Optical assay of erythrocyte function in banked blood.
Ontology highlight
ABSTRACT:
SUBMITTER: Bhaduri B
PROVIDER: S-EPMC4650916 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:BackgroundFecal occult blood testing (FOBT) has historically relied on methods to detect hemoglobin with no fundamental innovations in decades.AimTo examine microRNA (miRNA) as a new marker class for FOBT.MethodsCandidate miRNA markers were identified by small RNA sequencing of human whole blood compared to colorectal epithelia. Markers were tested in human blood cell subsets and blood from non-human species. We assessed assay linearity in blood spiking and marker stability in stool over incubation experiments. Levels of candidate erythrocyte markers were explored in stools from colorectal cancer (CRC) cases and controls.ResultsBased on small RNA sequencing and validation RT-qPCR, expression level of each of the top blood-enriched markers (hsa-miR-144-3p, 144-5p, 451a, 486-5p, 363-3p, 20b-5p) could perfectly discriminate blood from colorectal epithelia. All six markers arose from and showed specificity to human erythrocytes. Marker levels increased linearly with erythrocyte concentration in saline or stool and demonstrated a broader dynamic range than did immunochemical test for hemoglobin. Degradation of markers occurred in stool but was reduced with preservative buffers. Erythrocyte marker candidates for stool testing were selected in an exploratory set of stools (20 CRC, 40 normal). Candidates were then further tested in a feasibility set (29 CRC, 31 advanced adenoma, and 115 normal); a miRNA panel (hsa-miR-451a, 144-5p, and 200b-3p as normalizer) yielded an AUC of 0.89 (95% CI 0.82-0.95, P < .0001) for CRC.ConclusionsA novel miRNA-based approach accurately quantifies fecal blood levels over a broad, clinically relevant range.
| S-EPMC6021130 | biostudies-literature
Project description:From the perspectives of disease transmission and sterility maintenance, the world's blood supplies are generally safe. However, in multiple clinical settings, red blood cell (RBC) transfusions are associated with adverse cardiovascular events and multiorgan injury. Because ?85 million units of blood are administered worldwide each year, transfusion-related morbidity and mortality is a major public health concern. Blood undergoes multiple biochemical changes during storage, but the relevance of these changes to unfavorable outcomes is unclear. Banked blood shows reduced levels of S-nitrosohemoglobin (SNO-Hb), which in turn impairs the ability of stored RBCs to effect hypoxic vasodilation. We therefore reasoned that transfusion of SNO-Hb-deficient blood may exacerbate, rather than correct, impairments in tissue oxygenation, and that restoration of SNO-Hb levels would improve transfusion efficacy. Notably in mice, administration of banked RBCs decreased skeletal muscle pO2, but infusion of renitrosylated cells maintained tissue oxygenation. In rats, hemorrhage-induced reductions in muscle pO2 were corrected by SNO-Hb-repleted RBCs, but not by control, stored RBCs. In anemic awake sheep, stored renitrosylated, but not control RBCs, produced sustained improvements in O2 delivery; in anesthetized sheep, decrements in hemodynamic status, renal blood flow, and kidney function incurred following transfusion of banked blood were also prevented by renitrosylation. Collectively, our findings lend support to the idea that transfusions may be causally linked to ischemic events and suggest a simple approach to prevention (i.e., SNO-Hb repletion). If these data are replicated in clinical trials, renitrosylation therapy could have significant therapeutic impact on the care of millions of patients.
| S-EPMC3710799 | biostudies-other
Project description:BackgroundTransfusion of red blood cells (RBCs) is a frequent health care practice. However, unfavorable consequences may occur from transfusions of stored RBCs and are associated with RBC changes during storage. Loss of S-nitrosohemoglobin (SNO-Hb) and other S-nitrosothiols (SNOs) during storage is implicated as a detriment to transfusion efficacy. It was hypothesized that restoring SNOs within banked RBCs would improve RBC functions relevant to successful transfusion outcomes, namely, increased deformability and decreased adhesivity.Study design and methodsStored human RBCs were incubated with nitric oxide (NO) donors PROLI/NO and DEA/NO (disodium 1-[2-(carboxylato)-pyrrolidin-1-yl]diazen-1-ium-1,2-diolate and diethylammonium (Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate) under varying experimental conditions (e.g., aerobic/anaerobic incubation, NO donor to RBC ratio). SNO restoration was evaluated in vitro and in vivo as a means to improve RBC function after storage.ResultsIncubation of RBCs with the NO donors resulted in 10-fold greater levels of SNO-Hb versus untreated control or sham RBCs, with significantly higher Hb-bound NO yields from an NO dose delivered by DEA/NO. RBC incubation with DEA/NO at a stoichiometry of 1:62.5 NO:Hb significantly increased RBC deformabilty and reduced adhesion to cultured endothelial cells. RBC incubation with DEA/NO also increased S-nitrosylation of RBC cytoskeletal and membrane proteins, including the β-spectrin chain. Renitrosylation attenuated both RBC sequestration in the lung and the mild blood oxygen saturation impairments seen with banked RBCs in a mouse model of transfusion.ConclusionsRBC renitrosylation using NO donors has promise for correcting deficient properties (e.g., adhesivity, rigidity, and SNO loss) of banked RBCs and in turn improving transfusion outcomes.
| S-EPMC4605860 | biostudies-literature
Project description:This laboratory has previously described a method for scoring the incidence of rodent blood Pig-a mutant phenotype erythrocytes using immunomagnetic separation in conjunction with flow cytometric analysis (In Vivo MutaFlow®). The current work extends this approach to human blood. The frequencies of CD59- and CD55-negative reticulocytes (RET(CD59-/CD55-)) and erythrocytes (RBC(CD59-/CD55-)) serve as phenotypic reporters of PIG-A gene mutation. Immunomagnetic separation was found to provide an effective means of increasing the number of reticulocytes and erythrocytes evaluated. Technical replicates were utilized to provide a sufficient number of cells for precise scoring while at the same time controlling for procedural accuracy by allowing comparison of replicate values. Cold whole blood samples could be held for at least one week without affecting reticulocyte, RET(CD59-/CD55-) or RBC(CD59-/CD55-) frequencies. Specimens from a total of 52 nonsmoking, self-reported healthy adult subjects were evaluated. The mean frequency of RET(CD59-/CD55-) and RBC(CD59-/CD55-) were 6.0 × 10(-6) and 2.9 × 10(-6), respectively. The difference is consistent with a modest selective pressure against mutant phenotype erythrocytes in the circulation, and suggests advantages of studying both populations of erythrocytes. Whereas intra-subject variability was low, inter-subject variability was relatively high, with RET(CD59-/CD55-) frequencies differing by more than 30-fold. There was an apparent correlation between age and mutant cell frequencies. Taken together, the results indicate that the frequency of human PIG-A mutant phenotype cells can be efficiently and reliably estimated using a labeling and analysis protocol that is well established for rodent-based studies. The applicability of the assay across species, its simplicity and statistical power, and the relatively non-invasive nature of the assay should benefit myriad research areas involving DNA damage, including studies of environmental factors that modify "spontaneous" mutation frequencies.
| S-EPMC4406781 | biostudies-literature
Project description:RBCs distribute oxygen to tissues, but, paradoxically, blood transfusion does not always improve oxygen delivery and is associated with ischemic events. We hypothesized that storage of blood would result in loss of NO bioactivity, impairing RBC vasodilation and thus compromising blood flow, and that repleting NO bioactivity would restore RBC function. We report that S-nitrosohemoglobin (SNO-Hb) concentrations declined rapidly after storage of fresh venous blood and that hypoxic vasodilation by banked RBCs correlated strongly with the amounts of SNO-Hb (r(2) = 0.90; P < 0.0005). Renitrosylation of banked blood during storage increased the SNO-Hb content and restored its vasodilatory activity. In addition, canine coronary blood flow was greater during infusion of renitrosylated RBCs than during infusion of S-nitrosothiol-depleted RBCs, and this difference in coronary flow was accentuated by hypoxemia (P < 0.001). Our findings indicate that NO bioactivity is depleted in banked blood, impairing the vasodilatory response to hypoxia, and they suggest that SNO-Hb repletion may improve transfusion efficacy.
| S-EPMC2040473 | biostudies-literature
Project description:It is an axiom that blood cellular immunity is provided by leukocytes. As to erythrocytes, it is generally accepted that their main function is respiration. Our research provides objective video and photo evidence regarding erythrocyte bactericidal function. Phase-contrast immersion vital microscopy of the blood of patients with bacteremia was performed, and the process of bacteria entrapping and killing by erythrocytes was shot by means of video camera. Video evidence demonstrates that human erythrocytes take active part in blood bactericidal action and can repeatedly engulf and kill bacteria of different species and size. Erythrocytes are extremely important integral part of human blood cellular immunity.Compared with phagocytic leukocytes, the erythrocytesa) are more numerous; b) are able to entrap and kill microorganisms repeatedly without being injured; c) are more resistant to infection and better withstand the attacks of pathogens; d) have longer life span and are produced faster; e) are inauspicious media for proliferation of microbes and do not support replication of chlamidiae, mycoplasmas, rickettsiae, viruses, etc.; and f) are more effective and uncompromised bacterial killers. Blood cellular immunity theory and traditional view regarding the function of erythrocytes in human blood should be revised.
| S-EPMC4029293 | biostudies-literature
Project description:The erythrocyte sedimentation rate (ESR) is one of the oldest medical diagnostic tools. However, currently there is some debate on the structure formed by the cells during the sedimentation process. While the conventional view is that erythrocytes sediment as separate aggregates, others have suggested that they form a percolating gel, similar to other colloidal suspensions. However, visualization of aggregated erythrocytes, which would settle the question, has always been challenging. Direct methods usually study erythrocytes in 2D situations or low hematocrit (∼1%). Indirect methods, such as scattering or electric measurements, provide insight on the suspension evolution, but cannot directly discriminate between open or percolating structures. Here, we achieved a direct probing of the structures formed by erythrocytes in blood at stasis. We focused on blood samples at rest with controlled hematocrit of 45%, from healthy donors, and report observations from three different optical imaging techniques: direct light transmission through thin samples, two-photon microscopy and light-sheet microscopy. The three techniques, used in geometries with thickness from 150 μm to 3 mm, highlight that erythrocytes form a continuous network with characteristic cracks, i.e., a colloidal gel. The characteristic distance between the main cracks is of the order of ∼100 μm. A complete description of the structure then requires a field of view of the order of ∼1 mm, in order to obtain a statistically relevant number of structural elements. A quantitative analysis of the erythrocyte related processes and interactions during the sedimentation need a further refinement of the experimental set-ups.
| S-EPMC8832033 | biostudies-literature
Project description:Two novel real-time PCR assays were developed for the detection of Rickettsia spp. One assay detects all tested Rickettsia spp.; the other is specific for Rickettsia rickettsii. Evaluation using DNA from human blood and tissue samples showed both assays to be more sensitive than nested PCR assays currently in use at the CDC.
| S-EPMC3536194 | biostudies-literature
Project description:Autologous blood transfusion (ABT) has been frequently abused in endurance sport and is prohibited since the mid-1980s by the International Olympic Committee. Apart from any significant performance-enhancing effects, the ABT may pose a serious health issue due to aging erythrocyte-derived "red cell storage lesions." The current study investigated the effect of blood storage in citrate phosphate dextrose adenine (CPDA1) on the red blood cell (RBC) membrane proteome. One unit of blood was collected in CPDA1 blood bags from 6 healthy female volunteers. RBC membrane protein samples were prepared on days 0, 14, and 35 of storage. Proteins were digested in gel and peptides separated by nanoliquid chromatography coupled to tandem mass spectrometry resulting in the confident identification of 33 proteins that quantitatively change during storage. Comparative proteomics suggested storage-induced translocation of cytoplasmic proteins to the membrane while redox proteomics analysis identified 14 proteins prone to storage-induced oxidation. The affected proteins are implicated in the RBC energy metabolism and membrane vesiculation and could contribute to the adverse posttransfusion outcomes. Spectrin alpha chain, band 3 protein, glyceraldehyde-3-phosphate dehydrogenase, and ankyrin-1 were the main proteins affected by storage. Although potential biomarkers of stored RBCs were identified, the stability and lifetime of these markers posttransfusion remain unknown. In summary, the study demonstrated the importance of studying storage-induced alterations in the erythrocyte membrane proteome and the need to understand the clearance kinetics of transfused erythrocytes and identified protein markers.
| S-EPMC6249999 | biostudies-literature
Project description:Erythrocyte invasion by Plasmodium falciparum merozoites is an essential step for parasite survival and hence the pathogenesis of malaria. Invasion has been studied intensively, but our cellular understanding has been limited by the fact that it occurs very rapidly: invasion is generally complete within 1 min, and shortly thereafter the merozoites, at least in in vitro culture, lose their invasive capacity. The rapid nature of the process, and hence the narrow time window in which measurements can be taken, have limited the tools available to quantitate invasion. Here we employ optical tweezers to study individual invasion events for what we believe is the first time, showing that newly released P. falciparum merozoites, delivered via optical tweezers to a target erythrocyte, retain their ability to invade. Even spent merozoites, which had lost the ability to invade, retain the ability to adhere to erythrocytes, and furthermore can still induce transient local membrane deformations in the erythrocyte membrane. We use this technology to measure the strength of the adhesive force between merozoites and erythrocytes, and to probe the cellular mode of action of known invasion inhibitory treatments. These data add to our understanding of the erythrocyte-merozoite interactions that occur during invasion, and demonstrate the power of optical tweezers technologies in unraveling the blood-stage biology of malaria.
| S-EPMC4142257 | biostudies-literature