Plasma Dilution Research Articles

91-Year-Old Man With Upper Extremity Ecchymoses

91-Year-Old Man With Upper Extremity Ecchymoses

The role of critical micellization concentration in efficacy and toxicity of supramolecular polymers

The inception and development of supramolecular chemistry have provided a vast library of supramolecular structures and materials for improved practice of medicine. In the context of therapeutic delivery, while supramolecular nanostructures offer a wide variety of morphologies as drug carriers for optimized targeting and controlled release, concerns are often raised as to how their morphological stability and structural integrity impact their in vivo performance. After intravenous (i.v.) administration, the intrinsic reversible and dynamic feature of supramolecular assemblies may lead them to dissociate upon plasma dilution to a concentration below their critical micellization concentration (CMC). As such, CMC represents an important characteristic for supramolecular biomaterials design, but its pharmaceutical role remains elusive. Here, we report the design of a series of self-assembling prodrugs (SAPDs) that spontaneously associate in aqueous solution into supramolecular polymers (SPs) with varying CMCs. Two hydrophobic camptothecin (CPT) molecules were conjugated onto oligoethylene-glycol (OEG)-decorated segments with various OEG repeat numbers (2, 4, 6, 8). Our studies show that the lower the CMC, the lower the maximum tolerated dose (MTD) in rodents. When administrated at the same dosage of 10 mg/kg (CPT equivalent), SAPD 1, the one with the lowest CMC, shows the best efficacy in tumor suppression. These observations can be explained by the circulation and dissociation of SAPD SPs and the difference in molecular and supramolecular distribution between excretion and organ uptake. We believe these findings offer important insight into the role of supramolecular stability in determining their therapeutic index and in vivo efficacy.

Investigating surrogate cerebrospinal fluid matrix compositions for use in quantitative LC-MS analysis of therapeutic antibodies in the cerebrospinal fluid

As quantitative analysis of biotherapeutics in cerebrospinal fluid (CSF) with LC-MS becomes increasingly widespread, there is a need for method developments towards higher sensitivity. By using artificial CSF (aCSF) in the development phase, the consumption of costly and sparsely available CSF can be limited. The aCSF compositions tested here were made from various dilutions of bovine serum albumin (BSA) or rat plasma to mimic the total protein concentration found in CSF. Focusing on monoclonal antibodies, the aCSF was spiked with human immunoglobulin (hIgG) and prepared with the bottom-up analysis technique using LC-MS. Assuming that the composition of the aCSF would affect the digest, the response from aCSF matrices was compared with CSF from rat, monkey, and dog in terms of estimated sample concentration and matrix effects. The samples were spiked with hIgG in the range of 10 to 1000 ng/mL and volumes of 10 μL were transferred to sample preparation. The results indicate that BSA dilutions from 300 to 2000 μg/mL and rat plasma dilutions of 0.5–2% provide the most accurate concentration estimates when compared with rat CSF. 1000 μg/mL BSA did not produce significantly different concentration estimates for 500 ng/mL samples when compared with CSF from rat, monkey, and dog, and can therefore be used as aCSF for several different species.

224 Superstimulation effect on invitro embryo production and relationship with anti-Müllerian hormone in buffaloes (Bubalus bubalis) aspirated 7 or 14 days after follicular ablation

The success of buffalo invitro embryo production (IVEP) depends, in part, on the number and competence of oocytes. Superstimulation with FSH has been linked to oocyte quality and embryo yield, and anti-Müllerian hormone (AMH) has been proposed as an indicator of ovarian reserve. The objective was to evaluate FSH stimulation in one ovum pick-up (OPU) cycle conducted 7 or 14 days after follicular ablation; we additionally analysed residual FSH treatment effect carried over to a subsequent aspiration cycle. Finally, we evaluated the relationship between plasma AMH and FSH response. Nine multiparous, cycling Murrah donors (603±7.0kg, body condition score=3.5) were subjected to complete follicular ablation followed by OPU 1 week later (T0 Control), and plasma AMH was determined at this point. They were then randomly allocated to FSH stimulation (3 days b.i.d. in descending doses, total 300mg of pFSH, Vetpharm, with the last injection 48h before OPU) with aspiration conducted 7 (T7 FSH, n=5) or 14 days (T14 FSH, n=4) post-follicular ablation. The residual effect (T Residual, n=5) was evaluated in one subsequent, nonstimulated OPU cycle conducted 1 week later (9 days after last FSH administration). For plasma AMH, concentration was measured using ELISA kit (cat. no. AL114, AnshLabs; sensitivity 1.0 pgmL−1, interassay variation <5%, additional validation conducted with serial dilutions of buffalo plasma, r=0.98 against standard curve). Continuous data were analysed with ANOVA and Tukey's post hoc comparisons; categorical data were analysed using Chi-squared test. Pearson coefficient was used to evaluate correlation between AMH, follicular reserve, and invitro performance. Superovulatory treatment resulted in a higher number of follicles and good quality oocytes (grades 1 and 2) per donor, irrespective of OPU interval (T7 FSH: 3.0±2.55 and T14 FSH: 1.5±0.5 vs. T0 Control: 0.45±0.38 and T Residual: 0.4±0.4; P<0.05). There were more cleaved embryos (T7 FSH: 26%, T14 FSH; 30% vs. T0 Control: 7% and T Residual: 20%) and blastocysts (T7 FSH: 21%, T14 FSH; 30% vs. T0 Control: 5% and T Residual: 0; P<0.05) from FSH-treated donors. There was a strong correlation (r=0.9439) between follicle count and plasma AMH, and weaker relationships for oocytes recovered (r=0.3975), cleaved embryos (r=0.2317), or blastocysts (r=0.0106). Results indicate that FSH treatment with a 48-h coasting period results in significant improvements in follicular population, oocyte quality, and embryonic development; these effects are observed independently of the interval between initial follicular ablation and OPU. No residual effects of FSH treatment were observed, indicating that the beneficial effects of superstimulation are not carried over to the following OPU cycle. In conclusion, a strong correlation coefficient was established between initial plasma AMH level and follicular response to FSH treatment, indicating that it may be used as a selection criterion for animals likely to exhibit a good response to superstimulatory treatment.

Population-based volume kinetics of crystalloids and colloids in healthy volunteers

We characterized the volume kinetics of crystalloid solutions (Ringer’s lactate solution and 5% dextrose water) and colloid solutions (6% tetrastarch and 10% pentastarch) by nonlinear mixed-effects modeling in healthy volunteers. We also assessed whether the bioelectrical impedance analysis parameters are significant covariates for volume kinetic parameters. Twelve male volunteers were randomly allocated to four groups, and each group received the four fluid solutions in specified sequences, separated by 1-week intervals to avoid any carryover effects. Volunteers received 40 ml/kg Ringer’s lactate solution, 20 ml/kg 5% dextrose water, 1000 ml 6% tetrastarch, and 1000 ml 10% pentastarch over 1 h. Arterial blood samples were collected to measure the hemoglobin concentration at different time points. Bioelectrical impedance spectroscopy (BIS, INBODY S10, InBody CO., LTD, Seoul, Korea) was also carried out at preset time points. In total, 671 hemoglobin-derived plasma dilution data points were used to determine the volume kinetic characteristics of each fluid. The changes in plasma dilution induced by administration of crystalloid and colloid solutions were well-described by the two-volume and one-volume models, respectively. Extracellular water was a significant covariate for the peripheral volume of distribution at baseline in the volume kinetic model of Ringer’s lactate solution. When the same amount was administered, the colloid solutions had ~4 times more plasma expansion effect than did the crystalloid solutions. Starches with larger molecular weights maintained the volume expansion effect longer than those with smaller molecular weights.

Simultaneous voltammetric determination of free tryptophan, uric acid, xanthine and hypoxanthine in plasma and urine

We have designed an improved electrochemical analysis of clinical samples in bodily fluids. The method allows reliable identification of several coexisting substances (tryptophan, uric acid, xanthine and hypoxanthine) present on different physiological levels. We prepared in situ electrogenerated graphite oxides (GrO) directly on the surface of a graphite pencil electrode. GrO presence enhances electrooxidation signals of small aromatic molecules and can shift their oxidation potentials. The oxidation potentials of tryptophan and tyrosine in their free and protein-bound forms, and free xanthine are very close to each other on common carbon-based materials. We found that free tryptophan’s oxidation signal is significantly more enhanced (about 11-times) than the free tyrosine signal (only negligibly in the given conditions). Distinguishing xanthine’s oxidation signal from that of the free tryptophan is allowed by their approximately 60 mV separation, sufficient for parallel determination by signal deconvolution. Additionally, we show that proteins containing tyrosine and/or tryptophan behave as electro-inactive and do not interfere with free amino acid determination on the GrO-modified graphite electrodes, while they strongly influence all species’ detection on other carbon-based materials. Determining all individual compounds was possible at corresponding physiological values after sole dilution of human plasma (urine) and direct measurement with no pre-separation steps.

P1838Thrombocytopenia under ECMO and Shear-induced shedding of platelet receptor Glycoprotein-(GP)Ialpha

Abstract Background Several mechanisms are suspected to thrombocytopenia under Extracorporeal Membrane Oxygenation (ECMO) such as platelet-consumption or sepsis. Shedding of glycoprotein-(GP)Ibα is a recently identified mechanism of platelet clearance. ECMO generates high shear stress forces that could impact GPIbα-shedding. We hypothesized that ECMO continuous-flow devices could directly induce thrombocytopenia through shear-induced GPIbα-shedding. Aims Determine if ECMO induce GpIb-shedding in vitro and in vivo and determinates the kinetic evolution of platelet-count and GpIb-shedding after patient's implantation. Methods Platelet GPIbα-shedding was first investigated in vitro using a high-shear pump loop model. Plasma with normal platelet count (plasma-NPC) was obtained by dilution of platelet-rich plasma obtained from healthy donors in fresh-frozen-plasma. Samples were collected before and after (5, 30, 60 and 180 min) perfusion at 37°C of plasma-NPC at intermediate and high speed (2.6 and 3.6 L min–1 respectively, n=4 each). Platelet count and GPIbα-shedding were next investigated in 20 ECMO patients before/after implantation (WITECMO trial) and in 20 healthy volunteers. The geometric mean-fluorescence-intensity (gMFI) of platelet GPIbα (PE-staining) and GPIX (FITC-staining) was measured with a Navios flow cytometer (Beckman Coulter, Miami, FL). Results are expressed as GPIbα/GPIX gMFI-ratio. Results A significant time-dependent loss of GPIbα/GPIX gMFI-ratio was already apparent after 30 min in vitro and was significantly more pronounced at high-speed compared to intermediate-speed (pANOVA<0.001 and p<0.01 at 180 min respectively). GPIbα/GPIX gMFI-ratio was significantly increased in ECMO patients compared to healthy subjects 1- and 24-hour after implantation (p<0.001). A significantly lower platelet count was observed 1 hour after ECMO implantation (−23% vs baseline, p<0.01) with a further significant decrease at 24-hours (−53% vs baseline, p<0.0001). Figure 1. A. Significant time-dependent loss of platelet GPIbα/GPIX gMFI-ratio (pANOVA <0.001) assessed by flow-cytometry after 30 min of perfusion at 3.6 L/min with a high-shear continuous-flow device in vitro. B. Representative experiment showing the apparition of a platelet sub-population with loss of GPIbα expression after 30 min of perfusion at 3.6 L/minwith a high-shear continuous-flow device in vitro.

Bioinspired nanoplatform for enhanced delivery efficiency of doxorubicin into nucleus with fast endocytosis, lysosomal pH-triggered drug release, and reduced efflux

A novel bioinspired nanoplatform capable of fast endocytosis, lysosomal pH-triggered drug release, and reduced drug efflux based on PBA-PEG-b-P(Glu-co-GluDA) copolymer was developed in this study. The synthesized copolymer could facilitate doxorubicin encapsulation with relatively high drug-loading content and efficiency. Inspired by mussel byssal threads, a core crosslinking strategy based on the coordination between catechol and ferric ions was introduced to improve the stability of nanomicelles and realize lysosomal pH-controlled drug release. This nanoplatform could maintain integrity even after being dissolved in a good solvent, demonstrating its the potential to withstand infinite dilution of plasma after intravenous injection. Moreover, this nanoplatform demonstrated lysosomal pH-triggered drug release, and the cumulative release amount of doxorubicin under a simulated lysosomal condition was 13 times higher than that under a simulated plasma condition. Moreover, as a result of the high binding capacity between phenylboronic acid (PBA) and sialic acid on the surface of human hepatoma cell line (HepG2), the fast and enhanced endocytosis in addition to lysosomal pH-triggered release property and significantly low efflux, this nanoplatform exhibits improved delivery efficiency of doxorubicin into the nucleus and notably outstanding antiproliferative effects compared with doxorubicin. Furthermore, the PBA modification remarkably increased the mean fluorescence intensity of this nanoplatform endocytosed by HepG2 cells to twice that of doxorubicin after one hour of incubation. The nanoplatform exhibited an inhibition rate of 70% against tumor growth. Thus, this novel nanoplatform based on PBA-PEG-b-P(Glu-co-GluDA) copolymer displayed multifunctionality and exhibited great potential as an intelligent nanoplatform for antitumor drug delivery.

High performance double-null plasmas under radiating divertor and mantle scenarios on DIII-D

Experiments to combine a radiating divertor or mantle with high power, high βN plasma operation have successfully reduced the divertor heat flux by 40% but have also encountered several challenging problems. For example, injecting either neon or argon seed impurities have led both to significant fuel dilution of the core plasma and to the emergence of harmful tearing mode activity which compromised plasma beta and energy confinement time. Increased divertor closure resulted in more effective control over the injected seed impurity inventory, although it was also observed that small radial variations in the placement of the outer separatrix strike point within the DIII-D ‘closed’ divertor could lead to clear differences in the impurity build-up inside the core plasma. Active particle pumping of argon seed impurities through the divertor leg on the high-field side of a double-null divertor configuration showed little effect on seed impurity inventory and only modest control over the deuterium fuel inventory. Typically, 80% or greater of injected argon was removed by the corresponding cryo-pump on the low-field (outboard) side of that divertor, with the remaining argon being removed by the outboard cryo-pump from the opposite divertor. For single-null divertor cases, the contribution of the inner (high-field) cryo-pump to impurity and main-ion control appears to be complicated by the levels of divertor recycling and degree of divertor detachment. In general, divertor design that impedes the escape of seed impurities, plasma shaping that allows a higher stability limit, and electron cyclotron deposition profiles that prevent tearing mode onset and impede impurity accumulation in the core are under consideration for addressing the issues presented in this paper.

Short Communication: Effect of Seminal Plasma on Functions of Monocytes and Granulocytes.

Most HIV-1 transmissions occur at mucosae and involve exposure to semen. Semen contains immunomodulatory factors, which inhibit anti-HIV-1 natural killer cell and T cell responses. We demonstrate high concentrations (1:2 dilution) of seminal plasma (SP) inhibit monocyte phagocytosis and anti-HIV-1 Fc-dependent functions of both neutrophils and monocytes. In addition, slightly lower SP concentrations (1:2-1:10 dilutions) inhibit granulocyte phagocytosis and oxidative burst of both monocytes and granulocytes. These observations may have implications for HIV-1 infectivity after mucosal exposure.

Effects of pH and the plasma or serum concentrations of total calcium, chloride, magnesium, l-lactate, and albumin on the plasma ionized calcium concentration in calves.

BackgroundThe plasma ionized calcium concentration (cCa2+) represents the biologically active form of calcium and is the preferred method for evaluating calcium status in animals. Different pH‐corrective equations have been developed for human plasma, but the validity of the equations for bovine plasma is unknown.HypothesisWe hypothesized that pH‐corrective equations for bovine plasma would be similar to those used for human plasma; cCa2+ was dependent on the plasma concentrations of total calcium (cTCa), chloride (cCl), L‐lactate (cLactate), and albumin (cAlbumin); and the in vitro and in vivo cCa2+‐pH relationships would differ.AnimalsTen healthy calves (in vitro study), 1426 critically ill calves.MethodsThe in vitro plasma log10(cCa2+)‐pH relationship was determined by CO2 tonometry of 465 plasma samples. Plasma cCl was altered by equivolume dilution of plasma with 3 electrolyte solutions of different cCl. The in vivo plasma cCa2+‐pH relationship was investigated and validated using clinicopathologic data extracted from the medical records of 950 (model development) and 476 (model validation) critically ill calves.ResultspH‐corrective equations for bovine plasma were similar to those used for human plasma. Plasma cCa2+ increased in vitro with increases in plasma cCl. Plasma cCa2+ in critically ill calves was associated with plasma cTCa, blood pH, plasma cCl, serum cMg, and cL‐lactate (R 2 = 0.69) but not plasma cAlbumin.Conclusions and Clinical ImportanceCalculation of cCa2+ from cTCa in calf plasma or serum requires adjustment for at least pH and cCl when 1 or both are outside the reference range.

Integrated power exhaust modelling for DEMO with lithium divertor

A DEMO reactor with a liquid lithium divertor setup is considered. The simulation is performed for EU DEMO 2018 configuration using COREDIV code which self-consistently solves core and scrape-off layer transport equations for plasma and impurity. Influence of the sputtering, prompt redeposition and evaporation of the liquid lithium divertor was taken into account. Two operation regimes were identified. In the first regime evaporation rate is suppressed by the cooling system and is of the order of the sputtering rate. Scrape-off layer plasma characterises with low radiation and high total power to the plates. In the second regime the evaporation rate is much higher than the sputtering rate. High Li influx reduces temperature in the divertor region. Lithium radiation is increased reducing power to the plates. Plasma parameters in the core are comparable for the two regimes and the fusion gain parameter Q drops only slightly. Both regimes characterise with high plasma dilution. Power across the separatrix stays above the L-H threshold.

Detection of Low-Abundant MicroRNAs with Hybridization Microchips.

The effect of low concentrations of miRNA on the ability of GeneChip miRNA 4.0 hybridization chips to evaluate their representation in the sample was studied. It is shown that the evaluation of the expression of 61 miRNAs is statistically significantly associated with the multiplicity of plasma dilution. Only 12 miRNAs showed very high Pearson correlation coefficient (>0.95) and they all decreased in response to dilution. High abundance of has-miR-4532 miRNA in plasma was demonstrated. This miRNA was never detected during sequencing of similar samples. It was concluded that in case of miRNA expression <1.12±0.33 units in log2 scale, dilution was not followed by further decrease in the signal intensity in GeneChip miRNA 4.0 chips.

Analyses of the influence of the recycling coefficient on He confinement in DEMO reactor

This work describes integrated numerical modelling applied to DEMO discharges with tungsten wall and divertor, using the COREDIV code, which self-consistently solves 1D radial transport equations of plasma and impurities in the core region and 2D multi-fluid transport in the SOL. The model is self-consistent with respect to both the effects of impurities on the α-power level and the interaction between seeded (Ar) and intrinsic impurities (tungsten, helium). This work is to analyse the influence of the helium and hydrogen recycling on the He confinement time in DEMO reactor. Simulations have been done for argon seeding puff 5.0 × 10211/s. It is found that recycling coefficient of helium have strong influence on the He confinement time, which increases from 7.25 s to 26.9 s going from lowest to highest recycling coefficient, but it has small influence on the effective charge state and radiation in the SOL. Alpha power decreases only by about 20%, which is the effect of main plasma dilution.

Continuous-flow mechanical circulatory support induces shedding of platelet adhesion receptors GpIb and GpVI

Hemorrhagic complications, especially mucosal bleedings, are very common for using continuous-flow mechanical circulatory supports (CF-MCS) including Extracorporeal Membrane Oxygenation (ECMO). CF-MCS could impact platelets by exposing this key player in primary hemostasis to high shear stress. A proteolytic cleavage (shedding) of platelet adhesion receptors GpIb and GpVI has been described under high shear stress and could contribute to the hemorrhagic complications in patients with CF-MCS. To assess whether CF-MCS promotes GpIb and GpVI shedding in vitro and in vivo and determine the kinetic evolution during the CF-MCS. First platelet shedding was investigated in vitro using a CF-MCS (Impella-CP®, Abiomed) loop model. Plasma with normal platelet count (plasma-NPC) was obtained by dilution of platelet-rich plasma collected from healthy donors in fresh frozen plasma. Sampling was performed before and after 2, 5, 30, 60 and 180 min perfusion of plasma-NPC in the loop model ( n = 4 runs). Platelet shedding was also investigated for the blood samples collected from 20 ECMO patients (WITECMO-h trial). Platelets of 20 healthy volunteers were evaluated as a control. GpIbα and GpVI shedding was analyzed by flow cytometry. A significant time-dependent decrease of GpIbα ( P < 0.001) and GpVI ( P < 0.001) MFI was observed after 180 min in the Impella®-CP loop model. In vivo baseline GpIbα and GpVI MFIs has been significantly decreased before ECMO implantation compared to the healthy subjects ( P < 0.001). After ECMO implantation, no significant variation of GpIbα and GpVI MFI was observed. CF-MCS induces platelet GpIb and GpVI shedding in vitro. Increased GpIb and GpVI shedding is already present before ECMO implantation and remain significantly elevated after the implantation. Loss of the platelet receptors GPIbα and GPVI in patients with CF-MCS may contribute to hemorrhagic complications observed in these patients.

Abstract P285: Development of a Simplified Method for the Measurement of Plasma Alkylresorcinols as a Biomarker of Whole Grain Intake and Application to a Human Clinical Trial Evaluating the Effect of Carbohydrate Quality on Cardiometabolic Risk Factors

Introduction: Consumption of whole grains is associated with improvements in cardiometabolic risk factors and decreased CVD risk. Quantification of whole grain intake is challenging due to the limitations of self-reported intake; diversity among and differences in the interpretation of the term “whole grain”. Alkylresorcinols (ARs) are phenolic lipids present in the outer layer of wheat and rye grains that are considered objective biomarkers of whole grain intake. Current methods for ARs quantification involve a multi-step separation, extraction and purification processes that hampers applicability in large-scale studies. Hypothesis: Our aim was to develop a single-step method to measure 5 predominant ARs (C17:0, C19:0, C21:0, C23:0 and C25:0) in human plasma, and to validate this method by measuring plasma ARs levels in subjects who participated in a randomized, cross-over trial evaluating the effect of carbohydrate quality on CVD risk factors. We hypothesized that the direct method would distinguish between low- and high-whole grain intake and be more rapid and cost effective than prior methods. Methods: A dilute-and-shoot strategy based on plasma (20 μL) dilution with methanol for protein precipitation, followed by centrifugation and direct injection into an ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometer (UHPLC/Q-TOF-MS; Agilent Technologies), using negative electrospray ionization. C19:0-D 4 was used as internal standard. Separation was performed using a C18 column. Run time was 11 minutes/sample. Method validation was based on the following criteria: linearity, working range, lowest limit of quantification (LOQ), accuracy and precision. This method was then used to quantify ARs in fasting plasma samples from postmenopausal women and men (N=11, 65±8 years, BMI 29.8±3.2 kg/m 2 , LDL-C ≥2.6 mmol/L) who consumed each of 3 isocaloric diets (60%E carbohydrate, 15%E protein, 25%E fat) enriched in either simple, refined, or unrefined carbohydrate-containing foods for 4.5 weeks in a randomized crossover design, with 2-week washout periods. Results: (1) Analytical: The method showed linearity (&gt;0.999) in the range 2 to 100 ng/mL, and had acceptable values for accuracy and precision with a LOQ of 2 ng/mL. (2) Applicability: Total fiber content of the simple, refined and unrefined carbohydrate diets was 8.6, 9.6 and 19.5g/1000kcal, respectively. This was reflected in plasma AR levels, being significantly higher (p&lt;0.05) after subjects consumed the unrefined (124.8±55.8 pmol/mL) compared to the simple (29.5±10.3 pmol/mL) and refined (26.8±9.0 pmol/mL) diets. C21:0 and C19:0 were the major ARs present in plasma. Conclusion: This simplified method offers a direct and rapid strategy to accurately measure AR in human plasma that can be scaled up for large studies, and provides an objective assessment of whole grain intake.

Rapid measurement of hydrogen sulphide in human blood plasma using a microfluidic method

Hydrogen sulfide (H2S) is emerging as an important gasotransmitter in both physiological and pathological states. Rapid measurement of H2S remains a challenge. We report a microfluidic method for rapid measurement of sulphide in blood plasma using Dansyl-Azide, a fluorescence (FL) based probe. We have measured known quantities of externally added (exogenous) H2S to both buffer and human blood plasma. Surprisingly, a decrease in FL intensity with increase in exogenous sulphide concentration in plasma was observed which is attributed to the interaction between the proteins and sulphide present in plasma underpinning our observation. The effects of mixing and incubation time, pH, and dilution of plasma on the FL intensity is studied which revealed that the FL assay required a mixing time of 2 min, incubation time of 5 min, a pH of 7.1 and performing the test within 10 min of sampling; these together constitute the optimal parameters at room temperature. A linear correlation (with R2 ≥ 0.95) and an excellent match was obtained when a comparison was done between the proposed microfluidic and conventional spectrofluorometric methods for known concentrations of H2S (range 0–100 µM). We have measured the baseline level of endogenous H2S in healthy volunteers which was found to lie in the range of 70 μM – 125 μM. The proposed microfluidic device with DNS-Az probe enables rapid and accurate estimation of a key gasotransmitter H2S in plasma in conditions closely mimicking real time clinical setting. The availability of this device as at the point of care, will help in understanding the role of H2S in health and disease.

Development and validation of LC-MS/MS methods for the quantification of the novel anticancer agent guadecitabine and its active metabolite β‑decitabine in human plasma, whole blood and urine

Guadecitabine (SGI-110), a dinucleotide of β‑decitabine and deoxyguanosine, is currently being evaluated in phase II/III clinical trials for the treatment of hematological malignancies and solid tumors. This article describes the development and validation of bioanalytical assays to quantify guadecitabine and its active metabolite β‑decitabine in human plasma, whole blood and urine using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Since β‑decitabine is rapidly metabolized further by cytidine deaminase, plasma and whole blood samples were kept on ice-water after collection and stabilized with tetrahydrouridine (THU) directly upon sample collection. Sample preparation consisted of protein precipitation for plasma and whole blood and dilution for urine samples and was further optimized for each matrix and analyte separately. Final extracts were injected onto a C6-phenyl column for guadecitabine analysis, or a Nova-Pak Silica column for β‑decitabine analysis. Gradient elution was applied for both analytes using the same eluents for each assay and detection was performed on triple quadrupole mass spectrometers operating in the positive ion mode (Sciex QTRAP 5500 and QTRAP 6500). The assay for guadecitabine was linear over a range of 1.0–200 ng/mL (plasma, whole blood) and 10–2000 ng/mL (urine). For β‑decitabine the assay was linear over a range of 0.5–100 ng/mL (plasma, whole blood) and 5–1000 ng/mL (urine). The presented methods were successfully validated according to the latest FDA and EMA guidelines for bioanalytical method validation and applied in a guadecitabine clinical mass balance trial in patients with advanced cancer.

Long-term preservation of echinoderm sperm under non-cryo condition for ecotoxicological bioassay

The preservation of sea urchin gametes was examined to make them more convenient material for research and education, especially on the embryology and ecotoxicology field. The possibility of egg preservation for enough period, two weeks to one month, were reported (Epel et al., 2004; Kiyomoto et al., 2014). The sperm storage is usually done without seawater (dry sperm) for several days. This storage period is lengthened by the addition of antibiotics up to 10 days (Hata, 1998). To maximize the preserved period, we examined the dilution or replacement of seminal plasma with seawater containing antibiotics. Because the activation of sperm is induced by dilution, the condition to inhibit flagella motility was also investigated. Neither high potassium nor low pH improved the period of sperm preservation. The dilution of dry sperm around 100 times with normal seawater containing antibiotics was enough to keep the motility and fertilization ability for longer period, where sperm motility was prevented possibly by the effect of carbon dioxide.

An establishment of the procedures to eliminate incorrect blood due to non-matched or mislabeled tubes

This study aims to establish a set of procedures to eliminate incorrect blood due to non-matched tubes or mislabeled tubes. The errors such as these are suspected to have occurred when upon first and second-detection. Under the identical laboratory conditions, the results of re-detection of blood bag samples and rare type antigen samples, as well as re-collected samples from donor, and plasma diluted are not consistent with the original results. Here, 20 antigen erythrocytes were detected for blood bag samples and original samples, in which no incorrect blood was mistakenly administered or mislabeled. Samples taken under identical laboratory conditions were not found to have incorrect blood administered to a non-matched tube. The results showed nonlinear changes by HBsAg ELSIA after plasma dilution. The study suggests that the second-detection taken shortly after first detection is the most appropriate method to detect errors at the earliest time point. Blood bag samples are identical to those of the original antigen identification group, which means that the probability of the samples coming from the same donor is extremely highly. At same time, space and plasma diluted, re-detection can effectively exclude incorrect blood being added to a non-matched tube.