Red Blood Cell Samples Research Articles

Elastic property of sickle cell anemia and sickle cell trait red blood cells.

.Significance: We introduce a model for better calibration of the trapping force using an equal but oppositely directed drag force acting on a trapped red blood cell (RBC). We demonstrate this approach by studying RBCs’ elastic properties from deidentified sickle cell anemia (SCA) and sickle cell trait (SCT) blood samples.Aim: A laser trapping (LT) force was formulated and analytically calculated in a cylindrical model. Using this trapping force relative percent difference, the maximum (longitudinal) and minimum (transverse) radius rate and stiffness were used to study the elasticity.Approach: The elastic property of SCA and SCT RBCs was analyzed using LT technique with computer controlled piezo-driven stage, in order to trap and stretch the RBCs.Results: For all parameters, the results show that the SCT RBC samples have higher elastic property than the SCA RBCs. The higher rigidity in the SCA cell may be due to the lipid composition of the membrane, which was affected by the cholesterol concentration.Conclusions: By developing a theoretical model for different trapping forces, we have also studied the elasticity of RBCs in SCT (with hemoglobin type HbAS) and in SCA (with hemoglobin type HbSS). The results for the quantities describing the elasticity of the cells consistently showed that the RBCs in the SCT display lower rigidity and higher deformability than the RBCs with SCA.

Impaired age-dependent increases in phosphoglycerate kinase activity in red blood cells of Parkinson's disease patients

BackgroundImpaired bioenergetics are partially involved in the pathogenesis of Parkinson's disease (PD). Phosphoglycerate kinase (PGK), an essential enzyme for glycolysis, has recently attracted attention due to its pathogenic role in PD and as a target for disease-modifying therapies. This study is aimed to evaluate the profiles of PGK activity in red blood cells (RBCs) of PD patients and controls. MethodsSixty-eight PD patients and thirty-four age-matched unrelated controls were enrolled. PGK activities of RBCs were measured by the established colorimetric assay and standardized by the same RBC samples. ResultsPGK activity of the PD group was significantly higher than that of the control group in participants aged sixty-five years or younger, whereas it was not significantly different between the two groups at any age. PGK activity was positively correlated with aging in the control group, but this was not noted in the PD group. On multivariable analysis by partial correlation in the PD group, PGK activity was negatively correlated with the specific binding ratio of dopamine transporter scintigraphy in the striatum. The levodopa-equivalent daily dose was not significantly correlated with the enzyme activity. ConclusionThe results support the following: 1) elevation of PGK activities in RBCs can be detected in relatively young PD patients and with normal aging; 2) the degree of striatonigral degeneration is associated with elevated PGK activities. These are important considerations when the PGK assay is applied as a diagnostic biomarker of PD and to therapeutically monitor PGK-enhancing treatments.

Gold-Immobilized Photonic Crystal Fiber-Based SPR Biosensor for Detection of Malaria Disease in Human Body

This article presents the photonic crystal fiber (PCF) based surface plasmon resonance (SPR) biosensor for early detection of malaria disease in humans by measurement of the variation of red blood cells (RBCs). In the proposed PCF, two layers of air holes are arranged in a hexagonal lattice structure and a thin film of gold-coating is used over PCF for the occurrence of SPR phenomena. It occurs when surface plasmon polariton (SPP)-mode coupled with the core-mode during phase-matching conditions. Malaria infected RBCs samples are filled into the PCF, which have own refractive index (RI) that shift the SPR resonance wavelength during confinement loss measurement. The resonance wavelength of malaria-infected RBCs samples is different from their normal RBCs samples due to the difference in RI of infected and normal RBCs samples. The proposed work is helpful in the detection of different stages of malaria-infected RBCs such as ring phase, trophozoite phase and Schizont phase by measuring the shift in resonance wavelength. The calculated wavelength sensitivities of the proposed sensor for the ring phase, trophozoite phase and Schizont phase RBCs are 13714.29 nm/RIU, 9789.47 nm/RIU, and 8068.97 nm/RIU, respectively in x-polarized direction and 14285.71 nm/RIU, 10000 nm/RIU, and 8206.9 nm/RIU, respectively in y-polarized direction with the maximum detection limit of 0.029. The proposed PCF-based SPR biosensor is suitable for the early diagnosis of malaria disease due to its enhanced sensing performance (low detection limit and high sensitivity).

Defected Photonic Crystal Array Using Porous GaN as Malaria Sensor

A defective one-dimensional photonic crystal is investigated as a biosensor to detect malaria disease. The proposed photonic structure is air/(GaN/Porous GaN)N/Sample/(GaN/Porous GaN)N/Substrate. The red blood cells sample of the human being is used as a sample defect in the proposed optical device. The pioneer transfer matrix method is used to analyze the transmittance spectra. A change in sample refractive index highly affects the transmittance resonant peak and this shift in the peak plays a key role in the operation of the device. The relatively high figure of merit of 1022 RIU−1 with a sensitivity of 1472 nm/RIU and quality factor of 1076 is detected. The proposed sensor is relatively better than others available to detect malaria disease.

A New Centrifugation-Free Approach for Reducing IgA Levels in Units of Stored Red Blood Cells

Introduction: Washed red blood cells (RBCs) are indicated for immunoglobulin A (IgA) deficient recipients when RBCs from IgA-deficient donors are not available. Centrifugation-based cell processors commonly used by hospital blood banks cannot consistently reduce IgA to levels suitable for IgA-deficient recipients in a single washing cycle, hence double washing is frequently required. Additionally, cell processors are designed to wash one RBC unit at a time, making washing several units for a multiple-unit transfusion a laborious, lengthy and logistically difficult process. Importantly, these conventional cell washers are complex, expensive machines that may not be available in smaller medical facilities, thus limiting availability of washed RBCs on demand. To address these limitations, we developed a simple, portable, disposable system capable of washing stored RBC units at practical volumetric throughput without the use of centrifugation, while reducing IgA below 0.05 mg/dL in a single run. This new washing system consists of two core modules: (i) a coil of narrow-bore plastic tubing with three sequential bifurcations for high-throughput separation of washed RBCs from washing solution, and (ii) a highly-engineered membrane-hydrogel composite for concentrating the suspension of washed RBCs to the desired level of hematocrit.Materials and Methods: Several units of stored RBCs (n = 4, leukoreduced, 4-6 weeks storage duration) were purchased from the Gulf Coast Regional Blood Center (Houston, TX). Samples of stored RBCs were withdrawn from the units and diluted with 0.9% saline to a hematocrit (Hct) of 10%, to match the dilution typically used in the conventional centrifugation-based washing procedure (2000 mL of saline per typical 350 mL RBC unit with a 60-65% initial Hct). The diluted RBC samples were then washed either by our new washing system, or via conventional centrifugation. The efficiency of the two washing methods was quantified and compared by measuring several key in vitro quality metrics.Results and Discussion: Stored RBCs were diluted to 10% Hct with normal saline and passed through the new washing system at 5.0 mL/min to produce washed RBCs with output Hct of about 42%, and Hct of washing solution (waste) of about 2%. Overall, washing performed by either of the two methods substantially reduced hemolysis, lowered the concentration of free hemoglobin (by >4-fold) and total free protein (by >10-fold), while keeping the intracellular ATP unchanged (Table 1). Washing increased the fraction of well-preserved cells (discocytes, echinocytes 1) from 62.9 ± 14.3% in the initial RBC units to 74.1 ± 6.9% for our experimental device, which was a more pronounced improvement than for conventional centrifugation (67.8 ± 5.1%), although the difference was not statistically significant. Importantly, our new washing system reduced the level of IgA to well below 0.05 mg/dL required by AABB in a single pass, significantly outperforming the traditional, centrifugation-based washing procedure.Conclusions: This study demonstrated the feasibility of washing stored RBCs in normal saline for transfusion to patients with selective IgA deficiency without the use of centrifugation. Our inexpensive, portable, disposal system was significantly better than centrifugation in reducing the IgA level, and performed on par with the conventional procedure for all other key in vitro metrics of RBC quality. The ability of this new washing system to operate in line with existing infusion equipment at bedside could be particularly attractive in remote or resource-limited settings, when conventional centrifugation-based cell washers are unavailable.Acknowledgments: This work was supported by the 2012 NIH Director's Transformative Research Award (R01HL117329). [Display omitted] DisclosuresShevkoplyas:Halcyon Biomedical Incorporated: Employment, Equity Ownership, Patents & Royalties: U.S. Patent Appl. 61/929,357, Research Funding; New Health Sciences, Inc.: Consultancy, Research Funding.

A fluorometric erythrophagocytosis assay using differentiated monocytic THP-1 cells to assess the clinical significance of antibodies to red blood cells.

The significance of antibodies to red blood cells (RBCs) is variable and cannot be predicted solely by serological testing. A flow cytometry-based erythrophagocytosis assay was established using phorbol 12-myristate 13-acetate (PMA)-treated THP-1 cells and RBCs labelled with PKH26 to assess allo- and autoantibodies to RBCs. THP-1 cells were differentiated into macrophage-like cells by treatment with PMA. RBC samples coated with alloantibodies or autoantibodies were obtained from 16 patients with autoimmune haemolytic anaemia of warm type (wAIHA) as well as from five pregnant women with warm autoantibodies. RBCs from healthy blood donors were used as controls. RBCs were labelled with the red lipophilic fluorescent dye PKH26 and incubated with PMA-treated THP-1 cells. After removal of nonadherent RBCs by washing and haemolysis of adherent RBCs, erythrophagocytosis was quantified by flow cytometry. We observed significant phagocytosis of RBCs coated with clinically relevant alloantibodies (i.e. anti-D and anti-K) or autoantibodies from patients with active wAIHA, but not of those coated with alloantibodies (anti-Ch) or autoantibodies from patients and pregnant women without haemolysis. The flow cytometry-based erythrophagocytosis test described here is quantitative, highly reliable, and may be helpful for the assessment of the clinical significance of antibodies to RBCs.

Correlation of Oxygen‐offloading rate from Red Blood Cells with Body Mass

The diffusion of O2 across red blood cell (RBC) membranes is critically important for the delivery of O2 to peripheral tissues. Although the traditional view had been that all O2 moves through RBC membranes by dissolving in and diffusing through membrane lipid, our recent work on murine RBCs indicates that most O2 traversing RBC membranes moves via protein channels (bioRxiv. doi:10.1101/2020.08.28.265066). In stopped-flow (SF) kinetics experiments, we use the absorbance spectrum of hemoglobin (Hb) to monitor the rate constant for O2 offloading from Hb (kHbO2). In RBCs from mice genetically deficient in both aquaporin-1 (AQP1) and the Rh complex (mainly RhAG), kHbO2 is 1/3 lower than in RBCs from wild-type (WT) mice. According to mathematical simulations, this 1/3 decrease in kHbO2 corresponds to a ~55% decrease in O2 permeability of RBC membranes (PM,O2). The combination of the double-knockout and pCMBS (nonspecific inhibitor of membrane proteins that is nonetheless excluded from the RBC interior) reduces PM,O2 by ~91%. Control experiments—hematology, RBC morphology, proteomics (abundance of individual proteins), lipidomics (abundance of individual membrane lipids)—rule out other potential explanations for our striking SF data. Our next challenge is to identify the membrane protein(s) responsible for the 91% – 55% = 36% of PM,O2 not accounted for by AQP1 and the Rh complex, as well as to establish the role of membrane lipids. Comparative physiology could be an important tool for achieving these goals. For each RBC sample, we correct the raw kHbO2 data for modest degrees of hemolysis that occur inside the SF reaction chamber. We assess this hemolysis using our novel SF assay, which is based on the release of carbonic anhydrase (CA) enzymes from RBCs. We find that, compared to RBCs from our lab-standard C57BL/6 WT mice, bovine RBCs have a significantly lower hemolysis (~2.5% vs. ~5%, N=3, p <0.01). More interestingly, we find a 71% lower CA activity in bovine RBCs compared to that in mouse RBCs (N=3, p <0.01). This observation could be important as we explore CO2 permeability in the near future. The kHbO2 is 41% lower for bovine RBCs than for RBCs from WT mice (N=3, p <0.01). Hematological analyses of our limited number of bovine samples do not yet show a significant difference from extensive murine samples in either mean corpuscular volume (MCV) or mean corpuscular hemoglobin concentration (MCHC). A further evaluation of hematology, morphology, proteomics, lipidomics, and genomics (for deduced amino-acid sequences of proteins), will provide valuable insight to the contribution of specific membrane proteins and lipids to the relatively low kHbO2 of bovine RBCs. Our comparative analysis would benefit greatly from access to other mammals with a wide range of body mass (e.g., up to elephant size), from which we would use a small amount of blood (<1 ml) in the assays described above.

Concurrent Assessment of Deformability and Adhesiveness of Sickle Red Blood Cells by Measuring Perfusion of an Adhesive Artificial Microvascular Network.

Biomarker development is a key clinical research need in sickle cell disease (SCD). Hemorheological parameters are excellent candidates as abnormal red blood cell (RBC) rheology plays a critical role in SCD pathophysiology. Here we describe a microfluidic device capable of evaluating RBC deformability and adhesiveness concurrently, by measuring their effect on perfusion of an artificial microvascular network (AMVN) that combines microchannels small enough to require RBC deformation, and laminin (LN) coating on channel walls to model intravascular adhesion. Each AMVN device consists of three identical capillary networks, which can be coated with LN (adhesive) or left uncoated (non-adhesive) independently. The perfusion rate for sickle RBCs in the LN-coated networks (0.18 ± 0.02 nL/s) was significantly slower than in non-adhesive networks (0.20 ± 0.02 nL/s), and both were significantly slower than the perfusion rate for normal RBCs in the LN-coated networks (0.22 ± 0.01 nL/s). Importantly, there was no overlap between the ranges of perfusion rates obtained for sickle and normal RBC samples in the LN-coated networks. Interestingly, treatment with poloxamer 188 decreased the perfusion rate for sickle RBCs in LN-coated networks in a dose-dependent manner, contrary to previous studies with conventional assays, but in agreement with the latest clinical trial which showed no clinical benefit. Overall, these findings suggest the potential utility of the adhesive AMVN device for evaluating the effect of novel curative and palliative therapies on the hemorheological status of SCD patients during clinical trials and in post-market clinical practice.

Red blood cells serve as a vehicle for PEDV transmission

As the most abundant cell type in the blood, red blood cells (RBCs) are serving for transporting oxygen. However, the mechanism by which RBCs binding virus remains largely unknown. Here, we demonstrated that porcine epidemic diarrhea virus (PEDV), a kind of coronavirus, could hijack RBCs and cause typical diarrhea in neonatal piglets. In an epidemiology investigation of PEDV, the RBCs samples from diarrheic pigs in several pig farms were found to be PEDV-positive. PEDV could bind to neonatal RBCs through CD71 and clathrin-mediated endocytosis, and its viability was maintained for 12 h. PEDV-loaded RBCs could transfer the virus to CD3+ T cells by conjugation and reach the intestine mucosa, where it caused infection. Finally, a further animal challenge revealed that transfusing with PEDV-loaded RBCs could cause intestinal epithelial cells (IECs) infection and typical diarrhea symptom. Therefore, our studies illustrated the mechanism by which PEDV could cause intestinal infection through hijacking RBCs, further providing a novel insight into the role of RBCs as potential cells for viral transmission in coronavirus pathogenesis.

Is the blood of a surgeon performing HIPEC contaminated by irinotecan, its major metabolites and platinum compounds?

ObjectivesHyperthermic intraperitoneal chemotherapy (HIPEC) is a beneficial surgical technique for patients, but the surgeons are being exposed to cytotoxic drugs. Few biomonitoring studies were led on blood samples in the context of HIPEC. This study aimed to evaluate the surgeon’s plasmatic and red blood cell (RBC) contamination by irinotecan, two of its major metabolites and platinum compounds.MethodsHIPEC procedures performed using the coliseum techniques were observed between September 2015 and April 2018 in a French comprehensive cancer center. Irinotecan and its metabolites SN-38 and APC were dosed by UHPLC with a limit of quantification determined at 50 pg/mL. Platinum compounds were dosed by inductively coupled plasma mass spectrometry with a limit of quantification determined at 16 pg/mL.ResultsDespite collective and personal protective equipment, 80% of plasma samples were contaminated by irinotecan and 33% by platinum compounds out of 21. The results showed that the surgeon was contaminated after HIPEC and even after a period of HIPEC inactivity. Nineteen percent of plasmatic samples and 45% of RBC samples were contaminated by SN-38, the active metabolite of irinotecan. APC was only found in some RBC samples (33%).ConclusionsEven if this study shows blood contamination by irinotecan, two of its major metabolites (including active SN-38) and platinum compounds both in the plasma and RBC of a surgeon performing the HIPEC procedures, further studies should be performed to confirm these results. Additional studies should be carried out to further investigate the contamination in the context of HIPEC and more broadly in the hospital.

Differential DNA methylation in somatic and sperm cells of hatchery vs wild (natural-origin) steelhead trout populations.

Environmental factors such as nutrition, stress, and toxicants can influence epigenetic programming and phenotypes of a wide variety of species from plants to humans. The current study was designed to investigate the impacts of hatchery spawning and rearing on steelhead trout (Oncorhynchus mykiss) vs the wild fish on a molecular level. Additionally, epigenetic differences between feeding practices that allow slow growth (2 years) and fast growth (1 year) hatchery trout were investigated. The sperm and red blood cells (RBC) from adult male slow growth/maturation hatchery steelhead, fast growth/maturation hatchery steelhead, and wild (natural-origin) steelhead were collected for DNA preparation to investigate potential alterations in differential DNA methylation regions (DMRs) and genetic mutations, involving copy number variations (CNVs). The sperm and RBC DNA both had a large number of DMRs when comparing the hatchery vs wild steelhead trout populations. The DMRs were cell type specific with negligible overlap. Slow growth/maturation compared to fast growth/maturation steelhead also had a larger number of DMRs in the RBC samples. A number of the DMRs had associated genes that were correlated to various biological processes and pathologies. Observations demonstrate a major epigenetic programming difference between the hatchery and wild natural-origin fish populations, but negligible genetic differences. Therefore, hatchery conditions and growth/maturation rate can alter the epigenetic developmental programming of the steelhead trout. Interestingly, epigenetic alterations in the sperm allow for potential epigenetic transgenerational inheritance of phenotypic variation to future generations. The impacts of hatchery exposures are not only important to consider on the fish exposed, but also on future generations and evolutionary trajectory of fish in the river populations.

Design and analysis of a single elliptical channel photonic crystal fiber sensor for potential malaria detection

Malaria is a mosquito-borne disease caused by unicellular hemoparasites of the genus Plasmodium that results in the death of over one million people worldwide every year. Early diagnosis plays a key role in the treatment of infected patients and can reduce the mortality rate. This work proposes a simple designed photonic crystal fiber (PCF) sensor for detecting malarial infection using the refractive index (RI) of red blood cells (RBCs). The initial structure of the PCF sensor consists of double loops of circular air holes arranged in a hexagonal formation. A horizontal elliptical channel in the center of the fiber contains the RBCs sample. The sensor’s response was observed from the shift of the transmission spectra due to change in the RI of RBCs during different life stages of the parasite. Model parameters (transmission length, pitch, air hole diameter, and eccentricity of the elliptical channel) of the proposed sensor were optimized to obtain the best possible response. The highest spectral sensitivities were obtained about 11,428.57 nm/RIU, 9473.68 nm/RIU, and 9655.17 nm/RIU for the ring, trophozoite, and schizont phases of the parasite, respectively. Due to its high sensitivity, easy identification capability, and short transmission length, this sensor can be utilized as a cost-effective and useful device for malaria diagnosis.

In vitro alteration on erythrocytes mechanical properties by propofol, remifentanil and vecuronium

Several studies report flow disturbance and microcirculation disorders upon anesthesia treatment. These alterations are often related to blood rheology changes. In this work, it was attempted to make a detailed description of the alterations in erythrocyte mechanical properties by the action of propofol, remifentanil, and vecuronium. For this, an in vitro study was performed on red blood cell samples from healthy donors incubated with solutions of propofol (4 μg/mL whole blood), remifentanil (10 ng/mL plasma), and vecuronium (0.15 μg/mL plasma). Erythrocyte viscoelastic parameters were determined by octuplicate using a Reómetro Eritrocitario. Also, a Wilcoxon signed rank-test with Yates correction for continuity was performed to analyze the overall alteration in the mechanical properties of erythrocytes. Statistical analysis showed that the three studied anesthetics changed the erythrocyte mechanical properties at different parts of the membrane. These results would imply an interaction of these anesthetics with the erythrocyte membrane. Finally, this could conduce to alterations in microcirculation.

Self-aligned sequential lateral field non-uniformities over channel depth for high throughput dielectrophoretic cell deflection.

Dielectrophoresis (DEP) enables the separation of cells based on subtle subcellular phenotypic differences by controlling the frequency of the applied field. However, current electrode-based geometries extend over a limited depth of the sample channel, thereby reducing the throughput of the manipulated sample (sub-μL min-1 flow rates and <105 cells per mL). We present a flow through device with self-aligned sequential field non-uniformities extending laterally across the sample channel width (100 μm) that are created by metal patterned over the entire depth (50 μm) of the sample channel sidewall using a single lithography step. This enables single-cell streamlines to undergo progressive DEP deflection with minimal dependence on the cell starting position, its orientation versus the field and intercellular interactions. Phenotype-specific cell separation is validated (>μL min-1 flow and >106 cells per mL) using heterogeneous samples of healthy and glutaraldehyde-fixed red blood cells, with single-cell impedance cytometry showing that the DEP collected fractions are intact and exhibit electrical opacity differences consistent with their capacitance-based DEP crossover frequency. This geometry can address the vision of an "all electric" selective cell isolation and cytometry system for quantifying phenotypic heterogeneity of cellular systems.

Characterization of Iron Complexes in the Red Blood Cells of β-thalassaemia patients using 57Fe Mössbauer Spectroscopy

Red Blood Cells (RBCs) samples from healthy volunteers, β-thalassaemia major and β-thalassaemia intermedia patients were studied by means of 57Fe Mössbauer Spectroscopy (MS). The MS spectra from the RBCs of the thalassaemia patients showed the existence of significant amounts of ferritin-like iron, and in particular in the 82% of the β-thalassaemia intermedia ones. Also, β-thalassaemia intermedia patients were found to have higher Liver Iron Concentration (LIC) when compared to the β-thalassaemia major patients, as assessed by MRI exams, even though they had on an average about half the serum ferritin levels.

Patients with Gaucher disease display systemic oxidative stress dependent on therapy status

Gaucher disease is an autosomal recessive metabolic disorder caused by mutations in GBA1, which encodes for the lysosomal hydrolase enzyme, β-glucocerebrosidase. The resulting misfolded protein can trigger endoplasmic reticulum stress and an unfolded protein response within the affected cells. The enzyme deficiency leads to accumulation of its substrates, glucosylceramide and glucosylsphingosine, within macrophage lysosomes and with prominent disease manifestations in macrophage rich tissues. Resultant lysosomal pathology and impaired autophagy leads to redox imbalance, mitochondrial dysfunction and intracellular oxidative stress. Here we have systematically examined a role for oxidative stress in individuals affected by Gaucher disease. We compared multiple oxidative stress biomarkers in plasma and red blood cell samples from patients who are currently untreated, with those who are stable on standard-of-care therapy, and with healthy controls. We found significant differences in key oxidative stress biomarkers in untreated patients compared to healthy control. In treated patients, results generally fell between the controls and the untreated patients. Interestingly, even asymptomatic and minimally symptomatic untreated patients had evidence of significant systemic oxidative stress. We conclude that underlying oxidative stress may contribute to Gaucher disease pathophysiology including long-term adverse outcomes such as Parkinsonism and malignancies. Therapies targeting oxidative stress may prove useful as adjuvant treatments for Gaucher disease and other lysosomal storage disorders.

Effect of fasting status and other pre-analytical variables on quantitation of long-chain fatty acids in red blood cells

Long-chain fatty acids (LCFAs), including omega-3 and omega-6 fatty acids, play essential roles in health maintenance and outcomes. Insufficient intake or the inability to absorb LCFAs from the diet can cause a number of health problems. Evaluation of fatty acid profiles in plasma, serum or red blood cells (RBCs) is routinely used to monitor patients at risk of developing deficiency. Quantitation of LCFAs in RBCs offers advantages over serum/plasma due to low intra-individual variability. Fatty acid composition in RBCs also reflects long-term dietary intake, providing additional information about the patient's nutritional status. However, the literature does not currently address the impact of pre-analytical factors (conditions of RBC collection, sample handling and short-term storage) on LCFA measurements. This study evaluated the effect of several anticoagulants, interferents, different storage conditions and fasting status on quantitation of the twenty-one most abundant LCFAs in RBCs by gas chromatography negative chemical ionization-mass spectrometry (GCNCI-MS). LCFA results were assessed quantitatively (nmol/mL) or as a percent of total. Most common tube types (containing citrate, sodium heparin or EDTA) were all appropriate for blood collection. Whole blood and lysed RBCs were stable at least 24 h at room temperature and up to 7 days refrigerated. Lysed RBCs were also stable for up to three freeze/thaw cycles. The presence of icterus or lipemia did not affect results. LCFAs concentrations in RBCs did not change ~4 h after high-fat intake when the lipid concentration in circulation reaches a peak, while plasma levels of most fatty acids increased up to 40% in response. In summary, RBCs are a reliable sample type for LCFA quantitation in the clinical laboratory. In contrast to plasma or serum, RBCs isolated from non-fasting, hemolyzed or lipemic whole blood specimens are all acceptable for testing. Therefore, RBCs might be a preferable sample type for evaluation of nutritional status of young pediatric patients and in patients with conditions associated with hemolytic anemia or hyperlipidemia.

Verification of exposure to sarin nerve agent through the chemical analysis of red blood cell samples

Organophosphorus nerve agents (OPNAs) are an essential class of internationally banned chemical warfare agents that broadly target a family of enzymes known as serine hydrolases. In the body, OPNAs bind to butyrylcholinesterase (BChE), acetylcholinesterase (AChE), albumin and other proteins to form nerve agent adducts. These adducts can develop reliable, long-term protein biomarkers for human exposure to nerve agents. Red blood cells (RBC) are known targets of OPNAs toxicity effects. Since erythrocytes have a relatively long half-life, they may serve as a valuable tool for OPNA exposure verification. In the present report, a new, simple, and less expensive approach for detection of nerve agent exposure using alkaline hydrolysis of RBC membrane proteins, is evaluated. In this method, the hemoglobin-free ghost of human erythrocyte is prepared from 6 mL of sarin (GB) spiked RBC sample, and then treated with sodium hydroxide solution . In the presence of sodium hydroxide and elevated temperature, isopropyl methylphosphonic acid (IMPA) is released from RBC protein-sarin adducts. Two dimensional Solid-phase extraction (SPE) is employed for clean-up. The monitoring of IMPA using LC-MS/MS demonstrates that GB is added to RBC proteins. In the quantitative analysis of GB adducts, the calibration curve was found to be linear over a range of 10–100 µg L−1, and the limit of detection was 1 µg L−1. Our results indicate that membrane proteins of human erythrocyte could be exploited as a surrogate biomarker of nerve agent exposure.

Malaria Detection Using Image Processing

Malaria is one of a serious infectious disease in the world caused by a peripheral blood parasite of the genus Plasmodium. Traditional microscopy method, for malaria diagnosis is old method and has been occasionally proved inefficient since it is time consuming and results are difficult to reproduce. As it constitutes a serious global health problem, the evaluation process is of high importance. In this work, an accurate, easy, rapid and affordable model of malaria diagnosis using stained thin blood smear images was developed. The method makes use of the intensity features of Plasmodium parasites and also erythrocyte or red blood cell images. Images of infected and non-infected red blood cells were taken, pre-processed, and then relevant features were extracted from them and eventually diagnosis was made based on the features extracted from the images. A set of features based on intensity have been proposed, and the performance of these features on the red blood cell samples from the database have been evaluated using an artificial neural network (ANN) classifier. The results have shown that these features could be extremely successful if used for malaria parasite detection.

An unusual variant glycophorin expressing protease‐resistant M antigen encoded by the GYPB‐E(2‐4)‐B hybrid gene

MNS is a highly polymorphic blood group comprising 49 antigens recognized by International Society of Blood Transfusion, some of which may have been generated by genomic recombination among the closely linked genes GYPA, GYPB and GYPE. The GYPE gene has an almost identical sequence to GYPA*01 allele in exon 2 (99% homology), which accounts for M antigen. We investigated an unusual glycophorin molecule with protease-resistant M antigen. Blood samples were screened by an automated blood typing system (PK7300) using bromelain-treated red blood cells (RBCs) and murine monoclonal anti-M. The M-positive RBC samples were analysed by immunoblotting using anti-M as the primary antibody. GYPA, GYPB and GYPE genes were analysed by polymerase chain reaction (PCR), cloning and sequencing using reticulocyte mRNA and genomic DNA. Serological tests and immunoblotting revealed that 103 of the 193009 individuals (0·0534%) expressed protease-resistant M-active glycophorin having a molecular weight of 20kDa. All the 103 individuals were S+s- or S-s+. When reticulocyte mRNA from the individuals with M-active glycophorin (20kDa) was examined by PCR and cloning followed by sequencing, a novel GYPE-B hybrid transcript was identified. Long-range PCR and sequencing using genomic DNA revealed that the individuals had a GYPB-E(2-4)-B hybrid gene. This hybrid gene was predicted to encode a 59-amino-acid mature glycoprotein that expresses no S or s antigens CONCLUSIONS: The prevalence of the M-active glycophorin (20kDa) in the Japanese population is 0·0534%. This glycophorin is predicted to be a 59 amino acids polypeptide encoded by the novel GYPB-E(2-4)-B hybrid gene.