Protocol for one-step selective lysis of red blood cells and platelets with long-term preservation of white blood cells (human) at ambient temperature.
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ABSTRACT:
SUBMITTER: Chaurasia RK
PROVIDER: S-EPMC8449125 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Red blood cells (RBCs) and platelets derived from stem cells are possible solutions to the increasing demand for blood transfusion. Based on the availability of stem cells, their relatively defined differentiation mechanisms, and the massive exploration of induction systems, the generation of RBCs or platelets in vitro from cord blood hematopoietic stem/progenitor cells (CB-HSPCs) has potential for clinical applications. However, information on the clinical translation of stem cell-derived RBCs and platelets in the literature and at the ClinicalTrials.gov website is very limited. The only clinical trial on cultured RBCs, which aimed to assess the lifespan of RBCs cultured in vivo, was reported by Luc Douay and colleagues. Of note, the cultured RBCs they used were derived from autologous peripheral blood HSPCs, and no cultured platelets have been applied clinically to date. However, CB-HSPC-derived megakaryocytes, platelet precursors, have been used in the treatment of thrombocytopenia. A successful phase I trial was reported, followed by phase II and III clinical trials conducted in China. In this review, the gap between the many basic studies and limited clinical trials on stem cell-derived RBCs and platelets is summarized. The possible reasons and solutions for this gap are discussed. Further technological improvements for blood cell expansion and maturation ex vivo and the establishment of biological standards for stem cell derivatives might help to facilitate the therapeutic applications of cultured RBCs and platelets derived from CB-HSPCs in the near future.
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Project description:Conventional storage of blood-derived fractions relies on cold. However, lately, ambient temperature preservation has been evaluated by several independent institutions that see economic and logistic advantages in getting rid of the cold chain. Here we validated a novel procedure for ambient temperature preservation of DNA in white blood cell and buffy coat lysates based on the confinement of the desiccated biospecimens under anoxic and anhydrous atmosphere in original hermetic minicapsules. For this validation we stored encapsulated samples either at ambient temperature or at several elevated temperatures to accelerate aging. We found that DNA extracted from stored samples was of good quality with a yield of extraction as expected. Degradation rates were estimated from the average fragment size of denatured DNA run on agarose gels and from qPCR reactions. At ambient temperature, these rates were too low to be measured but the degradation rate dependence on temperature followed Arrhenius' law, making it possible to extrapolate degradation rates at 25°C. According to these values, the DNA stored in the encapsulated blood products would remain larger than 20 kb after one century at ambient temperature. At last, qPCR experiments demonstrated the compatibility of extracted DNA with routine DNA downstream analyses. Altogether, these results showed that this novel storage method provides an adequate environment for ambient temperature long term storage of high molecular weight DNA in dehydrated lysates of white blood cells and buffy coats.
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Project description:A transition in hemoglobin behavior at close to body temperature has been discovered recently by micropipette aspiration experiments on single red blood cells (RBCs) and circular dichroism spectroscopy on hemoglobin solutions. The transition temperature was directly correlated to the body temperatures of a variety of species. In an exploration of the molecular basis for the transition, we present neutron scattering measurements of the temperature dependence of hemoglobin dynamics in whole human RBCs in vivo. The data reveal a change in the geometry of internal protein motions at 36.9 degrees C, at human body temperature. Above that temperature, amino acid side-chain motions occupy larger volumes than expected from normal temperature dependence, indicating partial unfolding of the protein. Global protein diffusion in RBCs was also measured and the findings compared favorably with theoretical predictions for short-time self-diffusion of noncharged hard-sphere colloids. The results demonstrated that changes in molecular dynamics in the picosecond time range and angstrom length scale might well be connected to a macroscopic effect on whole RBCs that occurs at body temperature.
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Project description:BackgroundThe relationships between the ratios of transfused allogeneic blood products and clinical outcomes in patients with acute intraoperative hemorrhage are poorly defined.MethodsTo better define these ratios, we undertook a single-center, observational cohort study of all surgical patients (≥18 years) who received rapid transfusion defined by a critical administration threshold of 3 or more units of red blood cells (RBCs) intraoperatively within 1 hour between January 1, 2011 and December 31, 2015. Multivariable regression analyses were used to assess relationships between ratios of plasma to RBCs and platelets to RBCs at 3, 12, and 24 hours and clinical outcomes. The primary outcome was hospital mortality, with secondary outcomes of intensive care unit and hospital-free days.ResultsThe study included 2385 patients, of whom 14.9% had a plasma-to-RBC ratio of 1.0+, and 47.6% had a platelet-to-RBC ratio of 1.0+. Higher plasma-to-RBC and platelet-to-RBC ratios were observed for patients who underwent cardiac, transplant, and vascular surgery and in patients with greater derangements in hemostatic laboratory values. Ratios did not differ by patient age or severity of illness. Higher ratios were not associated with improved clinical outcomes. Mortality differed by platelet-to-RBC but not plasma-to-RBC ratio, with the highest mortality observed with a platelet-to-RBC ratio of 0.1-0.9 at 24 hours (odds ratio, 3.34 [1.62-6.88]) versus no platelets (P= .001). Higher plasma-to-RBC ratios were associated with decreased hospital-free days, although differences in clinical outcomes were not significant after exclusion of patients receiving only RBCs without component therapies.ConclusionsTransfusion ratios in surgical patients with critical intraoperative hemorrhage were largely related to surgical and hemostatic features rather than baseline patient characteristics. Higher ratios were not associated with improved outcomes.
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2009-01-15 | E-MEXP-1989 | biostudies-arrayexpress
Project description:This study reports a microfluidic device for whole blood processing. The device uses the bifurcation law, cross-flow method, and hydrodynamic flow for simultaneous extraction of plasma, red blood cells, and on-chip white blood cell trapping. The results demonstrate successful plasma and red blood cell collection with a minimum dilution factor (0.76x) and low haemolysis effect. The extracted red blood cells can also be applied for blood type tests. Moreover, the device can trap up to ~1,800 white blood cells in 20?minutes. The three components can be collected simultaneously using only 6??L of whole blood without any sample preparation processes. Based on these features, the microfluidic device enables low-cost, rapid, and efficient whole blood processing functionality that could potentially be applied for blood analysis in resource-limited environments or point-of-care settings.
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Project description:Temperature compensation and period determination by casein kinase 1 (CK1) are conserved features of eukaryotic circadian rhythms, whereas the clock gene transcription factors that facilitate daily gene expression rhythms differ between phylogenetic kingdoms. Human red blood cells (RBCs) exhibit temperature-compensated circadian rhythms, which, because RBCs lack nuclei, must occur in the absence of a circadian transcription-translation feedback loop. We tested whether period determination and temperature compensation are dependent on CKs in RBCs. As with nucleated cell types, broad-spectrum kinase inhibition with staurosporine lengthened the period of the RBC clock at 37°C, with more specific inhibition of CK1 and CK2 also eliciting robust changes in circadian period. Strikingly, inhibition of CK1 abolished temperature compensation and increased the Q10 for the period of oscillation in RBCs, similar to observations in nucleated cells. This indicates that CK1 activity is essential for circadian rhythms irrespective of the presence or absence of clock gene expression cycles.
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Project description:Mice lacking perilipin-2 (Plin2-null) are resistant to obesity, insulin resistance, and fatty liver induced by Western or high-fat diets. In the current study, we found that, compared with WT mice on Western diet, Plin2-null adipose tissue was more insulin sensitive and inguinal subcutaneous white adipose tissue (iWAT) exhibited profound browning and robust induction of thermogenic and carbohydrate-responsive genetic programs at room temperature. Surprisingly, these Plin2-null responses correlated with the content of simple carbohydrates, rather than fat, in the diet, and were independent of adipose Plin2 expression. To define Plin2 and sugar effects on adipose browning, WT and Plin2-null mice were placed on chow diets containing 20% sucrose in their drinking water for 6 weeks. Compared with WT mice, iWAT of Plin2-null mice exhibited pronounced browning and striking increases in the expression of thermogenic and insulin-responsive genes on this diet. Significantly, Plin2-null iWAT browning was associated with reduced sucrose intake and elevated serum fibroblast growth factor (FGF)21 levels, which correlated with greatly enhanced hepatic FGF21 production. These data identify Plin2 actions as novel mediators of sugar-induced adipose browning through indirect effects of hepatic FGF21 expression, and suggest that adipose browning mechanisms may contribute to Plin2-null resistance to obesity.
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Project description:Organ preservation solutions are designed to minimize organ damage during transplantation. A novel preservation solution, WMO-II, was developed to have a low viscosity and to improve microvasculature perfusion for kidneys. In an autologous canine transplantation model, kidney function and recovery were evaluated after organs were flushed and cold-stored with WMO-II or HTK solution, a perfusate currently approved for clinical use. The average number of red blood cells remaining in a single glomerulus after flushing with WMO-II was significantly reduced when compared with HTK solution. Additionally, WMO-II reduced the number of apoptotic bodies in stored kidneys compared to HTK treated tissue after 48 h of cold storage by reducing expression of Caspase-9, BiP, Chop, and Caspase-12. WMO-II solution reduced serum creatinine levels and serum potassium in kidneys stored for 48 h when compared to HTK perfusion. WMO-II preserves kidney function as evidenced by the reduction in serum creatinine and potassium during graft transplantation.
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