Aliquots Of Blood Research Articles

Superficial Venous-Associated Inflammation from Direct IV Administration of RRx-001 in Rats.

RRx-001 is a small molecule NLRP3 inflammasome inhibitor with anti-CD47 and antiangiogenic/vascular normalization properties in a Phase 3 clinical trial that has been designated as a drug-device combination by the FDA. In the Phase 1 first-in-man dose escalation clinical trial, where RRx-001 was given by direct intravenous (IV) infusion, the main adverse event was a sterile painful infusion phlebitis (IP). Less pain was experienced when RRx-001 was infused at a slower rate over multiple hours which was impractical on an outpatient basis. In Phase 2, for reasons of convenience and safety, RRx-001 was co-administered with an aliquot of autologous blood from an ex-vivo device called the eLOOP on the premise that RRx-001 binds to hemoglobin on red blood cells (RBCs), making it unavailable to directly interact with venous nociceptors. Phlebitis has the potential to progress to deep venous thrombosis or septic thrombophlebitis or post-thrombotic syndrome in hypercoagulable and immunosuppressed cancer patients. In this 13-week toxicology study of once weekly IV RRx-001 administration to Wistar Han rats followed by a recovery period of 28 days. The main observed toxicity was a significant inflammatory response in the vein wall, consistent with superficial venous thrombosis observed in man. Due to this development, direct IV infusion of RRx-001 is relatively contraindicated in favor of co-administration with autologous blood.

Purpose, Partnership, and Possibilities: The Implementation of the Dog Aging Project Biobank.

Biobanks have been supporting longitudinal prospective and retrospective studies by providing standardized services for the acquisition, transport, processing, storage, and distribution of high-quality biological material and associated data. Here, we describe how the Dog Aging Project (DAP), a large-scale longitudinal study of the domestic dog (Canis familiaris) with translational applications for humans, developed a biobank of canine biospecimens and associated data. This was accomplished by working with the Cornell Veterinary Biobank, the first biobank in the world to receive accreditation to ISO 20387:2018-General Requirements for Biobanking. The biobank research team was involved in the early collection stages of the DAP, contributing to the development of appropriate workflows and processing fit-for-purpose biospecimens. In support of a dynamic strategy for real-time adjustment of processes, a pilot phase was implemented to develop, test, and optimize the biospecimen workflows, followed by an early phase of collection, processing, and banking of specimens from DAP participants. During the pilot and early phases of collection, the DAP Biobank stored 164 aliquots of whole blood, 273 aliquots of peripheral blood mononuclear cells, 130 aliquots of plasma, and 70 aliquots of serum, and extracted high molecular weight genomic DNA suitable for whole-genome sequencing from 109 whole blood specimens. These specimens, along with their associated preanalytical data, have been made available for distribution to researchers. We discuss the challenges and opportunities encountered during the implementation of the DAP Biobank, along with novel strategies for promoting biobanking sustainability such as partnering with a DAP quality assurance manager and a DAP marketing and communication specialist and developing a pilot grant structure to fund small innovative research projects.

57. A rare case of near-tetraploidy in CLL: An investigation by MLPA, FISH, and chromosome analysis

Chronic lymphocytic leukemia (CLL) represents approximately 70% of all lymphoid leukemias. The majority of cases originate from B-cells, and four abnormalities are known to be of prognostic value: trisomy 12, deletion 13q, 11q, and 17p. At CHEO, an aliquot of peripheral blood is used for B-cell selection/enrichment and Multiplex Ligation-dependent Probe Amplification (MLPA), whereas the remaining blood sample is cultured for 24-48h for reflex interphase FISH for deletions 17p and 11q to exclude low level poor prognosis abnormalities.

Citrate dose for continuous hemofiltration: effect on calcium and magnesium balance, parathormone and vitamin D status, a randomized controlled trial

BackgroundRegional citrate anticoagulation may cause a negative calcium balance, systemic hypocalcemia and parathormone (PTH) activation but randomzed studies are not available. Aim was to determine the effect of citrate dose on calcium (Ca) and magnesium (Mg) balance, PTH and Vitamin D.MethodsSingle center prospective randomized study. Patients, requiring continuous venovenous hemofiltration (CVVH) with citrate, randomized to low dose citrate (2.5 mmol/L) vs. high dose (4.5 mmol/L) for 24 h, targeting post-filter ionized calcium (pfiCa) of 0.325–0.4 mmol/L vs. 0.2–0.275 mmol/L, using the Prismaflex® algorithm with 100% postfilter calcium replacement. Extra physician-ordered Ca and Mg supplementation was performed aiming at systemic iCa > 1.0 mmol/L. Arterial blood, effluent and post-filter aliquots were taken for balance calculations (area under the curve), intact (i), oxidized (ox) and non-oxidized (nox) PTH, 25-hydroxy-Vitamin D (25D) and 1,25-dihydroxy-Vitamin D (1,25D).Results35 patients were analyzed, 17 to high, 18 to low citrate. Mean 24-h Ca balance was - 9.72 mmol/d (standard error 1.70) in the high vs − 1.18 mmol/d (se 1.70)) (p = 0.002) in the low citrate group and 24-h Mg-balance was − 25.99 (se 2.10) mmol/d vs. -17.63 (se 2.10) mmol/d (p = 0.008) respectively. Physician-ordered Ca supplementation, higher in the high citrate group, resulted in a positive Ca-balance in both groups. iPTH, oxPTH or noxPTH were not different between groups. Over 24 h, median PTH decreased from 222 (25th–75th percentile 140–384) to 162 (111–265) pg/ml (p = 0.002); oxPTH from 192 (124–353) to 154 pg/ml (87–231), p = 0.002. NoxPTH did not change significantly. Mean 25 D (standard deviation), decreased from 36.5 (11.8) to 33.3 (11.2) nmol/l (p = 0.003), 1,25D rose from 40.9 pg/ml (30.7) to 43.2 (30.7) pg/ml (p = 0.046), without differences between groups.ConclusionsA higher citrate dose caused a more negative CVVH Ca balance than a lower dose, due to a higher effluent Calcium loss. Physician-ordered Ca supplementation, targeting a systemic iCa > 1.0 mmol/L, higher in the high citrate group, resulted in a positive Ca-balance in both groups. iPTH and oxPTH declined, suggesting decreased oxidative stress, while noxPTH did not change. 25D decreased while 1,25-D rose. Mg balance was negative in both groups, more so in the high citrate group.Trial registrationClinicalTrials.gov: NCT02194569. Registered 18 July 2014.

Human non-CpG methylation patterns display both tissue-specific and inter-individual differences suggestive of underlying function

ABSTRACT DNA methylation (DNAm) in mammals is mostly examined within the context of CpG dinucleotides. Non-CpG DNAm is also widespread across the human genome, but the functional relevance, tissue-specific disposition, and inter-individual variability has not been widely studied. Our aim was to examine non-CpG DNAm in the wider methylome across multiple tissues from the same individuals to better understand non-CpG DNAm distribution within different tissues and individuals and in relation to known genomic regulatory features. DNA methylation in umbilical cord and cord blood at birth, and peripheral venous blood at age 12–13 y from 20 individuals from the Southampton Women’s Survey cohort was assessed by Agilent SureSelect methyl-seq. Hierarchical cluster analysis (HCA) was performed on CpG and non-CpG sites and stratified by specific cytosine environment. Analysis of tissue and inter-individual variation was then conducted in a second dataset of 12 samples: eight muscle tissues, and four aliquots of cord blood pooled from two individuals. HCA using methylated non-CpG sites showed different clustering patterns specific to the three base-pair triplicate (CNN) sequence. Analysis of CAC sites with non-zero methylation showed that samples clustered first by tissue type, then by individual (as observed for CpG methylation), while analysis using non-zero methylation at CAT sites showed samples grouped predominantly by individual. These clustering patterns were validated in an independent dataset using cord blood and muscle tissue. This research suggests that CAC methylation can have tissue-specific patterns, and that individual effects, either genetic or unmeasured environmental factors, can influence CAT methylation.

Mitochondria-Targeted Effects of Water-Soluble Polyol-Methanofullerenes on Human Whole Blood Platelets, Lymphocytes and Granulocytes Ex Vivo

The mitochondrial membrane potential (ΔΨm) drives many functions including cellular energetics and signaling being a major determinant and indicator of cell fate. Mitochondria are considered as promising therapeutic targets because they are involved as either a cause or consequence of many chronic diseases. Previously, we reported on two novel water-soluble polyol-methanofullerenes С60[С9H10O4((OH)4]6 and С60[C13H18O4((OH)4]6 that concentraion-dependently dissipate ΔΨm in Yarrowia lipolytica yeast cells (Kalacheva et al., 2015). This observation justifies further studies of those fullerene derivatives as mitochondria-targeted biological response modifiers. Here, we proceed by exploring methanofullerenols-driven individual mitochondrial responses of human whole blood platelets, lymphocytes and granulocytes ex vivo .A cytofluorimetric JC-1-based ratiometric assay was used to quantify ΔΨm in human heparinized whole blood cells using “no lyse-no wash” protocol minimizing activation artifacts caused by cell isolation. Briefly, heparinized blood (50 µl) obtained from healthy volunteers (n= 5) was incubated with JC-1 (20 µM final concentration, Biotium, Inc) and either 5 µl anti-CD45-PerCP (BD Biosciences) or 5 µl anti-CD31-Alexa Fluor 647 (BD Biosciences) in the presence of methanofullerenols С60[С9H10O4((OH)4]6 and С60[C13H18O4((OH)4]6 (4 µM and 40 µM) for 30 min at 37 C°, 5% CO2. Immediately after the end of incubation 5 µl whole blood aliquots were taken up from each sample, mixed with 1000 µl CellWash buffer (BD Biosciences) and run on a FACSCalibur cytometer with a CellQuest Pro software. CD45-negative events and CD31-negative events were excluded throughout acquisition by appropriate thresholds. The normalized mitochondrial potential ΔΨm was expressed in arbitrary units as the FL2/FL1 ratio of orange (JC-1 dimers, em 590 nm) to green (JC-1 monomers, em 529 nm) fluorescence signals calculated and plotted by the Weasel v2.2.3 analysis software.Both methanofullerenols applied at concentrations of 4 µM and 40 µM after 2 h incubation period did not change ΔΨm, cell size and granularity of SSClowCD45high lymphocytes and SSChighCD45med granulocytes. The С60[C9H10O4((OH)4]6 methanofullerenol at concentration of 4 µM hampered by 32,5 % (P<0.01) time-dependent proapoptotic transition from CD31+ΔΨmhigh platelet subset(29,8 %) to CD31+ΔΨmlow platelet subset(20,1 %). An example representative graph is shown in Figure 1. However its more hydrophobic homologue С60[C13H18O4((OH)4]6 did not prevent platelet Ψm dissipation. We believe that different cytoprotective activity of methanofullerenols studied is attributed to diverse structure, antioxidant potential and target cell type.Conclusively, the provided “no lyse-no wash” proof-of-principle protocol offers a tool for personalized diagnostics and bioenergetic therapy monitoring of mitochondrial dysfunctions in clinical settings. The С60[C9H10O4((OH)4]6 methanofullerenol appears to be a promising candidate for mitochondria-targeted prevention of apoptosis of banked platelet concentrates.This study was supported by the Russian Government Program of Competitive Growth of Kazan Federal University. RAA was supported by state assignment 20.5175.2017/6.7 of the Ministry of Education and Science of Russian Federation.Figure 1. Cytofluorimetric quantification of mitochondrial membrane potential ΔΨm in whole blood platelets. (A) Gating of intact single platelets with physiological size (FSCmedCD31+ events) to exclude platelet microparticles (FSClow), platelet-WBC and platelet-RBC conjugates (FSChigh). (B) The C60[C9H10O4((OH)4]6 methanofullerenol (F1) did not affect platelet size and CD31 expression. (C) The normalized ΔΨm expressed as FL2/FL1 ratio in : (1) control platelets, (2) C60[C9H10O4((OH)4]6-treated platelets (4 µM), (3) protonophore CCCP-treated platelets (1 mM). Note bimodal ΔΨm distributions and clear-cut differences in platelet ΔΨm low-subset size. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Comparison of different preparation techniques of dried blood spot quality controls in newborn screening for congenital adrenal hyperplasia.

In newborn screening, samples suspected for congenital adrenal hyperplasia (CAH), a potentially lethal inborn error of steroid biosynthesis, need to be confirmed using liquid chromatography–tandem mass spectrometry. Daily quality controls (QCs) for the 2nd-tier CAH assay are not commercially available and are therefore generally prepared within the laboratory. For the first time, we aimed to compare five different QC preparation approaches used in routine diagnostics for CAH on the concentrations of cortisol, 21-deoxycortisol, 11-deoxycortisol, 4-androstenedione and 17-hydroxyprogesterone in dried blood spots. The techniques from Prep1 to Prep5 were tested at two analyte concentrations by spiking aliquots of a steroid-depleted blood, derived from washed erythrocyte suspension and steroid-depleted serum. The preparation processes differed in the sequence of the preparation steps and whether freeze-thaw cycles were used to facilitate blood homogeneity. The five types of dried blood spot QCs were assayed and quantitated in duplicate on five different days using a single calibration row per day. Inter-assay variations less than 15% and concentrations within ±15% of the nominal values were considered acceptable. Results obtained by means of the four dried blood spot QC preparation techniques (Prep1, Prep2, Prep4 and Prep5) were statistically similar and remained within the ±15% ranges in terms of both reproducibility and nominal values. However, concentration results for Prep3 (spiking prior to three freeze-thaw cycles) were significantly lower than the nominal values in this setting, with differences exceeding the ±15% range in many cases despite acceptable inter-assay variations. These findings have implications for the in-house preparation of QC samples in laboratory developed tests for CAH, including 2nd-tier assays in newborn screening.

N-Acetylcysteine Interference with a Glucose Dehydrogenase Linked Glucose Meter.

Our objective was to determine the effect of therapeutic concentrations of N-acetylcysteine, following intravenous infusion, on the measurement of blood glucose using a Roche Diagnostics glucose dehydrogenase-linked glucose meter compared to hospital laboratory methods. N-acetylcysteine was added to aliquots of blood, with glucose promptly measured by the glucose meter, blood gas analyzer (glucose oxidase comparative method) and following centrifugation, plasma glucose measured with a hexokinase spectrophotometric comparative method. Glucose results were evaluated with linear regression and Bland Altman plots. In the presence of NAC, at concentrations greater than 5 mg/dL (0.31 mmol/L), positively biased glucose meter results were compared to the clinical laboratory results. Multivariate linear regression revealed that NAC-mediated meter results are influenced by NAC and glucose concentrations. The addition of therapeutic concentrations of NAC to blood produces statistically significant positive biases when measured with the glucose dehydrogenase linked glucose meter device.

A field method for cryopreservation of whole blood from a finger prick for later analysis with flow cytometry.

Flow cytometry is a powerful tool for investigating immune function, allowing for the quantification of leukocytes by subtype. Yet it has not been used extensively for field work due to perishable reagents and the need for immediate analysis of samples. To make flow cytometry more accessible, we devise and evaluate a field protocol for freezing capillary blood. We collected finger prick blood samples from 110 volunteers, age 18 to 42. Blood samples were analyzed immediately for 18 cell surface markers. Aliquots of whole blood were frozen in the vapor phase of a liquid nitrogen tank with 10% dimethyl sulfoxide in medium. Samples were analyzed on a Guava EasyCyte HT flow cytometer after 2, 4, or 14 weeks. Major lymphocyte fractions in frozen samples were correlated with fresh values (T-cells: r = 0.82; Natural Killer [NK] cells: r = 0.64; CD4: r = 0.67; CD8: r = 0.82; Naïve CD4: r = 0.73, Naïve CD8: r = 0.71; B-cells: r = 0.73; all p < 0.001), and mean values were similar to those from fresh samples. However, correlations for smaller subsets of CD4 and B cells were generally poor. Some differences resulted from changes in non-specific binding for some antibody-conjugate pairs. Cryopreservation also resulted in a reduction in granulocytes more than lymphocytes. Our results suggest that antibody/fluorochrome combinations should be validated before use on frozen samples, and that functional changes in cells may affect some cell markers. However, this simple freezing protocol utilizing finger pricks, whole blood, and a liquid nitrogen shipping tank is viable for obtaining samples for flow cytometry under field conditions.

Design and Computational Modeling of Spiral Microfluidic Channel for Sorting and Separating the Biomolecules

Background: Microfluidic technologies are a very challenging area today in the field of biomolecule analysis. This has become feasible with the today’s advanced technologies by designing and fabricating the microfluidic channel. Materials and Methods: Initially, microfluidic channels are used to separate large molecules, where the molecular dimension of the fluidic filter is greater than the gap size. In this work, separation of biomolecules (like RBC, WBC and platelets) that are smaller than the microfluidic filter gap size is demonstrated. Results and Conclusion: Due to the curvilinear nature of the spiral, there exists two vortices called dean vortices within the channel and this is influenced by dean flow, centrifugal flow and tubular pinch effect. While flowing a small aliquot of blood in the channel, due to these three effects, molecules attain equilibrium position at one point. The position of equilibrium will be different for different sized biomolecules and this varies with different input velocities. Conclusion: The obtained computational modeling results show how the equilibrium positions influence the separation efficiency of biomolecules in passive based microfluidic filter. Compared with the traditional random nanoporous materials such as gel or polymer monolith, spiral based microfluidic channels can be made precisely to have a pre-determined loop count and Dean Flow number (De).

Methodological aspects of the micronutrient assessment in the Brazilian National Survey on Child Nutrition (ENANI-2019): a population-based household survey.

This article aims to present methodological aspects on the collection, analyses, coverage, challenges, and the lessons learned from laboratory assessment of micronutrients on the Brazilian National Survey on Child Nutrition (ENANI-2019). This is a household survey on a probability sample of children under five years of age from 123 Brazilian municipalities in all 26 states and the Federal District. Blood samples were drawn by venipuncture at the homes of children 6 to 59 months of age. This procedure was performed by experienced phlebotomists from the laboratories located in the selected municipalities and scheduled in advance. Blood and serum levels were measured for biomarkers of nutritional status, using the services of a clinical test laboratory with nationwide coverage, for the following micronutrients: iron (hemoglobin and ferritin), zinc, selenium, folic acid, and vitamins A, B1, B6, B12, D, and E. C-reactive protein was analyzed as a marker of inflammation. A barcode identifier was used to track the blood samples and to link the biochemical test results to the other data collected in the survey. A total of 14,558 children were studied. Of the 12,598 eligible children, 8,829 (70.1%) had blood samples drawn. Of the total number of children who had samples drawn, 91.8% (n = 8,025) have results for at least nine of the 12 analyses performed. Coverage of the analysis varied from 95% (for vitamins A and E) to 84.2% (for folic acid). Aliquots of whole blood and serum were stored in a biorepository for future analyses. The results of this pioneering study in the country will back the formulation and, when necessary, the reorientation of public policies in food and nutrition.

The use of Ocimum gratissimum L. essential oil during the transport of Lophiosilurus alexandri: Water quality, hematology, blood biochemistry and oxidative stress

This study evaluated the effects of the essential oil of Ocimum gratissimum L. (EOOG) on water quality, blood characteristics and tissue damage during transport of Lophiosilurus alexandri. One hundred and sixty juveniles of L. alexandri (123.44 ± 1.95 g and 23.71 ± 1.95 cm) were fasted for 24 h before transportation. Fish were then submitted to one of three treatments of transport water with five replicates each: 0 (control), 5 and 10 mg/L EOOG. The animals were transported for 4 h in plastic bags at a density of 1 juvenile/L and 10 animals per bag. The ventilatory frequency of one fish from each bag was determined after 3 h of transport by direct observation and counting of opercular beats. Blood, brain and liver samples were collected prior to (basal group) and at the end of transport from 10 animals each. Blood aliquots were used to determine hematocrit and hemoglobin, total plasma protein, glucose, aspartate aminotransferase and alanine aminotransferase levels. Samples of liver and brain were used to determine parameters and oxidative stress (reactive oxygen species, glutathione peroxidase, superoxide dismutase, glutathione S-transferase, lipid peroxidation and protein carbonyl formation). No mortality was observed 24 h after transport. The addition of EOOG to transport water appeared to reduce animal metabolism, thus ensuring higher dissolved oxygen and lower ammonia levels. Juveniles transported with 10 mg/L EOOG also had reduced opercular beats, lower levels of hemoglobin and blood aspartate aminotransferase and glucose. This concentration also reduced lipid and protein oxidation levels in tissues, thereby avoiding lipid peroxidation and the formation of carbonyl protein. The use of 10 mg/L EOOG during transport promotes a sedative effect in L. alexandri, with reduced oxygen consumption, ammonia excretion and biochemical changes and enhanced protection against oxidative damage.

Acute postprandial effect of yacon syrup ingestion on appetite: A double blind randomized crossover clinical trial

Yacon syrup is a rich source of fructooligosaccharides (FOS); however, its diet supplementation effect on subjective sensation and appetite biomarkers in human is still unknown. This study aimed to evaluate the acute postprandial effect of yacon syrup ingestion on appetite. The double-blind crossover clinical trial was carried out with 40 adult women: 20 eutrophic and 20 obese grade I. On each day, the first blood collection was performed after a 12-h fast. Then, the volunteers ingested either intervention A (breakfast + 40 g of placebo) or intervention B (breakfast + 40 g of yacon syrup, containing 14 g of FOS). New aliquots of blood were collected at 45, 60, 90, 120, and 180 min. Appetite was assessed by estimating ghrelin and glucagon-like peptide-1 (GLP-1) levels and by assessing subjective appetite sensation. Analysis was performed using two-way ANOVA, followed by Bonferroni's multiple comparison test. No effect of yacon syrup was observed on postprandial ghrelin and GLP-1 levels at all times evaluated. Similar observations were made after stratifying the analysis by BMI (body mass index) (eutrophic and obese). The effect of yacon syrup on postprandial subjective sensations of hunger, satiety, fullness, and desire to eat was not evident in the total group of women evaluated and even after BMI stratification. We concluded that yacon syrup had no effect on postprandial ghrelin and GLP-1 levels and on the subjective appetite sensation in young adult women.

Supplementation of Curcuma longa hydrolate improves immunomodulatory response in Nile tilapia reared in a recirculation aquaculture system

ABSTRACT The objective of this study was to evaluate the effects of dietary supplementation with different doses of Curcuma longa hydrolate on the hematological, immunological and zootechnical parameters of Nile tilapia cultivated in a recirculation system (RAS). Nile tilapia (Oreochromis niloticus) were used, distributed in 16 polyethylene boxes, divided into four treatments: 0.0%; 2.5%; 7.5% and 10.0% of Curcuma longa hydrolate, in quadruplicate. After 45 days of treatment, four fish per experimental unit were anesthetized to remove blood aliquot for hematological and immunological analyzes and dissect the liver to evaluate the hepatosomatic index and final biometry. In the haematological analysis, the fish fed with 2.5% had a higher number of leukocytes, monocytes and lymphocytes than control, while the doses of 7.5% and 10.0% did not differ. Antimicrobial activity showed a significant decrease as the dose of C. longa hydrolate increased. The other hematological, immunological, hepatosomatic index and zootechnical data did not differ between treatments. Thus, supplementation of the hydrolate of Curcuma longa at a dosage of 2.5%, improved and maintained blood-immune homeostasis parameters in these animals, being suggested for further studies.

High-Dose Dabigatran Is an Effective Anticoagulant for Simulated Cardiopulmonary Bypass Using Human Blood.

Currently no ideal alternative exists for heparin for cardiopulmonary bypass (CPB). Dabigatran is a direct thrombin inhibitor for which a reversal agent exists. The primary end point of the study was to explore whether Dabigatran was an effective anticoagulant for 120 minutes of simulated CPB. The study was designed in 2 sequential steps. Throughout, human blood from healthy donors was used for each experimental step. Initially, increasing concentrations of Dabigatran were added to aliquots of fresh whole blood, and the anticoagulant effect measured using kaolin/tissue factor-activated thromboelastography (rapidTEG). The dynamics of all thromboelastography (TEG) measurements were studied with repeated measures analysis of variance (ANOVA). Based on these data, aliquots of blood were treated with high-concentration Dabigatran and placed in a Chandler loop as a simple ex vivo bypass model to assess whether Dabigatran had sufficient anticoagulant effects to maintain blood fluidity for 2 hours of continuous contact with the artificial surface of the PVC tubing. Idarucizumab, humanized monoclonal antibody fragment, was used to verify the reversibility of Dabigatran effects. Finally, 3 doses of Dabigatran were tested in a simulated CPB setup using a heart-lung machine and a commercially available bypass circuit with an arteriovenous (A-V) loop. The primary outcome was the successful completion of 120 minutes of simulated CPB with dabigatran anticoagulation, defined as lack of visible thrombus. Thromboelastographic reaction (R) time was measured repeatedly in each bypass simulation, and the circuits were continuously observed for clot. Scanning Electron Microscopy (SEM) was used to visualize fibrin formation in the filters meshes during CPB. In in vitro blood samples, Dabigatran prolonged R time and reduced the dynamics of clot propagation (as measured by speed of clot formation [Angle], maximum rate of thrombus generation [MRTG], and time to maximum rate of thrombus generation [TMRTG]) in a dose-dependent manner. In the Chandler Loop, high doses of Dabigatran prevented clot formation for 120 minutes, but only at doses higher than expected. Idarucizumab decreased R time and reversed anticoagulation in both in vitro and Chandler Loops settings. In the A-V loop bypass simulation, Dabigatran prevented gross thrombus generation for 120 minutes of simulated CPB. Using sequential experimental approaches, we showed that direct thrombin inhibitor Dabigatran in high doses maintained anticoagulation of blood for simulated CPB. Idarucizumab reduced time for clot formation reversing the anticoagulation action of Dabigatran.

The Lyme Disease Biobank: Characterization of 550 Patient and Control Samples from the East Coast and Upper Midwest of the United States.

Lyme disease (LD) is an increasing public health problem. Current laboratory testing is insensitive in early infection, the stage at which appropriate treatment is most effective in preventing disease sequelae. The Lyme Disease Biobank (LDB) collects samples from individuals with symptoms consistent with early LD presenting with or without erythema migrans (EM) or an annular, expanding skin lesion and uninfected individuals from areas of endemicity. Samples were collected from 550 participants (298 cases and 252 controls) according to institutional review board-approved protocols and shipped to a centralized biorepository. Testing was performed to confirm the presence of tick-borne pathogens by real-time PCR, and a subset of samples was tested for Borrelia burgdorferi by culture. Serology was performed on all samples using the CDC's standard two-tiered testing algorithm (STTTA) for LD. LD diagnosis was supported by laboratory testing in 82 cases, including positive results by use of the STTTA, PCR, or culture or positive results by two enzyme-linked immunosorbent assays for cases presenting with EM lesion sizes of >5 cm. The remaining 216 cases had negative laboratory testing results. For the controls, 43 were positive by at least one of the tiers and 6 were positive by use of the STTTA. The results obtained with this collection highlight and reinforce the known limitations of serologic testing in early LD, with only 29% of individuals presenting with EM lesion sizes of >5 cm yielding a positive result using the STTTA. Aliquots of whole blood, serum, and urine from clinically characterized patients with and without LD are available to investigators in academia and industry for evaluation or development of novel diagnostic assays for LD, to continue to improve upon currently available methods.

Esculin reduces P2X7 and reverses mitochondrial dysfunction in the renal cortex of diabetic rats

AimsTo evaluate the effects of esculin treatment on P2X7 receptor and mitochondrial dysfunction in the renal cortex of diabetic rats. Main methodsMale Wistar rats, 7 weeks old, were unilaterally nephrectomized. Part of these animals were induced to diabetes using streptozotocin (60 mg/kg). Diabetes was confirmed 48 h after induction, with blood glucose levels ≥200 mg/dL. Part of control and diabetic animals were selected to receive daily doses of esculin (50 mg/kg), during 8 weeks. The animals were placed in metabolic cages at the eighth week of protocol for 24 h urine collection and a small aliquot of blood was collected for biochemical analysis. After this procedure, the animals were euthanized and the remaining kidney was stored for histopathological analysis, Western blotting and mitochondrial high-resolution respirometry. Key findingsAlthough esculin did not change metabolic parameters, renal biochemical function, neither TBARS in DM rats, esculin reduced P2X7 levels in these animals and restored mitochondrial function via glycolysis substrates and β-oxidation. Besides, at the histological analysis, we observed that esculin reduced inflammatory infiltrates and collagen IV deposits as compared to diabetic group. SignificanceEsculin attenuated the development of renal injuries caused by hyperglycemia, proinflammatory and oxidative mechanisms mediated by P2X7 receptor, as seen by histological findings and improved mitochondrial function in diabetic animals. This suggests that esculin could be used as an adjuvant therapy to prevent the diabetic nephropathy.

MON-101 Costa Rican National Primary Congenital Hypothyroidism Screening Program Evaluation. Retrospective Cohort Trial Between 2015 and 2017

Congenital hypothyroidism (CH) is one of the leading causes of intellectual impairment worldwide in infancy. The newborn screening has been able to prevent this mental disability, by a prompt initiation of therapy. Over the last years the incidence of HC has been increasing, mainly by lowering the screening cut-off level that leads to detection of milder cases. There is conflicting evidence if children with mild CH without treatment may develop neurological impairment in the future.Costa Rican newborn screening program in divided in three stages, measuring serum TSH concentrations from a heel prick aliquot of capillary blood dried onto a filter paper. Each test has different TSH cut-off values to determine if the newborn needs a clinical evaluation by an endocrinologist, needs another screening test or rules out hypothyroidism.We developed an observational, descriptive, retrospective study, based on medical records, to evaluate our newborn screening program performance. The study included the total national population of screened newborns from 2015 to 2017. Descriptive analysis and analytical analysis of variables were done, and test’s sensitivity and specificity were determined.The study analyzed 204.241 screened newborns, and 145 children referred to the Endocrinology Department of the National Children Hospital. This population represents 97% of births in these 3 years. The recall rate for a first positive test was 0.3%. Congenital hypothyroidism was confirmed in 73% of children referred to the Endocrinology Department because of a positive screening. Incidence was 1/1926 births. Detection rate was independent of birth weight nor gestational age. 45.3% of children diagnosed with CH were detected after the first screening test, 52.8% after the second screening test and 1.9% after the third screening test. Screening test analysis showed high sensitivity and specificity, with diagnostic accuracy above 90%, except for the third screening test. Free thyroxine measurements alone weren’t useful to predict CH diagnosis.The coverage of the national neonatal screening program extends almost to the entire population. Our test specificity is within international standards. Incidence of CH in Costa Rica is similar to that reported in medical literature. With current detection cut-off level, there is no need of performing a second mandatory evaluation to preterm and low weight newborns. The third screening test has a low performance rate, does not improve detection of children with CH significantly, and delays clinical evaluation by the endocrinologist. Prognosis of treated children with CH is excellent, with no evidence of severe cognitive deficit.

Essential oils in diets for Nile tilapia juveniles: Productive performance and plasmatic biochemistry

The objective of this study was to evaluate the inclusion of increasing levels of a commercial mix of essential oils (EOs) from cashew (Anacardium occidentale) and castor bean (Ricinus communis) as an additive in diets for Nile tilapia. Thus, 240 juveniles with an initial weight of 35.06 ± 1.02 g were distributed in a completely randomized design (four treatments and six replicates) in 24 cylindrical-tapered tanks; the fish were fed for 60 days, four times a day, diets containing increasing levels of EO (0.10, 0.15 and 0.20%) and a control diet (without the additive). After the experimental period, the animals were desensitized, and an aliquot of blood was collected for the biochemical analysis; they were subsequently euthanized for the evaluation of productive performance and fillet chemical composition. No significant differences (p > .05) were observed in the analyses of productive performance, fillet chemical composition, and triglycerides, VLDL, total proteins, urea and glucose. However, significant differences (p < .05) were observed in the analyses of total cholesterol, HDL and LDL. Inclusion above 0.15 of the additive showed an improvement in the levels of lipoprotein carriers, increasing HDL levels and decreasing LDL values, without interfering with the fish productive performance.

Monitoring of CMV-specific cell-mediated immunity with a commercial ELISA-based interferon-γ release assay in kidney transplant recipients treated with antithymocyte globulin.

Monitoring for cytomegalovirus (CMV)-specific cell-mediated immunity (CMV-CMI) may be useful for individualizing valganciclovir (VGCV) prophylaxis after kidney transplantation (KT). We performed a commercial ELISA-based interferon (IFN)-γ release assay (QTF-CMV) from posttransplant months 2-5 (362 points) in 120 CMV-seropositive KT recipients that received antithymocyte globulin as induction therapy and VGCV prophylaxis (median of 92days). Forty-seven patients (39.3%) had CMV infection after discontinuation of prophylaxis. The QTF-CMV assay was reactive, nonreactive, and indeterminate in 264 (72.9%), 90 (24.9%), and 8 points (2.2%). The QTF-CMV assay at prophylaxis discontinuation exhibited suboptimal accuracy for predicting protective CMV-CMI (sensitivity: 77.4%; specificity: 34.3%; positive predictive value [PPV]: 64.1%; negative predictive value [NPV]: 50.0%), with no differences in 1-year CMV infection rates between patients with negative (nonreactive or indeterminate) or reactive results (45.8% vs 36.1%; P=.244). Specificity and PPV to predict protective CMV-CMI improved by elevating the IFN-γ cutoff value to 1.13IU/mL (65.7% and 71.4%) and 7.0IU/mL (85.7% and 76.2%), although NPVs decreased. The QTF-CMV assay as per manufacturer's interpretative criteria performed poorly to predict protection from CMV infection following discontinuation of VGCV prophylaxis among ATG-treated CMV-seropositive KT recipients. This performance is slightly improved by modifying the IFN-γ positivity threshold.