Red Blood Cell Samples Research Articles

Development of a flow standard to enable highly reproducible measurements of deformability of stored red blood cells in a microfluidic device.

Great deformability allows red blood cells (RBCs) to flow through narrow capillaries in tissues. A number of microfluidic devices with capillary-like microchannels have been developed to monitor storage-related impairment of RBC deformability during blood banking operations. This proof-of-concept study describes a new method to standardize and improve reproducibility of the RBC deformability measurements using one of these devices. The rate of RBC flow through the microfluidic capillary network of the microvascular analyzer (MVA) device made of polydimethylsiloxane was measured to assess RBC deformability. A suspension of microbeads in a solution of glycerol in phosphate-buffered saline was developed to be used as an internal flow rate reference alongside RBC samples in the same device. RBC deformability and other in vitro quality markers were assessed weekly in six leukoreduced RBC concentrates (RCCs) dispersed in saline-adenine-glucose-mannitol additive solution and stored over 42 days at 4°C. The use of flow reference reduced device-to-device measurement variability from 10% to 2%. Repeated-measure analysis using the generalized estimating equation (GEE) method showed a significant monotonic decrease in relative RBC flow rate with storage from Week 0. By the end of storage, relative RBC flow rate decreased by 22 ± 6% on average. The suspension of microbeads was successfully used as a flow reference to increase reproducibility of RBC deformability measurements using the MVA. Deformability results suggest an early and late aging phase for stored RCCs, with significant decreases between successive weeks suggesting a highly sensitive measurement method.

Three-dimensional tomography of red blood cells using deep learning

We accurately reconstruct three-dimensional (3-D) refractive index (RI) distributions from highly ill-posed two-dimensional (2-D) measurements using a deep neural network (DNN). Strong distortions are introduced on reconstructions obtained by the Wolf transform inversion method due to the ill-posed measurements acquired from the limited numerical apertures (NAs) of the optical system. Despite the recent success of DNNs in solving ill-posed inverse problems, the application to 3-D optical imaging is particularly challenging due to the lack of the ground truth. We overcome this limitation by generating digital phantoms that serve as samples for the discrete dipole approximation (DDA) to generate multiple 2-D projection maps for a limited range of illumination angles. The presented samples are red blood cells (RBCs), which are highly affected by the ill-posed problems due to their morphology. The trained network using synthetic measurements from the digital phantoms successfully eliminates the introduced distortions. Most importantly, we obtain high fidelity reconstructions from experimentally recorded projections of real RBC sample using the network that was trained on digitally generated RBC phantoms. Finally, we confirm the reconstruction accuracy using the DDA to calculate the 2-D projections of the 3-D reconstructions and compare them to the experimentally recorded projections.

High-throughput dynamical analysis of dielectrophoretic frequency dispersion of single cells based on deflected flow streamlines.

Phenotypic quantification of cells based on their plasma membrane capacitance and cytoplasmic conductivity, as determined by their dielectrophoretic frequency dispersion, is often used as a marker for their biological function. However, due to the prevalence of phenotypic heterogeneity in many biological systems of interest, there is a need for methods capable of determining the dielectrophoretic dispersion of single cells at high throughput and without the need for sample dilution. We present a microfluidic device methodology wherein localized constrictions in the microchannel are used to enhance the field delivered by adjoining planar electrodes, so that the dielectrophoresis level and direction on flow-focused cells can be determined on each traversing cell in a high-throughput manner based on their deflected flow streamlines. Using a sample of human red blood cells diluted to 2.25 × 108 cells/mL, the dielectrophoretic translation of single cells traversing at a flow rate of 1.68μL/min is measured at a throughput of 1.1 × 105 cells/min, to distinguish positive versus negative dielectrophoresis and determine their crossover frequency in media of differing conductivity for validation of the computed membrane capacitance to that from prior methods. We envision application of this dynamic dielectrophoresis (Dy-DEP) method towards high-throughput measurement of the dielectric dispersion of single cells to stratify phenotypic heterogeneity of a particular sample based on their DEP crossover frequency, without the need for significant sample dilution. Grapical abstract.

Adherence to Azathioprine/6-Mercaptopurine in Children and Adolescents with Inflammatory Bowel Diseases: A Multimethod Study

Background Measurement of the degree of adherence is a key element for the evaluation of treatment efficacy and safety; thus, adherence plays an important role in clinical research and practice. The aim of this study was to investigate medication adherence in children with inflammatory bowel disease (IBD) utilizing a multimethod assessment approach. A further aim was to examine factors that can influence adherence within this population. Methods Medication adherence in 47 children (age range 3 to 17 years) with IBD in three centers in Northern Ireland and Jordan was assessed via subjective (parent and child versions of the Medication Adherence Report Scale (MARS) specific questionnaire) and objective methods, that is, high-performance liquid chromatography (HPLC) determination of the 6-mercaptopurine (6-MP) and azathioprine (AZA) metabolites in packed red blood cell samples taken during a clinic visit. Beliefs about prescribed medicines were also assessed in parents/guardians using the Beliefs about Medicines Questionnaire (BMQ). Results An overall nonadherence to AZA/6-MP therapy in children with IBD was found to be 36.17% (17 out of 47 patients were classified as nonadherent using at least one of the assessment methods). A total of 41 patients (91.1%) were classified as adherent to AZA or 6-MP using the blood sampling, while adherence rates using the MARS questionnaire completed by children and parents/guardians were 60.6% and 72.7%, respectively. The latter provides a more longitudinal measure of adherence. Child self-reported nonadherence rates were significantly higher than parent/guardian reported rates (p=0.013). Binary logistic regression analysis identified age to be independently predictive of adherence, with adolescents (children aged ≥ 13 years old) more likely to be classified as nonadherent. Regarding the BMQ, when parental/guardian necessity beliefs outweighed concerns, that is, higher scores in the necessity-concern differential (NCD), adolescents were more likely to be classified as adherent. Conclusion Results provide evidence for ongoing adherence challenges in the paediatric population with IBD. It is recommended that parents/guardians (particularly of older children) and older children themselves, should receive enhanced counselling and education about their prescribed medicines.

Laboratory and clinical management of leukocytospermia and hematospermia: a review.

Leukocytospermia and hematospermia are defined as the presence of abnormally high white blood cell and red blood cell concentration in the semen, respectively. Numerous etiologies and various implications on fertility have been identified. In a small proportion of men, the presence of white blood cells or red blood cells can adversely affect sperm quality by the production of reactive oxygen species. Several methods have been used to assess the presence of white blood cells and red blood cells in samples, such as identification of round cells, immunohistochemical staining using monoclonal antibodies, the Endtz test, the peroxidase test, and flow cytometry or microscopy. In addition, techniques have been identified to separate sperm samples from white blood cells and red blood cells for cryopreservation to improve outcomes in assisted reproductive technology. In this review, laboratory and clinical management of leukocytospermia and hematospermia are discussed. Currently available diagnostic methods and treatment options are outlined, and available optimal cryopreservation techniques for samples with white blood cells or red blood cells are summarized.

Flow Cytometry Measurement of Reactive Oxygen Species Generation: Some Caveats and Their Solution

The redox state is very important in normal physiology and pathology. Yet, its measurement is not practiced within the clinic. The explanation is principally methodological—the inadequacy of the methods for application in the clinical laboratory. Flow cytometry is routinely utilized in hemato-oncology and immunology. Measuring of the cellular fluorescence by flow cytometry following loading with 2′7′-dichlorodihydrofluorescin diacetate (DCFDA) has become a standard experimental method for quantification of reactive oxygen species (ROS). However, the DCF fluorescence depends on the probe uptake and esterification as well as the presence of intracellular quenchers. Cells of various types, stages of differentiation or senescence, or under different conditions (e.g., pathological vs. normal) may differ in these respects. A one-time measurement does not take into consideration these differences. Herein, we describe a protocol that overcomes these caveats by determining the change in DCF fluorescence with time as a measure of the rate of ROS generation. Normal (n = 20) and thalassemia (n = 20) red blood cells (RBCs) samples were loaded with DCFDA, washed, and then incubated in a DCFDA-free medium. DCF fluorescence was measured at different times of incubation by flow cytometry and the mean fluorescence (MFC) calculated. The results showed that the rate of increase in the DCF fluorescence was linear in both types of RBCs, regardless of their basal level and hemoglobin content, with thalassemia RBCs demonstrating a 4.5-fold higher rate (265/h) compared with normal RBCs (58/h) (p < 0.001). In conclusion, this pulse-chase procedure may be used for flow cytometry clinical evaluation of general and cell type-specific oxidative stress under normal and pathological conditions.

The capture of antibodies by antibody-binding proteins for ABO blood typing using SPR imaging-based sensing technology

In this study, a wavelength-interrogated surface plasmon resonance imaging-based immunosensor was developed for ABO blood typing. To construct the immunosensor, the detection line array was formed by the site-directed immobilization of blood group antibodies on SPR chip modified with antibody-binding proteins. Red blood cell (RBC) samples were flowed into the multichannel flow cell that was orthogonal to the detection line arrays. By serially analyzing the interaction between RBCs, antibody A, and antibody B through the observed changes in the resonance dip, blood typing of four RBC samples was achieved in a single run. Detailed investigations focused on optimizing the conditions that affected the efficiency and sensitivity of immunoassay. Under the optimized conditions, the RBC samples were appropriately grouped in less than 1 h, including chip modification time. The lowest detection limit for RBC-A and RBC-B was 2×106 cells/ml. The immunosensor developed through this study provided a simple, rapid, high-throughput, and sensitive immunology strategy for classification of ABO blood group.

1832. Development of an Ultrasensitive Field-Applicable Plasmodium falciparum Assay for Malaria Diagnosis and Eradication

BackgroundMalaria control and eradication have been hampered by asymptomatic carriage which serves as a parasite reservoir. Low-density infections (< 100 parasites/microliter) frequently fall below the limit of detection (LOD) of microscopy and rapid diagnostic tests (RDT) which are antigen-based tests. Molecular methods such as polymerase chain reaction are capable of higher sensitivity yet remain impractical for resource-limited settings. We describe development of an isothermal assay using the nucleic acid detection platform SHERLOCK (Specific High-Sensitivity Enzymatic Reporter UnLOCKing), which may also be increasingly important as there has been rising detection of histidine-rich protein 2 (HRP2) gene deletions in Plasmodium spp. HRP2 is the most commonly used antigen in RDTs and deletion of this gene would render many RDTs obsolete.MethodsSHERLOCK leverages the endonucleases of CRISPR-associated microbial adaptive immunity. Cas12a is an RNA-guided, DNA-cleaving enzyme, which can be programmed with guide RNAs to cleave nontarget reporter ssDNA. We exploit the nonspecific degradation of labeled ssDNA to detect the presence of the dsDNA target that activated Cas12a (Figure 1). Recombinase polymerase amplification (RPA) coupled with Cas12a detection enables a lower LOD. Plasmodium falciparum whole genomic DNA was compared with parasites cultured in red blood cells (RBCs) with known parasitemia and boiled at 95°C for 5 minutes for lysis of RBCs/parasites then diluted 1:2.5 to prevent solidification.ResultsThis SHERLOCK assay detected simulated Plasmodium falciparum infection at attomolar LODs when applied to whole genomic DNA and simulated samples of infected RBCs spiked into whole blood. The genomic assay detected down to 0.2 parasites/microliter and the simulated sample detected to 10 parasites/microliter in the final reaction volume. In comparison, LODs from the initial input volume was 5aM and 250aM, respectively (Figure 2).ConclusionWe demonstrate an isothermal nucleic acid detection platform capable of diagnosis in 60 minutes in a one-pot assay requiring minimal sample preparation and reaching an LOD recommended by the WHO for malaria eradication. In summary, we illustrate the utility of the SHERLOCK platform in application to malaria and global health. Disclosures All Authors: No reported Disclosures.

Dissipative particle dynamics for modeling micro-objects in microfluidics: application to dielectrophoresis.

The dissipative particle dynamics (DPD) technique is employed to model the trajectories of micro-objects in a practical microfluidic device. The simulation approach is first developed using an in-house Fortran code to model Stokes flow at Reynolds number of 0.01. The extremely low Reynolds number is achieved by adjusting the DPD parameters, such as force coefficients, thermal energies of the particles, and time steps. After matching the numerical flow profile with the analytical results, the technique is developed further to simulate the deflection of micro-objects under the effect of a deflecting external force in a rectangular microchannel. A mapping algorithm is introduced to establish the scaling relationship for the deflecting force between the physical device and the DPD domain. Dielectrophoresis is studied as a case study for the deflecting force, and the trajectory of a single red blood cell under the influence of the dielectrophoretic force is simulated. The device is fabricated using standard microfabrication techniques, and the experiments involving a dilute sample of red blood cells are performed at two different cases of the actuation voltage. Good agreement between the numerical and experimental results is achieved.

Laser Controlled Cavitation Deformation in Red Blood Cells

In this research work, we studied µTAS (µ total analysis system) method to measure the deformability of red blood cells .We took the sample of RBCs(red blood cells) in a micro fluidic based chamber, then allowed low intensity laser of infra red region, mostly at wavelength 1064 nm, to fall on the sample. This generated a cavitation bubble in the fluid because of evaporation mechanism involved. We tightly hold the slides containing blood sample so that the displacement of RBCs (red blood cells) stay confined in the viewing area of the microscope. Firstly the deformation due to expansion of cavitation bubble was sighted and then came the restoring of shape of RBCs (red blood cells) which was possible due to a focused and pulsed laser. The RBCs restored their shape after some time and was viewed with the help of a video recorded by a high- speed camera. In this way the plastic character and elastic properties of RBCs (red blood cells) was measured.

Feasibility of photoacoustic imaging for the non‐invasive quality management of stored blood bags

During the in vitro storage of red blood cells (RBCs), unfavourable changes (storage lesions) cause a rapid consumption of intracellular diphosphoglycerate. The latter deregulates the oxygen-haemoglobin binding potential, subsequently increasing oxygen saturation (SO2 ) and membrane degradation, transforming RBCs from biconcave discs to rigid spherical bodies (spheroechinocytes). Current laboratory techniques invasively extract RBC samples to assess the quality of red cell concentrate (RCC) units. Optical technologies could provide a means of assessing quality non-invasively. A photoacoustic (PA) imaging technique was developed for acquiring the SO2 of blood bags non-invasively. Seven RCC units were monitored every 3-5days until expiry (6weeks). Measurements were validated against a conventional blood gas analyzer (BGA). Using an image flow cytometry assay, morphological profile trends were compared against the SO2 trends during blood bag storage. A strong correlation (r2 ≥0·95) was found when comparing temporal data between PA and BGA SO2 measurements. Inter-sample PA variability was found to be similar to that produced by BGA (±0·8%). A strong correlation was found to exist between the temporal changes in SO2 and relative spheroechinocyte population (0·79≤r2 ≤0·97). This study suggests that PA imaging can non-invasively track the SO2 of stored RBCs non-invasively. By longitudinally monitoring the change in SO2 , it is possible to infer the effects of the storage lesion on RBC morphology. This non-invasive monitoring technique allows for the assessment of blood bags, without compromising sterility pre-transfusion.

Pharmacokinetics and hemorheology of phosphocreatine and creatine in rabbits: A directly comparative study between parent drug and active metabolite

This study is to investigate pharmacokinetics (PK) and hemorheology (HR) of exogenous phosphocreatine (PCr), a cardio-protective agent, and its active metabolite creatine (Cr), with particular focus on the PK and PD comparison between PCr and Cr. A specific ion-pair reversed-phase HPLC-UV assay was used to simultaneously measure PCr, Cr and ATP concentrations in plasma and red blood cells (RBC) samples of rabbits. PK and HR parameters were calculated based on concentration-time (C-T) curves and effect-time (E-T) curves, respectively, obtained after i.v. dosing. Meanwhile the apparent pharmacological activity ratio (Rapp) and real pharmacological activity ratio (Rreal) of Cr to PCr were calculated. The PCr disappeared from plasma rapidly and in a biphasic manner; plasma PCr was converted to Cr fast and largely with the elimination rate limited metabolite disposition in vivo (Km < K). The i.v. administration of PCr led to a markedly elevated and long-lasting ATP level in RBC. After i.v. administration of preformed Cr, plasma Cr displayed similar elimination kinetics behaviors to that of Cr generated metabolically after i.v. PCr. The Cr could also raise ATP level in RBC, but to less extent than PCr. Approximately 43% of PCr-derived ATP came from Cr-derived ATP in RBC. PCr could significantly reduce whole blood viscosity and RBC osmotic fragility and Cr could do so, but weakly with estimated Rapp of 0.53–0.68 and Rreal of 0.38–0.48. PCr also inhibited platelet aggregation significantly, as opposed to Cr. The PCr-caused improvement of HR is related to the rise in ATP level in RBC. Cr is likely to partially mediate HR effect of PCr.

Influence of lipids on stable isotope ratios in mammal hair: highlighting the importance of validation

AbstractCarbon (δ13C) and nitrogen (δ15N) stable isotope ratios are increasingly used in ecological studies to evaluate diet composition and trophic relationships. However, lipids may influence stable isotope ratios due to the depletion of 13C in adipose tissues relative to proteins and carbohydrates. δ13C values can be corrected by lipid extraction or normalization models. The aims of our study were to evaluate the effects of lipid extraction on stable isotope ratios in a terrestrial mammal, the caribou (Rangifer tarandus caribou), and to propose relevant lipid normalization models that are method‐ and tissue‐specific for δ13C values. We also evaluated whether four δ13C lipid normalization and correction models proposed in the literature were applicable to our study species. Stable isotope ratios were obtained for hair, plasma, and red blood cell samples of 44 caribou in the Gaspésie National Park (Québec, Canada). The effects of lipid extraction on stable isotope ratios were tested using a paired t‐test. A simple linear model was used to correct for the effects of lipid extraction and to assess its performance compared to that of published equations. Lipid content significantly influenced δ13C values in caribou hair. The four lipid normalization equations commonly used in the literature did not accurately predict δ13Clipid‐free values of caribou hair. Based on our results, we recommend controlling systematically for lipids in terrestrial systems and analyzing δ13C (lipid‐free) and δ15N (bulk) from two separate aliquots to reach a greater precision. We also recommend controlling for lipids in hair tissue. If not possible, we recommend using a lipid normalization model that is tissue‐, method‐, and species‐specific or applying a model that has been previously validated for the tissue and species of interest.

Selenoneine is a major selenium species in beluga skin and red blood cells of Inuit from Nunavik

Nunavimmiut (Inuit of Nunavik, Northern Quebec, Canada) exhibit a high selenium (Se) status because of their frequent consumption of marine mammal foods. Indirect evidence from our previous studies had suggested that selenoneine – a novel selenocompound – may be accumulating in the blood of Nunavimmiut. We used a liquid-chromatography/inductively coupled tandem mass spectrometry (LC-ICP-MS/MS) method to measure concentrations of selenoneine and its methylated metabolite Se-methylselenoneine in archived red blood cells (RBC) obtained from 210 Nunavimmiut living in communities along the Hudson Strait, where marine mammal hunting and consumption are most frequent in Nunavik. This method was adapted to quantify selenoneine and its methylated metabolite in beluga mattaaq, an Inuit delicacy consisting of the skin with the underlying layer of fat and the major dietary source of Se for Nunavimmiut. Total selenium concentration was also measured in RBC and beluga mattaaq samples by isotope dilution ICP-MS/MS. The median selenoneine concentration in RBC was 413 μg Se/L (range = 3.20–3230 μg Se/L), representing 54% (median) of total Se content (range = 1.6–91%). Quantification of selenoneine in five beluga mattaaq samples (skin layer) from Nunavik revealed a median concentration of 1.8 μg Se/g wet wt (range = 1.2–7.4 μg Se/g), constituting 54% (median) of the total Se content (range = 44–74%). Se-methylselenoneine was also detected in Inuit RBC but not in beluga mattaaq, suggesting that selenoneine undergoes methylation in humans. Selenoneine may protect Nunavimmiut from methylmecury toxicity by increasing its demethylation in RBC and in turn decreasing its distribution to target organs.

Extracellular vesicle concentration and procoagulant activity of canine haemoperitoneum fluid and packed red blood cells.

To compare the concentration of phosphatidylserine-positive extracellular vesicles and phosphatidylserine-derived procoagulant activity of canine haemoperitoneum fluids and packed red blood cell units. Ten dogs with haemoperitoneum (neoplasia = 7; trauma = 1; other = 2) were recruited, and five non-leukoreduced packed red blood cell units purchased. Supernatants were collected from haemoperitoneum samples and packed red blood cell units using a consistent centrifugation protocol. Phosphatidylserine-positive extracellular vesicle concentrations were measured by flow cytometry and phosphatidylserine-mediated procoagulant activity by a commercial thrombin generation assay. Phosphatidylserine-mediated procoagulant activity was significantly higher for supernatants collected from packed red blood cell units (median 54 nM, range 53 to 60 nM) than haemoperitoneum samples (median 43 nM; range 7 to 51 nM; P = 0.0007). By flow cytometry, the concentration of phosphatidylserine-positive extracellular vesicles was not significantly different between packed red blood cell (median: 415/μL, range 173 to 1331/μL) and haemoperitoneum samples (median: 314/μL, range 132 to 3880/μL; P = 0.77). This study does not suggest that shed abdominal blood contains more extracellular vesicles with phosphatidylserine-mediated procoagulant activity than donor packed red blood cell units. Clinical studies to compare the effects of autologous transfusion of shed abdominal blood and packed red blood cell units on coagulation status and clinical outcome are required.

Detection and quantification of 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB) from smoker albumin and its potential as a surrogate biomarker of tobacco-specific nitrosamines exposure and bioactivation

4-(Methylnitrosamino)-l-(3-pyridyl)-1-butanone (NNK) and N-nitrosonornicotine (NNN), two tobacco specific nitrosamine carcinogens, can form adducts with DNA and proteins via pyridyloxobutylation upon phase I enzyme-mediated bioactivation. Such DNA modifications have been proposed as the root cause to initiate carcinogenesis. Upon hydrolysis, both DNA and protein modifications would release 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB). The released HPB, being tobacco carcinogen specific, has the potential to serve as a surrogate biomarker for both tobacco exposure and carcinogen bioactivation. Because of its easy access, blood is a great source of such investigations with the potential in epidemiological application. HPB quantification from haemoglobin (Hb), however, has been demonstrated with limited success. To further explore this potentially paradigm-shift opportunity, we reported, for the first time, the detection and quantification of HPB from albumin (Alb) adducts formed by the tobacco-specific nitrosamines in mice and in human smokers. The time-course quantitative analysis of HPB from mouse Alb upon NNK exposure suggests that such an Alb adduct is stable. The amounts of HPB from Alb adducts in smoker plasma averaged 1.82 ± 0.19 pg/mg Alb (0.42 to 3.11 pg/mg Alb), which was 36 times the value in nonsmokers (0.05 ± 0.01 pg/mg Alb). Importantly, HPB level from Alb correlated positively with the level of human tobacco exposure estimated by urinary total nicotine equivalent (TNE) (R2 = 0.6170). For comparison, HPB level from Alb was 16.5 times that of Hb (0.12 ± 0.02 pg/mg Hb) in the plasma and red blood cell (RBC) samples of the same smokers. In addition, there was no significant correlation between HPB levels from Hb and TNE (R2 = 0.0719). These data overall suggest that HPB from Alb adducts can serve as a surrogate biomarker to monitor the level of tobacco exposure and carcinogenic nitrosamine bioactivation.

Chronic exposure to xenobiotic pollution leads to significantly higher total glutathione and lower reduced to oxidized glutathione ratio in red blood cells of children with autism

Analyses of reduced glutathione (GSH), oxidized glutathione (GSSG), and total glutathione (tGSH) in red blood cell samples from 30 children diagnosed with autism and 30 age, gender, and socioeconomic status matched controls were undertaken. The children's ages ranged from 2 to 9. Samples were obtained from subjects residing in Western Pennsylvania, an area of the United States greatly affected by high levels of mercury deposition and airborne PM 2.5 particulates. Liquid chromatography – mass spectrometry was utilized by following EPA Method 6800 for sample analyses. The children with autism had a significantly lower mean red blood cell (RBC) reduced to oxidized glutathione ratio (GSH/GSSG) compared to the control children (p = 0.025). In addition, compared to the controls, the children with autism had significantly higher RBC tGSH values (p = 0.0076) and GSH values (p = 0.022). These results suggest that exposure to toxic elements may prompt compensatory increases in production of GSH in children with autism in environments higher in toxins. The compensation did not fully correct the anti-oxidant properties of exposure to xenobiotics as demonstrated by the significantly lower GSH/GSSG in children with autism compared to controls. Out of a set of glutathione biomarkers, GSH/GSSG may best determine the degree of compensation for oxidative stress in children with autism.

Trace metals in green sea turtles (Chelonia mydas) inhabiting two southern California coastal estuaries

Foraging aggregations of east Pacific green sea turtles (Chelonia mydas) inhabit the Seal Beach National Wildlife Refuge (SBNWR) and San Diego Bay (SDB), two habitats in southern California, USA, located near urbanized areas. Both juvenile and adult green turtles forage in these areas and exhibit high site fidelity, which potentially exposes green turtles to anthropogenic contaminants. We assessed 21 trace metals (TM) bioaccumulated in green turtle scute and red blood cell (RBC) samples collected from SBNWR (n = 16 turtles) and SDB (n = 20 turtles) using acid digestion and inductively coupled plasma mass spectrometry. Principal component analyses of TM composition indicate that SBNWR and SDB turtles have location-specific contaminant signatures, characterized by differences in cadmium and selenium concentrations: SBNWR turtles had significantly more cadmium and selenium in RBC and more selenium in scute samples, than SDB turtles. Cadmium and selenium concentrations in RBC had a strong positive relationship, regardless of location. SBNWR turtles had higher selenium in RBCs than previously measured in other green turtle populations globally. Due to different retention times in blood vs. scute, these results suggest that SBNWR turtles have high long- and short-term selenium exposure. Turtles from SBNWR and SDB had higher trace metal concentrations than documented in green turtle populations that inhabit non-urbanized areas, supporting the hypothesis that coastal cities can increase trace metal exposure to local green turtles. Our study finds evidence that green turtle TM concentrations can differ between urbanized habitats and that long-term monitoring of these green turtles may be necessary.

Large Academic Hospital Laboratory Investigates a Major Pre-Analytical Challenge in Africa and Developing Countries

Background: Delay in serum separation from red blood cells in samples collected from most primary healthcare facilities and transported to a laboratory for analysis is of great concern. Standard guidelines state that serum or plasma should be separated from cells within 2 hours of collection. The aim was to determine effects of delayed sample separation on measured biochemical analytes. The objective was to store blood samples in primary collection tubes at 20-25°C post venesection, then separate, and analyse samples of selected analytes. Methods: Multiple sample tubes of whole blood were collected from one of the authors volunteer, and subjected to time delays in centrifugation. The baseline serum was separated from red blood cells within 30 minutes of post venesection to allow adequate coagulation. Twenty analytes were studied using 2 analytical platforms. Percentage variation and standard error method were used to evaluate time-dependent variability in analytes. Total change limit was used to measure significant changes within-run variability for both platforms. Results: Most analytes were stable up to day 3 to 4 on both platforms. Serum CO2, CL, Ca, ALT and ALB were stable up to 8 days when they were measured on Cobas 8000®. BUN, TRIG, TB, CHOL, AST, ALT and ALB were stable up to 10 days on Dimension® CCS. K showed significant changes at 2h on both platforms at initial measurements. It was out-of-range at day 10 on Dimension® CCS. Serum creatinine levels showed substantial changes at day 2 on Dimension® analyzer and on Cobas 8000® at day 3. Conclusions: The study showed stability of wide range of serum analytes at 20-25°C for several days. The acceptable results can be achieved if samples are centrifuged the same day and analyzed later for most of biochemical analytes.

Functional Properties of Hb S and Hb C in Stored Cpda-1 Red Blood Cells Concentrate — a Prospective Study

Most of the 1,300 human hemoglobin (Hb) variants are characterized by normal O2 binding, including here the highly prevalent Hbs S [HBB:c.20A>T; p.Glu6Val] and C [HBB:c.19G>A; p.Glu6Lys]. Hb S and Hb C carriers are generally asymptomatic and might be admitted as blood donors. However, the functional efficiency of these variants in standard blood bags - red blood cells (RBC) concentrates - under the interference of anticoagulant and storage is not well established. In this prospective study, three samples of RBC concentrates (phenotypes AA, AS and AC), with citrate phosphate dextrose adenine (CPDA-1) as anticoagulant, were analyzed for equilibrium tests and compared to fresh Hb A without anticoagulant. The Hb samples were extracted after 48 hours and 35 days of storage and purified by exclusion and ion exchange chromatography (Sephadex G-25 and Amberlite MB-3 columns in Hepes buffer without NaCl). O2 affinity was evaluated by spectrophotometry-tonometry method, by determining the p50 in stripped lysates ([Hb]=70 μM/Heme), in pHs of 6.5, 7.0, 7.5, 8.0 and 8.5, for Bohr effect calculations. Heme-heme cooperativity was determined by Hill coefficient (n) in all studied pHs. The activity of allosteric effector in the Hb-O2 ligation was also tested, using inositol hexaphosphate ([IHP]=1mM), a mimetic compound of the intraerythrocytic 2,3-BPG. The p50 of Hb A after 35 days in stored blood bag was decreased in all pHs, in both presence and absence of IHP, when compared to fresh Hb A, suggesting that the anticoagulant and the storage might affect HbA-O2 binding, increasing its affinity for the ligand. No other differences were observed: heme-heme cooperativity and Bohr effect were similar to fresh Hb A without anticoagulant. The same was found when stored Hb AS was compared to Hb A from blood bag (35 days storage, in the stripped state and under the interference of IHP). However, the stripped lysate containing Hb AC revealed an important reduction of the O2 affinity after both 48 hours and 35 days of storage, mainly at pHs ³ 7, thus interfering in the Bohr effect. The heme-heme cooperativity resulted in normal n coefficients, indicating that the Hb-O2 ligation remained cooperative, in spite of the significant reduction of O2 affinity. Similar results were observed in the presence of IHP (48 hours and 35 days), although Bohr effect was reestablished, indicating that the supplementation with a 2,3-BPG-like allosteric effector might restore some Hb-ion interactions, and therefore, Hb C function. As the 2,3-BPG is consumed with storage of RBC concentrates, Hb AC showed an even lower affinity for O2 after 35 days of storage. Although still preliminary, our results indicate a significant functional reduction of Hb C in RBC concentrates in standard blood bag (CPDA-1 anticoagulant), proportional to the time of storage. Considering that the screening for hemoglobinopathies in the blood banks routine is mainly focused in the detection of Hb S, a greater understanding about the properties of native Hb and its most frequent variants in stored RBC concentrates could also contribute with the determination of safe parameters in hemotherapy based on the functional efficiency of each hemoglobin. Financial Support: Fapesp, CNPq, Capes, Faepex-Unicamp. DisclosuresNo relevant conflicts of interest to declare.