Plasma Extracts Research Articles

Abstract PO2-18-11: Short-term Pre-OPerative Durvalumab (MEDI 4736) in early small triple negative breast cancer patients (POP-Durva)

Abstract Background: Immune checkpoint inhibitors (ICIs) are one of the major therapeutic advancements in cancer treatment. Anti-programmed cell death protein 1 (Anti-PD1)/PD-L1 ICIs have improved progression-free survival in patients with metastatic triple negative breast cancer (TNBC) and pathologic complete response (pCR) and event-free survival in patients with early TNBC. Nevertheless, some patients treated with anti-PD(L)1 ICIs experience recurrence or do not achieve sustained clinical benefit. In addition, very interesting data show the existence of a subgroup of patients with exceptional tumour responses to monotherapy with anti-PD(L)1 ICIs. These exceptional responses are observed in various tumour types, such as colorectal or breast cancer. Although in colorectal cancer, the determinants for this extreme sensitivity to immunotherapy treatment are identified (existence of MSI) in breast cancer, this is not the case. There is still a lack of knowledge about predictive biomarkers and mechanisms of action for ICIs. Trial Design: POP-Durva (NCT05215106) is a prospective, one-arm-only study aiming at determining the pCR rate after two administrations of Durvalumab monotherapy in patients with stage I TNBC. This window-of-opportunity study will recruit 195 consecutive cases of stage I TNBC (ER < 1%, PR < 1%, HER2 negative) and TILs≥5%, eligible for short-term treatment with durvalumab. Study treatment consists of two administrations of intravenous Durvalumab monotherapy, 10mg/kg, at two weeks intervals. After study treatment, patients will receive a standard treatment strategy (surgery or neoadjuvant systemic treatment) as per physician choice. The primary endpoint is the pCR rate after treatment with Durvalumab, defined as the absence of invasive disease in the breast and negative axillary nodes (ypT0/yTis ypN0). Assessment of the primary endpoint will be performed at the surgery or at the biopsy at the end of treatment) for patients undergoing neoadjuvant treatment. For patients in whom neo-adjuvant therapy is the first standard treatment strategy (i.e. after study treatment) a breast ultrasound-guided biopsy is mandatory at the end of the treatment visit. If the biopsy-proven residual disease is demonstrated, patients can receive standard neoadjuvant therapy at the discretion of the treating investigator. Patients with biopsy-proven residual disease will be considered as having no pCR. Those with a complete response may proceed directly to surgery. The expected pCR rate with Durvalumab monotherapy is 20%. The sample size of 195 patients will allow us to estimate this expected pCR rate with a 95% confidence interval of a precision of 6.2%. Secondary objectives are objective response rate and safety. Exploratory objectives are to: a. describe immune cell dynamics associated with exceptional responses to ICI (using spectral cytometry analysis), b. describe somatic genetic contributions to the determination of immune responsiveness (using whole exome sequencing (WES) and in a subset of patient's single-cell RNA-seq), c. characterize tumour cells – immune cells' interaction/spatial distribution(using imaging mass cytometry), d. explore the association between gut microbiome composition/signatures predictive of response to ICI (using 16s rRNA sequencing) and e. identify predictive tissue/blood-based biomarkers of response and to anti-PD(L)1 ICIs therapy. A total of two dedicated FFPE samples and two fresh biopsies will be collected at the time of inclusion and at the end of treatment biopsy or on the surgical specimen. In addition, we will collect stool samples pre and at the completion of all Durvalumab treatments and blood samples at the time of inclusion, during treatment (before each Durvalumab administration) and at the end of treatment for serum and plasma extraction and whole blood to serve as a control for WES. Citation Format: Joana Mourato Ribeiro, Isabelle Pic, Quentin Blampey, Elie Rassy, Nusaibah Ibrahimi, Laura Salabert, Olivier Trédan, Monica Arnedos, Semih Dogan, Kamar Serhal Serhal, Clementine Mahaut, Alessandro Viansone, Salim Laghouati, Barbara Pistilli, Charles-Antoine Dutertre, Magali Lacroix-Triki, Jean-Yves Scoazec, Lisa Derosa, Laurence Zitvogel, Corinne Balleyguier, Nadege Bercovici, Paul-Henry Cournede, Stefan Michiels, Fabrice André. Short-term Pre-OPerative Durvalumab (MEDI 4736) in early small triple negative breast cancer patients (POP-Durva) [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-18-11.

Microsampling Techniques Suitable for Therapeutic Drug Monitoring of Antipsychotics.

Therapeutic drug monitoring (TDM) of antipsychotics for dose titration or detection of noncompliance is not uncommon in daily practice. Normally, TDM implies measuring a drug concentration in venous blood samples. This technique is invasive and requires trained assistants and patients normally need to go to an outpatient clinic. Over the past decades, sensitivity of analytical equipment has improved leading to a growing interest in microsampling techniques. These techniques are minimally invasive, require a small volume (<100 μL), usually result in stable samples, and can be collected by the patient or a caregiver at home. Before a microsampling technique can be used in daily routine, proper method development and a clinical validation study should be performed. For this review, the databases of PubMed and Embase were systematically searched. Currently available microsampling techniques for antipsychotics in blood, serum, or plasma are summarized. Subsequently, it has also been assessed whether these techniques are sufficiently validated for TDM monitoring in daily practice. Several microsampling techniques are available today, for example, dried blood spot sampling, dried plasma extraction cards, and volumetric absorptive microsampling. Eighteen studies were identified in which a microsampling technique for 1 or a few antipsychotics was chemically analytically and clinically validated. However, the majority of these studies have relevant shortcomings that mean its usefulness for different antipsychotics is not yet well established. Microsampling for TDM can be recommended for patients using clozapine. For TDM of other antipsychotics, it is a very promising development.

Simultaneous determination of five constituents of areca nut extract in rat plasma using UPLC-MS/MS and its application in a pharmacokinetic study

Areca nuts have been used as a traditional Chinese medicine (TCM) for thousands of years. Recent studies have shown that it exhibits good pharmacological activity and toxicity. In this study, the pharmacokinetics of five major components of areca nut extract in rats were investigated using a highly sensitive ultra-performance liquid chromatography coupled with triple quadrupole mass spectrometry (UPLC-MS/MS) method. Arecoline, arecaidine, guvacoline, guvacine, and catechin were separated and quantified accurately using gradient elution with mobile phases of (A) water containing 0.1 % formic acid-10 mM ammonium formate, and (B) methanol. The constituents were detected under a timing switch between the positive and negative ion modes using multiple reaction monitoring (MRM). Each calibration curve had a high R2 value of >0.99. The method accuracies ranged −7.09–11.05 % and precision values were less than 14.36 %. The recovery, matrix effect, selectivity, stability, and carry-over of the method were in accordance with the relevant requirements. It was successfully applied for the investigation of the pharmacokinetics of these five constituents after oral administration of areca nut extract. Pharmacokinetic results indirectly indicated a metabolic relationship between the four areca nut alkaloids in rats. For further clarification of its pharmacodynamic basis, this study provided a theoretical reference.

Application of enzyme-linked immunosorbent assay in cancer detection

The enzyme-linked immunosorbent assay (ELISA) has emerged as an indispensable method in the realm of cancer detection., serving as a significant factor in the timely identification, prediction, and surveillance of many forms of cancer. ELISA as a highly sensitive immunological assay, utilizes the binding affinity between antibodies and antigens to detect and quantify cancer-associated biomolecules, such as tumor markers, oncogenic proteins, and specific antibodies. The recently adopted methodology facilitates the identification of cancer-associated biomarkers in various types of samples, such as plasma, urine, and tissue extracts. In the context of cancer diagnosis, ELISA aids in the early detection of malignancies by detecting cancer-specific biomarkers at low concentrations, allowing for timely intervention and improved patient outcomes. This research provides a concise overview of the multifaceted applications of ELISA in the field of oncology, especially in electrochemical ELISA and gold nanoparticle–based ELISA. It discusses the diverse biomarkers employed, and elucidates its significance in cancer research and clinical practice.

Utilizing droplet digital polymerase chain reaction for siRNA quantitation in rodent plasma and tissue via stem-loop reverse transcription.

Background: siRNA is a promising therapeutic modality highlighted by several US FDA approvals since 2018, with many more oligonucleotide assetsin clinical development. To support siRNA discovery and development, robust and sensitive quantitative platforms for bioanalysis must be established to assess pharmacokinetic/pharmacodynamic relationships and toxicology. Droplet digital PCR offers improved sensitivity and throughput, as well as reduced susceptibility to matrix effects, compared with other analytical platforms. Methodology: The authors developed a stem-loop reverse transcription droplet digital PCR method to measure siRNA in mouse plasma and liver extract using bioanalytical method qualification guidelines. Conclusion: This newly developed assay has been demonstrated to be a superior alternative to other platforms, with the added benefit of greater sensitivity, with dynamic range from 390 to 400,000 copies/reaction and readiness for FDA investigational new drug-enabling applications.

Development and Validation of Bioanalytical Method for Estimation of Rivaroxaban using RP-HPLC with Liquid liquid extraction in Human Blood Plasma and its application in Bioequivalence Study

Rivaroxaban is andirect acting oralanticoagulant and factor Xa inhibitor. A simple, selective, precise and rapid RP-HPLC method for estimation of Rivaroxaban (RIVA) in human blood plasma was developed and validated. The sample spike in plasma was extracted using liquid liquid extraction were extracted with the organic solvent ethyl acetate as organic solvent. Apixaban as an internal standard. The compounds were analysed by Agilent HPLC was used with control panel software using UV detector on a Inertsil ODS (250mm x 4.6mm ID;5μ) column with an Flow rate of 1.2mL/min, an isocratic mobile phase consisting of 0.02M Ammonium acetate buffer: Acetonitrile (70:30%v/v). Different sample pre-treatment techniques were evaluated, but Liquid Liquid extraction was found to be satisfactory, with good recovery values of 93.70% for RIVA. The developed method is validated by ICHM10 and USFDA guidelines over the concentration range of 5.00 to 200.00 ng/ml in human blood plasma with R² =0.9993. Within-day precisions and accuracy for RIVA were found in 0.36% to 4.73% and 92.58% to101.82% respectively. The validated RP-HPLC method has been used successfully for both preliminary pharmacokinetic studies and therapeutic drug monitoring

High-Throughput Microfluidic Extraction of Platelet-free Plasma for MicroRNA and Extracellular Vesicle Analysis.

Cell-free RNAs and extracellular vesicles (EVs) are valuable biomarkers in liquid biopsies, but they are prone to preanalytical variabilities such as nonstandardized centrifugation or ex vivo blood degradation. Herein, we report a high-throughput and label-free inertial microfluidic device (ExoArc) for isolation of platelet-free plasma from blood for RNA and EV analysis. Unlike conventional inertial microfluidic devices widely used for cell sorting, a submicrometer size cutoff (500 nm) was achieved which completely removed all leukocytes, RBCs, platelets, and cellular debris based on differential lateral migration induced by Dean vortices. The single-step operation also reduced platelet-associated miRNAs (∼2-fold) compared to centrifugation. We clinically validated ExoArc for plasma miRNA profiling (39 samples) and identified a 7-miRNA panel that detects non-small cell lung cancer with ∼90% sensitivity. ExoArc was also coupled with size exclusion chromatography (SEC) to isolate EVs within 50 min with ∼10-fold higher yield than ultracentrifugation. As a proof-of-concept for EV-based transcriptomics analysis, we performed miRNA analysis in healthy and type 2 diabetes mellitus (T2DM) subjects (n = 3 per group) by coupling ExoArc and ExoArc+SEC with quantitative polymerase chain reaction (RT-qPCR) assay. Among 293 miRNAs detected, plasmas and EVs showed distinct differentially expressed miRNAs in T2DM subjects. We further demonstrated automated in-line EV sorting from low volume culture media for continuous EV monitoring. Overall, the developed ExoArc offers a convenient centrifugation-free workflow to automate plasma and EV isolation for point-of-care diagnostics and quality control in EV manufacturing.

Uniformity optimization of ion beam sputtering coating equipment based on strong current ion source

The widespread application of ion beam sputtering coating, especially in optical devices, requires the improvement of beam current intensity and uniformity of large-area uniform coatings. The advent of high current Penning sources offers a potential solution. This study introduces an automated optimization simulation method based on a three-electrode extraction system to investigate its influence on ion beam quality and uniformity. Focusing on high current intensity and uniformity, our simulation explores the effects of plasma electrode, inhibition electrode, and extraction electrode angles and distances on ion beam performance. Evaluation metrics include average beam intensity density, average energy of a single particle, and reciprocal variance of each macro particle position, which are achieved through normalization functions, allowing comprehensive comparison of simulation results. To assess coating efficiency, we estimate sputtering yield and depth. The study identifies patterns among electrodes and emphasizes the influence of different ion ratios on beam extraction. The results indicate that optimizing the angle of the plasma electrode and the distance of the suppressed electrode yields a highly uniform ion beam for low charge ions. However, for highly charged ions, similar optimization will reduce the current strength, so compensation needs to be achieved through electrode shape optimization. This research provides a model for systematically optimizing the three-electrode extraction system, guiding researchers in achieving optimal solutions based on ion source characteristics and application requirements. Additionally, we introduce a method of estimating the sputtering depth of mixed ion beams. This study provides valuable insights for advancing ion beam sputtering coating technology and reference for making the decision on design and application of ion source.

Stability Indicating an LC-MS/MS Method Development and Validation for the Quantification of Capmatinib in Human Plasma

A linear and accurate liquid chromatographic tandem mass spectrometric (LC-MS/MS) technique for the quantitation of capmatinib in plasma was developed and subjected for validation. Canagliflogin was employed as an internal standard (IS). Extraction of plasma was executed utilizing 5 mL of ethyl acetate solvent. Analysis was performed Zorbax ODS (50 mm × 2.1 mm x 3 μ) stationary phase at room conditions and a movable solvent composition of 0.1% HCOOH, acetonitrile and methyl alcohol (15:50:35). The flowing rate of the movable phase was 0.4 mL/min. Drug and IS were identified in positive mode of ionization with electrospray mode to get the mass transitions of (m/z): Capmatinib, 413.15/386.14 and canagliflogin (IS), 445.15/267.12. The correlation between capmatinib concentrations and their respective peak proportions to canagliflogin was a straight line over 0.2 to 3200 ng/mL concentration levels. Intra day and inter-day precisions were ≤5.18% for capmatinib. Inter and intra day bias were within the range of −4.17 to 4.53%. The mean measured extraction recovery of capmatinib was 99.23%. Recovery of IS was 98.31%. Capmatinib was subjected for long-term, freeze thaw, bench top, short-term stability, auto-sampler, dry extract, and stock solution stability at low QC and high QC levels and it was stable at all these conditions. The established technique can be utilized to regularly quantitfy of capmatinib in plasma samples in industries, forensic labs, and clinical research organizations.

Influence of extraction voltage on electron and ion behavior characteristics

The characteristics of the extracted ion current have a significant impact on the design and testing of ion source performance. In this paper, a 2D in space and 3D in velocity space particle in cell (2D3V PIC) method is utilized to simulate plasma motion and ion extraction characteristics under various initial plasma velocity distributions and extraction voltages in a Cartesian coordinate system. The plasma density is of the order of 1015 m−3–1016 m−3 and the extraction voltage is of the order of 100 V–1000 V. The study investigates the impact of various extraction voltages on the velocity and density distributions of electrons and positive ions, and analyzes the influence of different initial plasma velocity distributions on the extraction current. The simulation results reveal that the main reason for the variation of extraction current is the space-charge force formed by the relative aggregation of positive and negative net charges. This lays the foundation for a deeper understanding of extraction beam characteristics.

Clinical pharmacokinetic evaluation of Withania somnifera (L.) Dunal root extract in healthy human volunteers: A non-randomized, single dose study utilizing UHPLC-MS/MS analysis

Ethnopharmacological relevanceWithania somnifera (L.) Dunal; (Solanaceae), commonly known as Ashwagandha, is one of the most significant medicinal herbs in ‘Ayurveda’, a traditional Indian medicine used for centuries with evidence in scriptures. Ashwagandha was mentioned in old Ayurvedic medical literature such as Charaka Samhita and Sushruta Samhita for improving weight and strength, with multiple citations for internal and exterior usage in emaciation and nourishing the body. Ethnopharmacological evidence revealed that it was used to relieve inflammation, reduce abdominal swelling, as a mild purgative, and treat swollen glands. The root was regarded as a tonic, aphrodisiac, and emmenagogue in the Unani tradition of the Indian medicinal system. Further, Ashwagandha has been also described as an Ayurvedic medicinal plant in the Ayurvedic Pharmacopoeia of India extending informed therapeutic usage and formulations. Despite the widespread ethnopharmacological usage of Ashwagandha, clinical pharmacokinetic parameters are lacking in the literature; hence, the findings of this study will be relevant for calculating doses for future clinical evaluations of Ashwagandha root extract. AimThis study aimed to develop a validated and highly sensitive bioanalytical method for quantifying withanosides and withanolides of the Ashwagandha root extract in human plasma to explore its bioaccessibility. Further to apply a developed method to perform pharmacokinetics of standardized Withania somnifera (L.) Dunal root extract (WSE; AgeVel®/Witholytin®) capsules in healthy human volunteers. MethodsA sensitive, reliable, and specific ultra-high pressure liquid chromatography-mass spectrometry (UHPLC-MS/MS) method was developed and validated for the simultaneous quantification of five major withanosides and withanolides (withanoside IV, withanoside V, withanolide A, withaferin A, and 12-deoxy-withastramonolide) in human plasma. Further for the study, eighteen healthy male volunteers (18–45 years) were enrolled in a non-randomized, open-label, single period, single treatment, clinical pharmacokinetic study and given a single dose (500 mg) of WSE (AgeVel®/Witholytin®) capsules containing not less than 7.5 mg of total withanolides under fasting condition. Later, pharmacokinetic profiles were assessed using the plasma concentration of each bioactive constituent Vs. time data. ResultsFor all five constituents, the bioanalytical method demonstrated high selectivity, specificity, and linearity. There was no carryover, and no matrix effect was observed. Furthermore, the inter-day and intra-day precision and accuracy results fulfilled the acceptance criteria. Upon oral administration of WSE capsules, Cmax was found to be 0.639 ± 0.211, 2.926 ± 1.317, 2.833 ± 0.981, and 5.498 ± 1.986 ng/mL for withanoside IV, withanolide A, withaferin A, and 12-deoxy-withastramonolide with Tmax of 1.639 ± 0.993, 1.361 ± 0.850, 0.903 ± 0.273, and 1.375 ± 0.510 h respectively. Further, withanoside V was also detected in plasma; but its concentration was found below LLOQ. ConclusionThe novel and first-time developed bioanalytical method was successfully applied for the quantification of five bio-active constituents in human volunteers following administration of WSE capsules, indicating that withanosides and withanolides were rapidly absorbed from the stomach, have high oral bioavailability, and an optimum half-life to produce significant pharmacological activity. Further, AgeVel®/Witholytin® was found safe and well tolerated after oral administration, with no adverse reaction observed at a 500 mg dose.

Simultaneous determination of five compounds of fried Radix Paeoniae Alba extract in beagle dogs plasma by Ultra Performance Liquid Chromatography Tandem Mass Spectrometry and its application in a pharmacokinetic study.

In this present study, we developed a reliable and simple ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) assay for the simultaneous quantification of paeoniflorin, albiflorin, oxypaeoniflorin, benzoylpaeoniflorin and isomaltopaeoniflorin in beagle dog plasma. We also analyzed the pharmacokinetics of those components after oral administration of fried Radix Paeoniae Alba (FRPA) in beagle dogs. Plasma samples were processed by protein precipitation with methanol. Chromatographic separation was performed with a Waters HSS-T3 C18 column (100 × 2.1mm, 1.8μm, kept at 40°C) using multiple reaction monitoring mode. A gradient elution procedure was used with solvent A (0.02% formic acid-water) and solvent B (0.02% formic acid-acetonitrile) as mobile phases. Method validation was performed as US Food and Drug Administration guidelines, and the results met the acceptance criteria. The method we establish in this experiment was successfully applied to the pharmacokinetic study after oral administration of FRPA extract to beagle dogs.

Liquid Chromatography-Tandem Mass Spectrometry-Based Therapeutic Monitoring of Plasma Atovaquone Concentrations in Pediatric Patients.

Atovaquone is an FDA-approved antiparasitic and antifungal therapeutic that is currently used as a prophylactic agent to prevent Pneumocystis carinii pneumonia (PCP) infections in acute myeloid leukemia (AML) patients after receiving hematopoietic stem cell transplantation (HSCT). Recent studies have shown that atovaquone has shown potential as an anticancer agent. The high variability in atovaquone bioavailability prompts the need for therapeutic drug monitoring, especially in pediatric patients. The goal of our study was to develop and validate the performance of an assay to quantify atovaquone plasma concentrations collected from pediatric cancer patients. Briefly, an organic-based solvent system is used to precipitate protein and extract the atovaquone content from each patient-derived plasma sample. After completing a second stage of sample dilution (5000-fold overall), a 2 μL volumeof the plasma extract is analyzed using the liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based bioanalytical method described.

Flow-Rate-Insensitive Plasma Extraction by the Stabilization and Acceleration of Secondary Flow in the Ultralow Aspect Ratio Spiral Channel.

Although microfluidic devices have made remarkable strides in blood cell separation, there is still a need for further development and improvement in this area. Herein, we present a novel ultralow aspect ratio (H/W = 1:36) spiral channel microfluidic device with ordered micro-obstacles for sheathless and flow-rate-insensitive blood cell separation. By introducing ordered micro-obstacles into the spiral microchannels, reduced magnitude fluctuations in secondary flow across different loops can be obtained through geometric confinement. As a result, the unique Dean-like secondary flow can effectively enhance the separation efficiency of particles in different sizes ranging from 3 to 15 μm. Compared to most existing microfluidic devices, our system offers several advantages of easy manufacturing, convenient operation, long-term stability, highly efficient performance (up to 99.70% rejection efficiency, including platelets), and most importantly, insensitivity to cell sizes as well as flow rates (allowing for efficient separation of different-sized blood cells in a wide flow rate from 1.00 to 2.50 mL/min). The unique characteristics, such as ultralow aspect ratio, sequential micro-obstacles, and controlled secondary flow, make our device a promising solution for practical plasma extraction in biomedical research and clinical applications.

1H-NMR based-metabolomics reveals alterations in the metabolite profiles of chickens infected with ascarids and concurrent histomonosis infection

BackgroundGut infections of chickens caused by Ascaridia galli and Heterakis gallinarum are associated with impaired host performance, particularly in high-performing genotypes. Heterakis gallinarum is also a vector of Histomonas meleagridis that is often co-involved with ascarid infections. Here, we provide a first insight into the alteration of the chicken plasma and liver metabolome as a result of gastrointestinal nematode infections with concomitant histomonosis. 1H nuclear magnetic resonance (1H-NMR) based-metabolomics coupled with a bioinformatics analysis was applied to explore the variation in the metabolite profiles of the liver (N = 105) and plasma samples from chickens (N = 108) experimentally infected with A. galli and H. gallinarum (+H. meleagridis). This was compared with uninfected chickens at different weeks post-infection (wpi 2, 4, 6, 10, 14, 18) representing different developmental stages of the worms.ResultsA total of 31 and 54 metabolites were quantified in plasma and aqueous liver extracts, respectively. Statistical analysis showed no significant differences (P > 0.05) in any of the 54 identified liver metabolites between infected and uninfected hens. In contrast, 20 plasma metabolites including, amino acids, sugars, and organic acids showed significantly elevated concentrations in the infected hens (P < 0.05). Alterations of plasma metabolites occurred particularly in wpi 2, 6 and 10, covering the pre-patent period of worm infections. Plasma metabolites with the highest variation at these time points included glutamate, succinate, trimethylamine-N-oxide, myo-inositol, and acetate. Differential pathway analysis suggested that infection induced changes in (1) phenylalanine, tyrosine, and tryptophan metabolism, (2) alanine, aspartate and glutamate metabolism; and 3) arginine and proline metabolism (Pathway impact > 0.1 with FDR adjusted P-value < 0.05).ConclusionIn conclusion, 1H-NMR based-metabolomics revealed significant alterations in the plasma metabolome of high performing chickens infected with gut pathogens—A. galli and H. gallinarum. The alterations suggested upregulation of key metabolic pathways mainly during the patency of infections. This approach extends our understanding of host interactions with gastrointestinal nematodes at the metabolic level.

High-Throughput Blood Plasma Extraction in a Dimension-Confined Double-Spiral Channel

High-Throughput Blood Plasma Extraction in a Dimension-Confined Double-Spiral Channel

A novel endogenous retention-index for minimizing retention-time variations in metabolomic analysis with reversed-phase ultrahigh-performance liquid-chromatography and mass spectrometry

Consistent retention time (tR) of metabolites is vital for identification in metabolomic analysis with ultrahigh-performance liquid-chromatography (UPLC). To minimize inter-experimental tR variations from the reversed-phase UPLC-MS, we developed an endogenous retention-index (endoRI) using in-sample straight-chain acylcarnitines with different chain-length (LC, C0–C26) without additives. The endoRI-corrections reduced the tR variations caused by the combined changes of mobile phases, gradients, flow-rates, elution time, columns and temperature from up to 5.1 min–0.2 min for most metabolites in a model metabolome consisting of 91 metabolites and multiple biological matrices including human serum, plasma, fecal, urine, A549 cells and rabbit liver extracts. The endoRI-corrections also reduced the inter-batch and inter-platform tR variations from 1.5 min to 0.15 min for 95 % of detected features in the above biological samples. We further established a quantitative model between tR and LC for predicting tR values of acylcarnitines when absent in samples. This makes it possible to compare metabolites’ tR from different tR databases and the UPLC-based metabolomic data from different batches.

Serum ve Plazma Örneklerinde İrisin Seviyesini Ölçmeden Önce Aprotinin İlave Etmek Gerekir mi?

Irisin is a myokine with 112 amino acids and its blood concentration is regulated by peroxisome proliferator-activated receptor-γ coactivator1-α (PGC1-α). It is released into circulation from skeletal muscle tissue after a photolytic cleavage of extracellular domain of Fibronectin type III domain-containing protein 5 (FNDC5), a type I integral membrane protein. Aprotinin is a polyvalent serin protease inhibitor. It is added to sample solutions such as serum, plasma or tissue extracts in order to inhibit serine proteases found in the sample medium. Hence, degradation of the proteins to be measured can be prevented. This study has been carried out to obtain a preliminary data if any irisin loss could be seen in the serum samples which are kept at -80°C for a long duration. For this purpose, blood samples of 10 men and 10 women volunteers aged between 25-40 has been used. Aprotinin has been added to the plasma and the serum samples have been kept at -80°C for 3 months. At the end of 3 months, irisin levels of the samples with and without aprotinin have been determined by ELISA. Statistical analysis has shown no difference between the plasma samples with or without aprotinin (p=0.525). However, a significant decrease between the serum samples with and without aprotinin (p=0.009). In conclusion, with the results of this study, no net decision could have been achieved to add aprotinin to the samples for irisin determination with ELISA in plasma and serum kept at -80°C for about 3 months.

B-163 Development and Validation of a New Quantitative LC-MS/MS Method for Vitamin K1 in Plasma and Serum

Abstract Background Vitamin K1 (phylloquinone) is a fat-soluble cofactor; its deficiency depresses synthesis of plasma prothrombin (factor II) and other factors, causes excessive bleeding, and increases the risk of bone fractures and osteoporosis. Biochemical and metabolic studies on vitamin K1 require both highly sensitive and specific techniques. Analytical challenges to quantitation include the presence of interfering lipids, molecular instability, and extremely low physiological levels. High performance liquid chromatography (HPLC) with fluorescence and electrochemical detection (FLD-ECD) methods used for vitamin K1 involve labor-intensive, liquid-liquid extraction procedures and extremely long run times. Our goal was to develop and validate a high throughput and accurate method for measuring vitamin K1 in plasma and serum using liquid chromatography with tandem mass spectrometry (LC-MS/MS). Methods Plasma or serum specimens were mixed with stable isotope internal standard in an extraction solution containing acetonitrile to release vitamin K1 and precipitate proteins. After centrifugation, the supernatant was then extracted using a HybridSPE® (Supelco) plate to remove the remaining precipitated proteins and phospholipids that may contribute to ion-suppression. Certified standards were used to derive a 6-point calibration curve. Extracts (plasma or serum) and calibrators were analyzed on a SCIEX 6500 + LC-MS/MS with a Thermo TX-4 HTLC multiplexed 4-channel system for increased throughput. Chromatographic separation was achieved on a Biphenyl analytical column. Precision, accuracy, linearity, recovery, and stability were evaluated. Random specimen discards, previously submitted for vitamin K1 testing, were used for a split-sample comparison study vs the current HPLC-FLD-ECD method. Results Compared with the HPLC-FLD-ECD method, the LC-MS/MS method employed a simplified automated hybrid solid phase extraction procedure, used less specimen volume (100 µL vs 700 µL), increased total throughput (4 h vs 32 h per 96-well plate), was more sensitive (limit of quantitation [LOQ] 6.1 pg/mL vs 25.0 pg/mL), and provided greater molecular specificity for vitamin K1 through multiple reaction monitoring (MRM). The intra-assay and total inter-assay precision coefficients of variation (CVW, CVT) in plasma samples (n = 100) were: (6.8%, 5.8%) at 100 pg/mL, (3.5%, 3.0%) at 800 pg/mL, and (3.0%, 3.9%) at 2000 pg/mL. In spiked plasma and serum samples (n = 10), the linearity range was 40–20 000 pg/mL and percent recovery were 98%–107%. Least-squares regression analysis comparing the results for split plasma extracts by LC-MS/MS vs HPLC-FLD-ECD (n = 100) yielded correlation coefficients for vitamin K1: 0.9917, y = 0.927x + 0.827, range 40–7000 pg/mL. Vitamin K1 was stable in plasma and in serum stored in amber plastic screw-capped vials for 8 days at 2–8 °C and 30 days at −20 °C. Conclusion We have developed and validated an LC-MS/MS method that provides rapid, sensitive, precise, and accurate evaluation of vitamin K1 in plasma and serum. Advantages over the existing HPLC-FLD-ECD method include increased throughput and improved sensitivity and specificity. Vitamin K1 results obtained using this method can be used by clinicians to guide clinical intervention to improve or normalize metabolism or prevent the potential adverse consequences associated with inadequate dietary intake.

Rapid prototyping of 3d printed micropillars using fused filament fabrication technique for biomedical applications

PurposeThe hygroscopic properties of 3D-printed filaments and moisture absorption itself during the process result in dimensional inaccuracy, particularly for nozzle movement along the x-axis and for micro-scale features. In view of that, this study aims to analyze in depth the dimensional errors and deviations of the fused filament fabrication (FFF)/fused deposition modeling (FDM) 3D-printed micropillars (MPs) from the reference values. A detailed analysis into the variability in printed dimensions below 1 mm in width without any deformations in the printed shape of the designed features, for challenging filaments like polymethyl methacrylate (PMMA) has been done. The study also explores whether the printed shape retains the designed structure.Design/methodology/approachA reference model for MPs of width 800 µm and height 2,000 µm is selected to generate a g-code model after pre-processing of slicing and meshing parameters for 3D printing of micro-scale structure with defined boundaries. Three SETs, SET-A, SET-B and SET-C, for nozzle diameter of 0.2 mm, 0.25 mm and 0.3 mm, respectively, have been prepared. The SETs containing the MPs were fabricated with the spacing (S) of 2,000 µm, 3,200 µm and 4,000 µm along the print head x-axis. The MPs were measured by taking three consecutive measurements (top, bottom and middle) for the width and one for the height.FindingsThe prominent highlight of this study is the successful FFF/FDM 3D printing of thin features (<1mm) without any deformation. The mathematical analysis of the variance of the optical microscopy measurements concluded that printed dimensions for micropillar widths did not vary significantly, retaining more than 65% of the recording within the first standard deviation (SD) (±1 s). The minimum value of SD is obtained from the samples of SET-B, that is, 31.96 µm and 35.865 µm, for height and width, respectively. The %RE for SET-B samples is 5.09% for S = 2,000µm, 3.86% for S = 3,200µm and 1.09% for S = 4,000µm. The error percentage is so small that it could be easily compensated by redesigning.Research limitations/implicationsThe study does not cover other 3D printing techniques of additive manufacturing like stereolithography, digital light processing and material jetting.Practical implicationsThe presented study can be potentially implemented for the rapid prototyping of microfluidics mixer, bioseparator and lab-on-chip devices, both for membrane-free bioseparation based on microfiltration, plasma extraction from whole blood, size-selection trapping of unwanted blood cells, and also for membrane-based plasma extraction that requires supporting microstructures. Our developed process may prove to be far more economical than the other existing techniques for such applications.Originality/valueFor the first time, this work presents a comprehensive analysis of the fabrication of micropillars using FDM/FFF 3D printing and PMMA in filament form. The primary focus of the study is to minimize the dimensional inaccuracies in the 3D printed devices containing thin features, especially in the area of biomedical engineering, by delivering benefits from the choice of the parameters. Thus, on the basis of errors and deviations, a thorough comparison of the three SETs of the fabricated micropillars has been done.