Concentrations In Serum Samples Research Articles

Effect of Hydrotherapy and Physical Activity on Uric Acid and Nitric Oxide Concentrations in Serum Samples

Hydrotherapy is highly recommended for older adults because it offers warmth and support, so people with pain or reduced mobility and balance can move and exercise more freely compared to regular physical activity. The high plasma antioxidant capacity of uric acid in neutralizing oxidative species and the importance of nitric oxide (NO) in vasodilation and inflammatory response are relevant in older adults. This work aimed to evaluate hydrotherapy's effect on uric acid and NO concentrations in a population aged ≥ 60 years before and after hydrotherapy. A randomized study with 37 individuals was divided into two groups: the experimental group, which was evaluated at two time points: T0-before treatment and T1-after 15-hydrotherapy sessions, and the control group, which did not practice any exercise. The determinations of uric acid and NO levels in serum were carried out by spectrophotometry. Statistics were carried out with SPSS. The experimental group showed a significant increase in the concentration of uric acid in T1 compared to T0. NO results did not show significant differences between T0 and T1. Regular hydrotherapy increases the concentration of serum uric acid, potentially beneficial in the performance of antioxidant functions, reducing oxidative stress, delaying aging, and improving the well-being of older adults.

Assessment of Phthalate Esters and Physiological Biomarkers in Bottlenose Dolphins (Tursiops truncatus) and Killer Whales (Orcinus orca).

There is growing concern about the potential adverse health effects of phthalates (PAEs) on human health and the environment due to their extensive use as plasticizers and additives in commercial and consumer products. In this study, we assessed PAE concentrations in serum samples from aquarium-based delphinids (Tursiops truncatus, n = 36; Orcinus orca, n = 42) from California, Florida, and Texas, USA. To better understand the physiological effects of phthalates on delphinids, we also explored potential correlations between phthalates and the biomarkers aldosterone, cortisol, corticosterone, hydrogen peroxide, and malondialdehyde while accounting for sex, age, and reproductive stage. All PAEs were detected in at least one of the individuals. ΣPAE ranges were 5.995-2743 ng·mL-1 in bottlenose dolphins and 5.372-88,675 ng·mL-1 in killer whales. Both species displayed higher mean concentrations of DEP and DEHP. PAEs were detected in newborn delphinids, indicating transference via placenta and/or lactation. Linear mixed model results indicated significant correlations between aldosterone, month, location, status, and ΣPAEs in killer whales, suggesting that aldosterone concentrations are likely affected by the cumulative effects of these variables. This study expands on the knowledge of delphinid physiological responses to PAEs and may influence management and conservation decisions on contamination discharge regulations near these species.

Measurement of Serum Vitamins B2 and B6 in Patients with Hashimoto’s Thyroiditis by LC–MS/MS Method

AbstractVitamin B2 and vitamin B6 are cofactors and coenzymes in many biochemical reactions, and their sufficient serum concentrations may be crucial for the proper functioning of the thyroid gland. The aim of the study was to develop and validate a novel high-performance liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the determination of vitamin B2 and vitamin B6 in the serum of patients with Hashimoto’s thyroiditis (HT). Detection of the analytes was performed on a triple-quadrupole MS with multiple reaction monitoring using electrospray ionization. The mobile phase consisted of water and methanol, both containing 0.1% (v/v) formic acid. The analytes were isolated from serum by the one-step sample preparation of the trichloroacetic acid precipitation. Validation parameters, including linearity, accuracy, precision, limits of detection and quantification, and stability, were determined and fulfilled the criteria for analyzing compounds in biological fluids. The method was applied for the determination of the above vitamin concentrations in serum samples of 74 patients with HT and 33 healthy controls. The concentration of vitamin B2 was significantly lower in patients with HT compared to the control group (7.20 ± 7.86 ng/mL vs. 12.03 ± 9.62 ng/mL) and the concentration of vitamin B6 was also lower, but without a statistically significant difference (30.09 ± 19.16 ng/mL vs. 37.29 ± 23.02 ng/mL). We concluded that the vitamins concentration measurement may be helpful in the diagnosis of vitamin deficiencies and the supplementation strategy.

Anti-IL-6 Receptor Treatment in Giant Cell Arteritis Patients Reduces Levels of IL-1β-Receptor Antagonist but Not IL-1β

This work aimed to investigate a potential link between serum IL-1β levels in patients with giant cell arteritis (GCA) and their responsiveness to combined anti-IL-6 receptor (IL-6R) and glucocorticoid (GC) treatments within the context of two separate clinical trials. IL-1β levels were analyzed in serum samples of two prospective clinical trials investigating tocilizumab in GCA patients using quantitative Polymerase Chain Reaction (qPCR) based Proximity Ligation Assays (PLA). In the phase II randomized controlled trial, serum samples from five patients were quantified at two critical time points: the commencement of the trial (Week 2) and the conclusion of the trial (Week 52). In the GUSTO trial, serum samples from nine patients were similarly analyzed using PLA at Day 0 and Week 52. Furthermore, for the GUSTO trial, serum samples from 18 patients were assessed for IL-1β and IL-1RN at six time points: days 0, 3, and 10, weeks 4, 24, and 52 by a second assay (Proximity Extension Assay, PEA). PLA results from both studies indicated that IL-1β levels were below 1 pg/mL in most of the patients, resulting in notable signal deviations within the same samples. In the analysis of the GUSTO trial, both PLA and PEA exhibited similar trends in IL-1β variations among patients from day 0 to week 52. Notably, the PEA analysis did not show significant variation over time. Furthermore, we did not find a correlation of IL-1β levels with active disease as compared to remission, but interestingly, the measurement of IL-1β receptor antagonist (IL-1RN) revealed a substantial decrease over time. Our study shows that IL-1RN but not IL-1β concentration in serum samples could be directly related to anti-IL-6R treatment in patients diagnosed with GCA.

Chronic rhinosinusitis possibly associated with decreased lung function in chronic cough patients

ObjectivesThe purpose of this study is to investigate the lung function in Chronic Rhinosinusitis (CRS) patients with Chronic Cough (CC). MethodsA total of 1413 CC patients were retrospectively screened and 109 CRS patients with CC were enrolled. Lung function, Lund-Mackay Computed Tomography (CT) score, smoking status, peripheral blood eosinophil count, and immunoglobulin E concentration in serum samples, and Sino-Nasal Outcome Test were examined. Normal control subjects are also recruited. ResultsThe Forced Expiratory Volume in 1 second (FEV1.0), Percent Predicted FEV1.0, and FEV1.0/Forced Vital Capacity (FVC) ratio in the patients were significantly low as compared with the control subjects. The FEV1.0/FVC ratio was negatively correlated with the Lund-Mackay CT scores of the patients with a high CT score. ConclusionsThe CRS patients with CC should be investigated with lung function. In addition, the multidisciplinary evaluation including a pulmonologist is needed to manage the CRS patients with CC. Level of evidenceLevel 4.

Developing a nanomaterial-based 3D-printed platform: Application as a cancer aptasensor via detection of heat shock protein 90 (HSP90)

Overexpressed heat shock protein 90 (HSP90) has been proven as a new cancer biomarker, and its detection can be used as an auxiliary diagnosis. In this research, we designed an electrochemical aptasensor to detect this biomarker. Initially, a 3D-printed electrode was designed and produced while the electrode surface (made from a conductive carbon black/ polylactic acid (CB-PLA) filament) was improved by CO2 laser ablation. Then, the electrode surface was modified with graphene oxide (GO) and an array of frost-like gold nanocrystals (FGNCs); the results showed that the final surface covered with gold contained nano/microstructures with a mean width of ∼ 65 nm and a mean length of ∼ 509 nm. Also, a significant increase in the electrochemically active surface area was observed for modified surfaces with GO and FGNCs. Efficient immobilization of aptamer strands on the gold surface could complete the assembly of this aptasensing platform, which could detect analyte molecules in a linear range from 0.1 to 200 ng mL−1. The estimated limit of detection (LOD) and the limit of quantification (LOQ) were as low as 0.1 ng mL−1 and 0.23 ng mL−1, respectively. The performance of reproducibility (5 times), regeneration (5 times), and stability (7 days) were satisfactory (low relative standard deviation (RSD)). Also, this aptasensor showed adequate performance for selective and accurate analyte capturing in the presence of interfering agents and spiked analyte concentrations in serum samples.

Highly sensitive and specific graphene oxide-based FRET aptasensor for quantitative detection of human soluble growth stimulating gene protein 2

Soluble growth stimulation expressed gene 2 (sST2) is a new generation biomarker in the diagnosis and prognosis of heart failure (HF). Here, the sST2-specific aptamers were selected from a random ssDNA library with the full length of 88 nucleotides (nt) via target-immobilized magnetic beads (MB)-based systematic evolution of ligands by exponential enrichment (SELEX) technology. After eight rounds of selection, six aptamers with the most enrichment were selected. Among, the aptamer L1 showed the high-affinity binding to sST2 with the lowest Kd value (77.3 ± 0.05 nM), which was chosen as the optimal aptamer for further molecular docking. Then, the aptamer L1 was used to construct a graphene oxide (GO) - based fluorescence resonance energy transfer (FRET) biosensor for sST2, which exhibits a linear detection range of 0.1–100 μg/ml and a detection limit of 3.7 ng/ml. The aptasensor was applied to detect sST2 in real samples, with a good correlation and agreement with the traditional enzyme-linked immunosorbent assay (ELISA) when quantitative analyzing the sST2 concentration in serum samples from HF patients. The results show that not only an efficient strategy for screening the practicable aptamer, but also a rapid and sensitive detection platform for sST2 were established.

Evaluation of serum nickel levels in some Syrian patients with gastrointestinal cancers

Gastrointestinal cancers describe malignant changes that affect the organs of the digestive system and its ancillary organs and are responsible for more cancers and cancer-related deaths than any other system in the body. Nickel is widely used in various industries, causing many human cancers through different mechanisms. Our present study is designed to estimate nickel concentrations in serum samples from patients with different types of gastrointestinal cancers. The study was carried out in 45 patients with many types of gastrointestinal cancers at Al-Biruni University Hospital for Cancerous Diseases in Syria compared with 35 healthy controls. All measurements of serum were done with a graphite atomic absorption spectrophotometer equipped with a deuterium lamp. Our results showed that patients with gastrointestinal cancers had significantly higher serum levels of Ni than healthy controls (p < 0.0001), and there were no statistically significant differences between the four types of gastrointestinal cancers included in the study (p > 0.05). The increase in serum nickel concentrations has a direct effect on the incidence of gastrointestinal cancers by influencing the mechanisms that induce cancer by nickel.

Presence of Elevated Interleukin – 6 (IL-6) in the Blood of COVID-19 Convalescent Patients (40 – 93 Days) Post Onset of Symptoms Could be an Indicator of Ongoing Activation of the Immune System

IL-6 concentrations rise with the onset of COVID-19 infection and is detected in 68% of patients on admission but is expected to reduce after the acute phase. IL-6 concentrations at time points: (2-7 days), (6-11 days), (11-15 days) and (13-20 days) after intensive care unit (ICU) admission showed the highest level of IL-6 concentrations at time point 2-7 days, we decided to characterize IL-6 concentrations in serum samples collected in the sera of COVID-19 convalescent patients (40 – 93 days) post onset of symptoms. The estimated IL- 6 concentrations detected ranged between 2.65 pg/ml and 29.01 pg/ml, with a median of 9.18 pg/ml. Nine out of the 28 (32.14%) patients had normal IL-6 levels (<7 pg/ml) and 19 of the 28 patients (68%) had IL-6 elevated concentrations (>7 pg/ml). The IL-6 concentrations were grouped normal (<7 pg.ml, n= 9), mildly elevated (7 – 10 pg/ml, n = 10) and elevated (>10 pg/ml n = 9). There was no relationship between IL-6 concentrations with gender, days from onset of symptoms, days from subsiding symptoms and COVID-19 IgG antibodies. There was some relationship with age, as the younger patient cohort produced much higher IL-6 concentrations than the older patient cohort. This study describes an increase in blood IL-6 concentrations in the sera of COVID-19 convalescent patients (40 – 93 days) post onset of symptoms like that seen in COVID patients on admission and higher levels seen in the younger patient cohort indicating that “cytokine storms” still occur in the recovery phase.

Identification of novel metabolites of abiraterone in human serum and their metabolic pathways.

Two novel abiraterone (Abi, 3β-OH-Abi) metabolites in human serum were identified as 3α-OH-Abi and Δ5-Abi (D5A). Both metabolites were confirmed by their retention times on LC/MS and their product-ion mass spectra on LC-MS/MS compared to those of authentic compounds, which were chemically synthesized. The plausible metabolic pathways of these two metabolites are as follows: Abi is first oxidized to D5A by 3β-hydroxysteroid dehydrogenase (3β-HSD) and then irreversibly converted to Δ4-Abi (D4A) by ∆5-∆4 isomerase. Presumably, D5A detection is difficult because of its rapid conversion to D4A and its low concentration in serum samples. In contrast, the low concentration 3α-OH-Abi was generated by reducing the remaining D5A using 3α-hydroxysteroid dehydrogenase (3α-HSD).

Label-free electrochemical immunosensor based on antibody-immobilized Fe-Cu layered double hydroxide nanosheetas an electrochemical probe for the detection of ultra trace amount of prostate cancer biomarker (PSA)

Here, a label-free immunosensor has been developed to detect prostate specific antigen as a diagnostic biomarker for prostate cancer. A pencil graphite electrode (PGE) coated with nanocomposites of iron-copper layered double hydroxide (Fe-Cu LDH) and reduced graphene oxide (rGO) was utilized as a sensor platform to capture the monoclonal anti-PSA antibody. The LDH/rGO nanocomposite can boost the electrical conductivity and enhance the surface area of the fabricated electrode; accordingly, it can efficiently amplify the electrode response and sensitivity in PSA detection. Electrochemical properties of the constructed sensor have been conducted utilizing cyclic voltammetry (CV), and electrochemical impedance spectroscopy(EIS). The PSA concentration level in serum samples was measured by monitoring the current changes of the Cu oxidation peak which was through the formation of antibody-PSA complexes on the surface of the electrode. The current response of the label-free immunosensor has been correlated to the concentration of PSA in a linear manner ranging from 100.0 fg/ml to 10.0 µg/ml with a limit of detection (LOD) of 63.24 fg/ml (S/N = 3). The morphology and structure of the synthesized Fe-Cu LDH/rGO nanocomposite was characterized using various analytical techniques such as field-emission scanning electron microscopy (FESEM), energy dispersive X-ray analysis (EDX), X-ray diffraction (XRD)and Fourier transform infrared (FTIR). The suggested immunosensor was employed to accurately and sensitively determine PSA concentration in serum samples with satisfactory results consistent with those achieved by enzyme-linked immunosorbent assay (ELISA) as a reference method. Finally, the obtained results indicate the promising application of the fabricated immunosensor in premature prostate cancer diagnostics.

SAT235 An Orally Administered Robotic Pill (RP) Reliably And Safely Delivers the Human Parathyroid Hormone Analog hPTH(1-34) (Teriparatide) With High Bioavailability in Healthy Human Volunteers: A Phase 1 Study

Abstract Disclosure: J.T. Myers: Employee; Self; Rani Therapeutics. Stock Owner; Self; Rani Therapeutics. A. Dasari: Employee; Self; Rani Therapeutics. Stock Owner; Self; Rani Therapeutics. A. Battiwala: Employee; Self; Rani Therapeutics. Stock Owner; Self; Rani Therapeutics. N. Patel: Employee; Self; Rani Therapeutics. Stock Owner; Self; Rani Therapeutics. A. Toledo Vo: Employee; Self; Rani Therapeutics. Stock Owner; Self; Rani Therapeutics. A. Yamaguchi: Employee; Self; Rani Therapeutics. Stock Owner; Self; Rani Therapeutics. K. Horlen: Employee; Self; Rani Therapeutics. Stock Owner; Self; Rani Therapeutics. L.C. Fung: Employee; Self; Rani Therapeutics. Stock Owner; Self; Rani Therapeutics. B. Syed: Employee; Self; Rani Therapeutics. Stock Owner; Self; Rani Therapeutics. S. Nguyen: Employee; Self; Rani Therapeutics. Stock Owner; Self; Rani Therapeutics. J. Van Dam: Employee; Self; Rani Therapeutics. Stock Owner; Self; Rani Therapeutics. M. Imran: Employee; Self; Rani Therapeutics. Stock Owner; Self; Rani Therapeutics. M. Hashim: Employee; Self; Rani Therapeutics. Stock Owner; Self; Rani Therapeutics. A.K. Dhalla: Employee; Self; Rani Therapeutics. Stock Owner; Self; Rani Therapeutics. Background/Purpose: Teriparatide, an effective osteoanabolic agent, requires a chronic regimen of daily subcutaneous (SC) injections, representing a burden for patients which may interfere with compliance and quality of life. We have developed a novel oral robotic pill (RP) to deliver biotherapeutic agents with bioavailability rivaling or surpassing that of parenteral injections. Here we present data from a Phase 1 study in healthy women volunteers designed to evaluate the safety, tolerability, and pharmacokinetics (PK) of 20- and 80 µg of hPTH (1-34) administered orally via RP (RT-102) at single doses. Methods: The study was approved by the Alfred Health HREC in Australia. Participants received a single dose of either RT-102 at 20 µg (RT-102/20 µg group; N=15) or 80 µg (RT-102/80 µg group; N=14) or a single 20 µg dose of the commercially available teriparatide (Forteo®) via SC injection (SC group; N=10). Fluoroscopic images were taken to track the transit/deployment of RT-102 and confirm excretion of remnants. Drug concentration in serum samples serially collected over 6 hours were measured by a validated ELISA assay. Results: All 29 participants in the RT-102 arms were able to swallow RT-102 and none experienced any adverse events (AEs) upon device deployment. Both doses of RT-102 were safe and well-tolerated with no serious adverse events (SAEs) reported. In the SC group, a total of six drug-related AEs were reported in four participants. There were no AEs reported in the RT-102/20 µg group. Mild and transient AEs attributed to the drug were observed in one participant in the RT-102/80 µg group. No AEs related to the RP were reported. Imaging confirmed excretion of all RP device remnants within an average of 2.7 ± 1.4 days. Based on serum analyses, drug was successfully delivered by RT-102 in 12 of 15 participants in the 20 µg group and in all 14 participants in the 80 µg group. PK analyses indicated that RT-102 delivered drug with more sustained peak concentrations than the SC group. The Cmax of RT-102 was 98 ± 10 pg/mL for the 20 µg dose and 971 ± 223 pg/mL for the 80µg dose compared to 128 ± 20 pg/mL for the 20 µg SC injection group. The Tmax for RT-102 delivery was 68 and 60 min for the 20 µg and 80 µg doses, respectively, compared to 13 min for the SC injection. Based on AUC calculations of the PK curves, the relative bioavailability of RT-102 dose groups was 3- to 4-fold higher compared to the SC injection group. Conclusions: This study provides the first clinical evidence of safe and successful delivery of hPTH (1-34) via an orally administered RP with high reliability and bioavailability. These data suggest that RT-102 can potentially offer an easier and more convenient treatment option to osteoporosis patients, and given the high bioavailability, RT-102 may be effective at doses lower than the currently approved 20 µg SC dose of teriparatide. Presentation: Saturday, June 17, 2023

Determination of Tiamulin Concentration in Sow Milk and in Sera of Suckling Piglets.

Although modern analytical methods developed for monitoring antibiotics in several biological matrices are easily available, none of them have been applied to evaluate the transfer of tiamulin into sow milk. Therefore, this work was intended to analyse the concentrations of tiamulin in milk samples collected from lactating sows during and after a treatment consisting of three consecutive intramuscular applications of the antibiotic. The second aim of this investigation was to determine tiamulin concentrations in serum samples obtained from suckling piglets ingesting milk contaminated with the compound. Ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) was used to quantify the analyte in both matrices. Our investigation proved tiamulin was transmitted into the milk of lactating sows. The mean concentration of the antibiotic among samples collected 3 h after administration was 1043 μg/L. The mean level of tiamulin on days 1 and 2 was 876 μg/L and 902 μg/L, respectively. The highest mean concentration of the antibiotic (1061 μg/L) was observed in samples collected on day 3. The mean concentration of the antibiotic in serum samples collected from 3-day-old piglets was 22.2 μg/L. The association between their body weight and serum tiamulin concentration was not statistically significant (p = 0.456).

A combined top-down and bottom-up LC-HRMS/MS method for the quantification of human growth hormone in plasma and serum

ObjectiveThe precise and accurate quantification of human growth hormone (GH) in plasma/ serum is crucial for the diagnosis and treatment of diseases like GH deficiency or acromegaly. However, the ligand-binding assays (LBAs) currently used for routine testing show considerable methodological variability. Here, we present a complementary, combined top-down and bottom-up LC-MS-based method to quantify (intact) GH in plasma and serum, which concurrently provides a basis for a MS-based analysis of GH in doping controls. DesignExtraction of GH from plasma/ serum was accomplished by protein precipitation, followed by an immunocapture step using protein A-coupled magnetic beads and a polyclonal anti-GH antibody. The intact protein was subsequently analyzed top-down on a 2D-LC-HRMS/MS system. In addition, sample extracts were digested with trypsin and analyzed for signal peptides corresponding to ‘total’, 22 kDa and 20 kDa GH (bottom-up). Both assays were validated according to current guidelines and compared to the GH isoform differential immunoassay used in routine doping control analysis. GH concentrations in serum samples of healthy adults, patients with acromegaly, and in samples obtained after administration of recombinant GH were analyzed as proof-of-principle. ResultsThe intact monomeric 22 kDa isoform of GH was selectively quantified in a representative working range of 0.5 to 10 ng/ml by top-down LC-HRMS/MS. Subsequent bottom-up analysis provided additional data on ‘total’ and 20 kDa GH. Top-down and bottom-up assay results for the 22 kDa isoform correlated well with the corresponding immunoassay results (R2 > 0.95). For a possible application of the method in an anti-doping context, the ratio between 22 kDa and ‘total’ GH was evaluated, indicating differences between the various donor groups, but only with limited significance. ConclusionThe top-down and bottom-up LC-HRMS/MS method developed here presents a valuable tool for the quantification of GH in plasma/ serum complementary to established LBAs used at present in clinical measurements. Albeit the examination of the GH isoform proportions by the LC-MS method does not yet allow for the assessment of GH abuse, the obtained findings provide an important basis to enable LC-MS-based GH analysis of doping control samples in the future.

A-382 Evaluation of the Labcorp TrueSpinTM for Decentralized Blood Sample Centrifugation

Abstract Background The increased demand for decentralized blood sample collection presents numerous operational challenges for diagnostics providers. Sample degradation including sample hemolysis due to time, temperature, and handling between collection and laboratory analysis leads to limited test menus and unreliable results. Here we evaluate the light-weight, portable Labcorp TrueSpin™ for rapid point-of-care blood separation using commercially available microvolume blood collection tubes. The TrueSpin is a class I FDA registered device designed for layperson use. The centrifuge runs on AA batteries and separates a blood sample in five minutes. Methods Here we show results of studies evaluating sample quality and analyte stability in serum samples collected into gel microtubes and processed using the TrueSpin. Hemolysis and residual red blood cell (RBC) concentration are compared between samples separated using the TrueSpin and conventional centrifugation. Additionally, we evaluate serum-based chemistry analyte stability in both separated and unseparated samples stored for three days refrigerated and unrefrigerated. Results No significant difference was seen in hemolysis or residual RBC concentration in serum samples prepared by TrueSpin compared to the reference centrifuge method. Furthermore, we show that many common serum-based chemistry analytes have limited (<1 day) stability if uncentrifuged, but improve to ≥3-day stability following TrueSpin separation and refrigerated or room temperature storage (see Table 1). Conclusions These findings suggest that the TrueSpin is a simple and effective solution for remote sample separation and may enable broader test menus and increased test result reliability for decentralized sample collection pursuits.

Evaluation of a Compact, Portable Centrifuge for Separating Microvolume Blood Samples at the Point of Collection.

The increased demand for decentralized blood sample collection presents numerous operational challenges for diagnostics providers. Sample degradation including sample hemolysis due to time, temperature, and handling between collection and laboratory analysis leads to limited test menus and unreliable results. Here we introduce the lightweight, portable Labcorp TrueSpin™ for rapid point-of-care blood separation using commercially available microvolume blood collection tubes. The TrueSpin is a class I FDA-registered device designed for untrained users. The centrifuge runs on AA batteries and separates a blood sample in 5 minutes. Here we describe a series of studies evaluating sample quality and analyte stability in serum samples collected into gel microtubes and processed using the TrueSpin. Hemolysis, residual red blood cell concentration, sample volume, and serum-based chemistry analyte stability were evaluated. No significant difference was seen in hemolysis or residual red blood cell concentration in serum samples prepared by TrueSpin compared to the reference method. Additionally, capillary and venous blood samples separated using the TrueSpin and exposed to International Safe Transit Association 3A-simulated shipping conditions were shown to yield acceptable sample volume and quality for laboratory analysis. Finally, we show that many common serum-based chemistry analytes have limited (< 1 day) stability if uncentrifuged but improve to ≥ 3-day stability following TrueSpin separation and refrigerated or room temperature storage. These findings suggest that the TrueSpin is a simple and effective solution for remote sample separation and may enable broader test menus and increased test result reliability for decentralized sample collection pursuits.

A monoclonal antibody-based sandwich ELISA for measuring canine Thymidine kinase 1 protein and its role as biomarker in canine lymphoma.

Dogs play an important role in society, which increased during the covid epidemics. This has led to a much higher workload for the veterinarians. Therefore, there is a need for efficient diagnostic tools to identify risk of malignant diseases. Here the development of a new test that can solve some of these problems is presented. It is based on serum Thymidine Kinase 1 (TK1), which is a biomarker for cell proliferation and cell lysis. Anti-TK1 monoclonal antibodies were produced against two different epitopes, the active site of the TK1 protein and the C-terminal region of canine TK1. The antibodies were developed with hybridoma technology and validated using dot blot, Quartz Crystal Microbalance (QCM) technology, western blots, immunoprecipitation (IP), and enzyme-linked immunosorbent assay (ELISA). Clinical evaluation of Canine TK1 ELISA was done by using sera from 131 healthy dogs and 93 dogs with lymphoma. The two selected Anti-TK1 monoclonal antibodies have Kd values in the range of 10-9 M and further analysis with dot and western blots confirmed the high affinity binding of these antibodies. A sandwich Canine TK1 ELISA was developed using the anti-TK1 antibodies, and TK1 concentrations in serum samples were determined using dog recombinant TK1 as a standard. Serum TK1 protein levels were significantly higher in dogs with lymphoma compared to those in healthy dogs (p < 0.0001). Receiver operating curve analysis showed that the canine TK1-ELISA obtain a sensitivity of 0.80, at a specificity of 0.95. Moreover, the Canine TK1 ELISA has a positive predictive value (PPV) of 97%, and the negative predictive value (NPV) of 83%, reflecting the proportion of test results that are truly positive and negative. Furthermore, Canine TK1 ELISA had significantly higher capacity to differentiate dogs with T-cell lymphoma from those with B-cell lymphoma compared to earlier used TK1 activity assays. These results demonstrate that the Canine TK1 ELISA can serve as an efficient tool in the diagnosis and management of dogs with lymphomas.

Colorimetric and photothermal dual readout biosensor for flap endonuclease 1 based on target-prevented gold nanoparticles aggregation

A colorimetric and photothermal dual readout biosensor for Flap endonuclease 1 (FEN1) quantification was developed on the basis of target-prevented gold nanoparticles (AuNPs) aggregation. The exposed 5′-flap of double-flap DNA substrate modified on SAMBs was firstly cleaved by FEN1. Large amount of cleaved 5′-flap remained in the supernatant after simple magnetic separation, which can adsorb on the surface of AuNPs and effectively prevent the dispersed AuNPs from aggregation under high ionic concentration, accompanied with the color changing of the system, which can be recognized by nake eyes easily. The absorption intensity at 528 nm shows a good linear relationship with the increasing FEN1 concentration from 5.0 × 10-3 to 3.1 × 10-2 U μL-1 with a LOD of 1.6 × 10-3 U μL-1 (S/N = 3). Given the aggregated AuNPs have higher photothermal effect than that of the dispersed AuNPs, the target-prevented AuNPs aggregation avoids a sharp increase of temperature for the system under the laser radiation. The temperature change is linearly correlated with the FEN1 concentration in the range of 3.1 × 10-3–6.1 × 10-2 U μL-1 with a LOD of 1.1 × 10-3 U μL-1. The whole detection process can be completed within 1 h. The proposed system had been applied to detect FEN1 concentration in serum samples with satisfied results, which can be applied in resource-limited area easily and quickly.

Establishment of 11-dehydro-thromboxane B2 time-resolved immunoassay and application in membranous nephropathy

Background11-Dehydro-thromboxane B2 (11-dehydro-TXB2) is the final stable metabolite of thromboxane A2 (TXA2) and is involved in thrombus formation. Patients with membranous nephropathy (MN) are prone to thromboembolism events. MethodsTime-resolved fluorescence immunoassay (TRFIA) for 11-dehydro-TXB2 was established by indirect competitive method. The coated 11-dehydro-TXB2-BSA conjugate was used to bind the 11-dehydro-TXB2 antibody competitively to the 11-dehydro-TXB2 antigen in the samples, followed by Eu3+-labeled goat anti-mouse IgG antibody, to detect 11-dehydro-TXB2. This study measured 11-dehydro-TXB2 concentrations in serum samples from healthy individuals and patients with MN. ResultsThe linear range of TRFIA was 16.38–2000 pg/mL, the sensitivity was 4.70 pg/mL, the average coefficients of variation from intra-assay and inter-assay were 3.50% and 4.95%, respectively, and the recovery was 99.38%. The serum level of 11-dehydro-TXB2 in patients with MN was significantly higher than that in healthy subjects (P < 0.05). The serum 11-dehydro-TXB2 concentration detected by TRFIA was highly consistent with that by ELISA (ρ = 0.900). DiscussionThis study successfully established a new highly sensitive method for the detection of 11-dehydro-TXB2 in serum. 11-Dehydro-TXB2 has great potential in evaluating the risk of thromboembolic events in patients with MN and is expected to be applied to other thromboembolic-related diseases.

403-P: Profiling of Circulating MicroRNAs (miRNAs) Associated with Progression to End Stage Kidney Disease (ESKD) in Diabetes—Preanalytical and Analytical Considerations

Background: miRNAs are short non-coding RNA molecules involved in gene regulation. Previously, we identified 17 plasma miRNAs (8 risk and 9 protective miRNAs) associated with progression to ESKD in diabetes (JASN 2021). Factors that influence the measurement of miRNA concentrations, and thus could impact replication of these findings, including extraction method, miRNA profiling method, and the miRNA source biofluid, have not been investigated. Methods: Plasma and serum were obtained from patients who participated in the Joslin Kidney Study. Extraction-free plasma and serum miRNA profiles were obtained using the HTG EdgeSeq platform. RNA was also extracted from plasma and used to measure miRNAs by RNA sequencing (RNASeq) and quantitative RT-PCR (qRT-PCR). Results: Out of 2,083 miRNAs on the EdgeSeq platform, almost all of them (96%) were detected in plasma without RNA extraction, while only 45% of these miRNAs were detected in RNA extracted from the same plasma specimens. The content of miRNAs detected in extracted RNA was significantly enriched for GC sequences in comparison with miRNAs that were lost during RNA extraction. In the specimens with RNA extracted, significantly lower number of miRNAs were detected by RNASeq or qRT-PCR (31% and 26%, respectively) in comparison with the EdgeSeq platform. Regarding the 17 miRNAs associated with ESKD, many of them had different concentrations in serum samples than in plasma and had lower concentration or were not detected in plasma samples with profiled following RNA extraction. Conclusion: This study demonstrates very strong effects of pre-analytical and analytical factors on concentration of circulating miRNAs. To replicate our findings for 17 circulating miRNAs, it is critical that plasma specimens be used and that miRNAs be measured using the EdgeSeq platform or other platforms that do not require prior extraction of RNA. Disclosure E.Satake: None. Z.Md dom: None. H.Kobayashi: Research Support; Bayer Inc. J.Ricca: None. K.O'neil: None. B.Krolewski: None. M.G.Pezzolesi: Research Support; Renalytix. A.Krolewski: None. Funding National Institutes of Health (DK131061-01)