screened donations) were confirmed WP donations. The median estimated parasite load in WP donations was 350 parasites/mL, no different than AFIA-positive and PCR-positive donors. Parasite loads in RBC samples from WP units ranged from 14 to 11,022 parasites/mL; RBC samples from three of 10 (30%) WP donations infected hamsters. The mean age of WP donors was 48 years (range, 17-75 years); three (20%) were female. WP donor demographics did not differ significantly from demographics of other donors. We report one per 15,000 B. microti WP infections in blood donors in endemic areas, demonstrating the importance of nucleic acid testing to mitigate the risk of transfusion-transmitted babesiosis.

Direct Electrochemical Determination of Erythrocyte Concentration and Morphology

Red blood cell (RBC) count in blood is an important clinical test, since erythrocytosis (high RBC count) and erythropenia (low RBC count) are features of severe diseases. For example, erythrocytosis occurs in certain tumors or in hypoxic conditions, while erythropenia accompanies blood loss, iron deficiency, or poisonings by hemolytic toxicants. In current routine clinical laboratory practice, the RBC count is determined automatically using a hemocytometer or via microscopy. The latter allows for both the RBC count and identification of sizes and morphologies of erythrocytes, although the method is quite complex and time-consuming. Electrochemical methods for determining erythrocytes concentration (RBC count) are known, although they are few and have not been utilized in routine clinical laboratories. The main advantages of the electrochemical methods are due to the simplicity of measurement procedure, high sensitivity, and high speed of analysis. However, existing electrochemical techniques were developed for use with animal erythrocytes. For example, a proposed electrochemical method [1] of determining red and white blood cell counts using a graphite electrode was developed for measurements in rabbit blood, and the effect of electrode potential on the behavior of blood cells was examined; however, the mechanism of the observed effects was unclear. Recently, an attempt was made to develop a method to determine the amount of peroxidase activity of sheep red blood cells on the pyrolytic graphite electrode [2]. This is an indirect electrochemical method that used the electroreduction of oxygen formed in a reaction of hydrogen peroxide with a sample of sheep erythrocytes to characterize the RBC sample. The present work explored the possibility of using direct electrooxidation of an RBC suspension on an optically transparent ITO electrode. Theoretically, the likelihood of the electrochemical activity of the blood cells is quite high, since the surface of RBC membranes contains electron-donor and electron-acceptor functional groups. Moreover, circumstantial evidence of erythrocyte electrochemical activity has been shown [3], manifesting as morphological changes of cells depending on electrode potential. The goal of the present work was to develop an electrochemical method for determining the number of erythrocytes and their morphologies in real time. A suspension of erythrocytes containing 8∙109 – 8∙1011 cells/L was prepared by diluting packed RBCs of apparently healthy donors with isotonic physiologic saline (0.15 M NaCl). A three-electrode electrochemical cell was used, and an optically transparent ITO electrode (Sigma-Aldrich, USA) with a surface area of 28.3 mm2formed the bottom of the cell. A carbon black electrode was used as the auxiliary electrode, with a silver/silver chloride electrode as the reference electrode. Polarization measurements were performed using an IPC Pro L potentiostat ("Kronas", Russia) in cyclic scan mode, with a scan rate of 10 mV/s. Erythrocyte morphology depending on the electrode potential was examined in real time using an Eclipse TS100 inverted microscope (Nikon, Japan), with 40X lens magnification. Polarization measurements on the ITO electrode were carried out in isotonic saline containing a suspension of erythrocytes. It was observed (Fig. 1A) that the suspension of erythrocytes under these conditions is electrochemically active at anodic potentials between +400 mV and +1,000 mV (Ag/AgCl). It should be emphasized that this is the first time that direct evidence of RBC anodic oxidation has been obtained. This phenomenon was used to determine the number (concentration) of erythrocytes in blood or other biological media, since erythrocyte electrooxidation produced a limiting current on the polarization curves. The dependence of limiting current on RBC concentration (Fig.1B) is linear across ca. two orders of magnitude of erythrocyte concentrations (8∙109 – 8∙1011 cells/L), with a correlation coefficient for the calibration curve in that range R 2= 0.99. Previously it was reported [3] that simultaneous microscopy studies allow for the morphology of red blood cells in the process of electrode polarization to be evaluated. In the potential range utilized in the present work, the morphological transformation of RBCs with normal discocyte morphology into stomatocytes was observed between electrode potentials of ca. +600 to +1,000 mV. Thus, the proposed electrochemical method with concurrent microscopy shows promise for the analytical determination of blood cell counts and their morphological analysis. This may have a wide range of applications, including determination of the effects of blood interaction with foreign materials or monitoring of stored blood quality. 1. Ci Y.-X., Li H.-N., Feng J. Electroanalysis 1998 10(13): 921-925. 2. Sepunaru L., Sokolov S.L., Holter J., Neil P. N.P., Compton R.G. Angew. Chem. Int. Ed. 2016 55(33): 9768-9771. 3. Khubutiya M.Sh., Evseev A.K., Mirzaeian M., Borovkova N.V., Goroncharovskaya I.V., Goldin M.M. 229th ECS Meeting, 2016, San Diego: Abs. 1632. Figure 1