Plasma Research Articles

A Study Into Systemic and Oral Levels of Proinflammatory Biomarkers Associated With Endpoints After Active Non-Surgical Periodontal Therapy.

To analyse whether some selected inflammatory biomarkers collected from venous blood and gingival crevicular fluid (GCF) were associated with the outcome of non-surgical periodontal therapy. Two-hundred and nine patients affected by periodontitis were enrolled in the study, who had undergone steps I and II therapy as well as a non-surgical re-instrumentation (NSRI) of periodontal pockets after 6 months. Serum (SE), plasma (PL) and GCF samples were quantitatively analysed for the following inflammatory biomarkers: active matrix metalloproteinase-8 (aMMP-8), prostaglandin E2 (PGE2) and surfactant protein D (SP-D). Therapy outcomes were evaluated using a 'treat-to-target' endpoint (T2T) at the patient level, defined as ≤ 4 sites with pocket depth ≥ 5 mm. Patients presented with 23 ± 6 teeth (mean ± SD) at baseline. After steps I and II therapy, 41.6% of the patients reached T2T and after NSRI 47.4%. Univariate analysis identified a potential association between high levels of PL-SP-D and more favourable treatment outcomes. Multivariate binary logistic regression adjusted for sex, mean baseline probing depth, diabetes and current smoking status confirmed an independent relationship between baseline PL-SP-D and the T2T after steps I and II therapy (aOR 0.432, p = 0.011), implying that a higher level PL-SP-D at baseline is associated with a > 50% reduced risk of failing T2T. However, no such association was found for PL-SP-D and NSRI. Higher baseline PL-SP-D levels might be associated with more favourable treatment outcomes after steps I and II therapy. This may be due to its role in the regulation of neutrophil function. However, further investigation is required to confirm this hypothesis. If proven, PL-SP-D could play a role as a biomarker for identifying individuals who respond differentially to primary therapy.

PSIV-29 Efficacy of a chute-side pregnancy test on early pregnancy determination in mature Rambouillet ewes

Abstract Distinguishing pregnant from non-pregnant ewes in early pregnancy allows for more efficient flock management by producers. While early pregnancy detection techniques like ultrasonography and hormone quantification are well established, these techniques are often not adaptable to field conditions or require specialized equipment and training. The objective of this study was to evaluate a validated bovine commercial on-farm pregnancy test on efficacy for detecting early pregnancy in distinct blood constituents of ewes. Rambouillet ewes [n = 30; average body weight (BW) = avg 60.9 ± 1.1 kg; age 2 to 8 yr) were estrous synchronized using a 5 d CIDR protocol followed by 2 injections of PGF2α at 0 and 4 h. A rattle-painted ram was subsequently placed with ewes for a 35-d breeding season. Blood constituents including whole blood (WB), plasma (PL), and serum (SE) were collected via jugular venipuncture prior to synchronization (d 0) and on d 20, 35, and 70 after breeding. Blood constituents were individually evaluated using the IDEXX Alertys OnFarm pregnancy test for pregnancy detection. Trans-abdominal ultrasonography was performed on d 35 and d 70 post-breeding. Serum progesterone (P4) was quantified on d 100 of gestation to confirm pregnancy status. Progesterone concentrations ≥ 4 ng/mL were considered pregnant. On d 20 of gestation, IDEXX tests agreed with P4 concentrations on d 100 (χ2 = 0.30) in WB, SE and PL at 35.7, 53.6, and 53.6%, respectively. Pregnancy detection accuracy increased on d 35 of gestation as IDEXX tests agreed with P4 at d 100 (χ2 = 1.00) in WB, SE, and PL at 60.7, 60.7 and 60.7% accuracy, respectively. On d 70 of gestation, IDEXX tests agreed with P4 at d 100 (χ2 = 0.81) in WB, SE, and PL at 90.7, 93.3 and 93.3% accuracy, respectively indicating similarity in pregnancy diagnosis in the two methods. Detection of pregnancy using ultrasonography at d 35 identified 33% of pregnant ewes, while d 70 ultrasound detected 93% of pregnant ewes when compared with d 100 serum P4. Results suggest that the IDEXX pregnancy test provides early diagnosis of pregnancy with d 70 results comparable to d 100 serum P4 quantification regardless of blood constituent type.

Performance evaluation and characterization of pre-analytical effects by sample types and storage conditions on tumor marker measurement by the point-of-care test platform IchromaTM II

Tumor markers should be measured regularly and accurately to prevent, diagnose, and monitor cancers efficiently. We aimed to characterize the pre-analytical factors effecting on the analytical performance of point-of-care test (POCT) platform IchromaTM II (Boditech Med Inc., Gangwon-do, Korea) for alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), and prostate specific antigen (PSA) and evaluate their consequences in clinical practice. Based on comprehensive evaluation for the analytical performance of IchromaTM II including precision, linearity, and method comparison performed according to CLSI guidelines, pre-analytical factors of sample types and conditions were extensively analyzed. A total of five sample types [serum, plasma (PL) and whole blood (WB) from EDTA tube, PL and WB from sodium heparin tube] from 40 patients were used for comparing among specimen types. Additionally, stability was assessed up to 21 h at room temperature, refrigerated for 8 days, and frozen for 16 weeks by using 4 levels of pooled patient samples which were measured in triplicate. Precision, linearity and correlation with central laboratory analyzers observed in all three tumor markers were within acceptable criteria. However, variable degrees of percent deviations were observed according to sample type and storage conditions. Only EDTA PL samples presented clinically acceptable percentage biases for all three tumor markers when stored at room temperature or refrigerated condition. Positive bias of CEA and PSA in storage duration until 16 weeks were observed when stored in frozen condition. While IchromaTM II showed an adequate analytical performance as a POCT platform with simple operating procedures for the measurement of tumor markers, clinical laboratories should be aware of stability issues when different types of blood specimens are practically utilized.

Subclinical peripheral inflammation has systemic effects impacting central nervous system proteome in budgerigars

Regulation of neuroimmune interactions varies across avian species. Little is presently known about the interplay between periphery and central nervous system (CNS) in parrots, birds sensitive to neuroinflammation. Here we investigated the systemic and CNS responses to dextran sulphate sodium (DSS)- and lipopolysaccharide (LPS)-induced subclinical acute peripheral inflammation in budgerigar (Melopsittacus undulatus). Three experimental treatment groups differing in DSS and LPS stimulation were compared to controls. Individuals treated with DSS showed significant histological intestinal damage. Through quantitative proteomics we described changes in plasma (PL) and cerebrospinal fluid (CSF) composition. In total, we identified 180 proteins in PL and 978 proteins in CSF, with moderate co-structure between the proteomes. Between treatments we detected differences in immune, coagulation and metabolic pathways. Proteomic variation was associated with the levels of pro-inflammatory cytokine mRNA expression in intestine and brain. Our findings shed light on systemic impacts of peripheral low-grade inflammation in birds.

Flushing fluid of post-bronchoscopic biopsy as a novel liquid biopsy specimen for genome profiling in advanced lung cancer.

e15038 Background: Tumour tissue is the primary substrate for molecular testing in advanced NSCLC. However, obtaining sufficient tissue samples from biopsy for molecular testing can often be challenging. Flushing fluid of post-bronchoscopic biopsy, which contains tumor-derived DNA, may serve as a supplement for genotyping. The aim of this study is to assess the efficacy and precision of detecting gene alterations in flushing fluid samples and evaluate their clinical potential. Methods: Flushing fluid, matched tissue (TIS), and whole blood were prospectively collected from 25 patients with stage III-IV NSCLC between June 2022 and December 2023. Ten milliliters of flushing fluid and whole blood were used to prepare flushing fluid supernatant (FFS) and plasma (PLA), respectively. Capture-based targeted sequencing of TIS, FFS and PLA samples was performed using a universal oncology panel. Results: In this cohort of 25 patients, there was a male predominance (22 out of 25) and the mean age was 60.2 years. The majority of patients were diagnosed with lung adenocarcinoma (23 out of 25), and smokers outnumbered non-smokers (16 out of 25). Furthermore, 18 out of 25 patients presented with metastatic disease at diagnosis. The performance of three sample types regarding key quality control indices was evaluated first. FFS exhibited a maximum allelic fraction similar to TIS, while PLA showed a significantly lower than TIS and FFS. Subsequently, we compared the positive detection rates, with TIS, FFS, and PLA achieving respective rates of 100%, 96%, and 80%. A total of 495 mutation events were detected from the three samples (FFP:209, FFS:196, PLA:90), Among these, 161 were concordant between TIS and FFS samples, and 72 were concordant between TIS and PLA samples. The sensitivity and positive predictive value (PPV) of detecting eight established driver alterations in NSCLC were compared using TIS as the gold standard with the two liquid biopsies. FFS demonstrated superior sensitivity and PPV compared to PLA (80.5% vs. 56.1%, 97% vs. 88.5%) and showed equivalent performance for EGFR mutation. Furthermore, estimates of tumor mutational burden (TMB) based on TIS and FFS were highly correlated (Pearson r= 0.97). Conclusions: Our study demonstrated that FFS performs comparably to matched TIS and superiorly to PLA for genotyping advanced NSCLC. FFS emerges as a valuable specimen source, especially in patients with insufficient tumor tissue.

Abstract 967: Clinical applicability of targeted panel sequencing in liquid biopsies for disease diagnosis and monitoring of patients with pediatric solid tumors

Abstract Liquid biopsy assays based on sequence analysis of cell-free DNA (cfDNA) have the potential to transform patient care. We recently validated a liquid biopsy platform, LBSeq4Kids, for patients with retinoblastoma, brain tumors and solid tumors using low pass whole genome sequencing (LP-WGS) of cfDNA from aqueous humor (AH), cerebrospinal fluid (CSF), and plasma (PL) to detect Copy Number Alterations (CNAs) in circulating tumor DNA (ctDNA). Herein, we present phase 2, a high-depth comprehensive Next-Generation Sequencing (NGS) panel using cfDNA that allows us to detect SNV’s, indels and gene fusions in clinically relevant genes for pediatric solid and brain tumors. Seventy-four cfDNA specimens have been analyzed to date, using plasma from 43 patients with solid tumors, CSF from 25 patients with brain tumors and AH from six patients with ocular tumors including retinoblastoma. cfDNA was extracted at diagnosis, during therapy, at remission and/or relapse. Sequencing libraries were constructed with the xGen Prism DNA Library Prep Kit using 5ng of cfDNA. For the detection of recurrent mutations and fusions we designed a custom panel using a hybridization-based capture method (Twist Bioscience) that included full coverage of the exons of 136 genes harboring recurrent mutations and selected introns for five genes with recurrent rearrangement breakpoints including BRAF, EWSR1, SYT, FOXO1 and NTRK1. Hybridized libraries were paired-end sequenced (Illumina NextSeq 500) at an average 1000x collapsed coverage. Mutations identified in cfDNA were compared with those in the primary or metastatic tumors profiled with our OncoKids® NGS panel. A total of 35/43 (81%) plasma specimens were positive for either an SNV, deletion or fusion with the most common events being a somatic missense variant in TP53 (13/35, 37%) or translocation involving EWSR1 (9/35, 26%). A total of 16/25 (64%) CSF specimens were positive for SNVs and fusions, most often involving KIAA1549::BRAF (7/25, 28%) in low grade gliomas. A germline variant in RB1 was detectable in two AH specimens from the left and right eye of a patient with bilateral retinoblastoma while plasma from a patient with metastatic retinoblastoma demonstrated clinically relevant variants in RB1 and DICER1. Serial cfDNA profiling revealed a clinically reported TP53 c.746G>C missense variant in the plasma of a patient with osteosarcoma at multiple timepoints through the disease course, with evidence for relapse prior to standard imaging. These data suggest that clinical implementation of a second version of LBSeq4Kids that combines CNA and mutation analysis allows for diagnosis, residual disease detection and risk stratification for pediatric patients with diverse tumor types, regardless of histologic grade, with a variety of pediatric solid and brain tumors. Citation Format: Eirini Christodoulou, Venkata Yellapantula, Katrina O'Halloran, Liya Xu, Jesse L. Berry, Fariba Navid, Dejerianne Ostrow, Jaclyn A. Biegel. Clinical applicability of targeted panel sequencing in liquid biopsies for disease diagnosis and monitoring of patients with pediatric solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 967.

WITHDRAWN: Psychoneuroimmunological characterization in schizophrenia requires health-related adjustments

Schizophrenia is severe and complex. It’s etiology, course, and severity are influe nced by biologica l (e.g., genetic) and psychosocial factors (e.g., urbanicity). Altera tions in cytokine s leve ls (im mune function bioma rkers) were reported in schizophrenia, which related to symptom severity, indicating a potentia l role of im mune activity in this dis order. Cytokine leve ls are influenced by life habits (e.g., smoking), personal characteristics (e.g., sex/ gender), and life conditions (e.g., care access).We used data from the Signature Biobank: 262 schizophrenic patients vis iting an emergency room in Montreal, Canada, and 145 healthy controls. We investigated pla sma concentrations of IL-6, TNF-α and CRP in relation to schizophrenia diagnosis and symptom severity and adjusted analyses with tobacco use, BMI, age and sex.Patients showed higher levels of IL-6 (t=2.67, p=0.008) than controls, along with females compared to ma le s (t=1.844, p=0.033), and smokers compared to non-smokers (t=2.304, p=0.029). Among non-smokers, IL-6 remained higher for patients (t=1.932, p(unila teral)=0.027), but not when controlling for age and BMI (F=2.849, p=0.093). Non-smoking female patients showed higher IL-6 levels, even when controlling for BMI and age (F=6.916, p=0.010). TNF-α correlated with depressive (r=0.162, p=0.010) and psychotic symptoms (r=0.213, p<0.001) among patients.Cytokine s in schizophrenia need contextua liza tion to refle ct life habits ’ influence. Each cytokine has a different role in im mune activity that relates differently with dia gnos is and symptom severity.Immunophenotyping among schizophre nic patients require s adjusting for individual characteristics. This characterizati

Crosstalk between oxidative stress, inflammation, angiogenesis and apoptosis in diabetic macular edema

Aims/Purpose: To evaluate molecules involved in oxidative stress (OS), inflammation, angiogenesis, and apoptosis, and discern which of these are more implicated in diabetic retinopathy (DR) and diabetic macular edema (DME) by investigating the correlation between them in the plasma (PLS) and vitreous humour (VIT), and examining data obtained from ophthalmological examinations.Methods: Type 2 diabetic (T2DM) patients with DR/DME (DRG/DMEG n = 112) and non‐DM subjects as the surrogate controls (SCG n = 48) were selected. Blood samples were processed to determine the glycaemic and lipid profiles, C reactive protein, malondialdehyde (MDA), 4‐hydroxynonenal (4HNE), superoxide dismutase (SOD), catalase (CAT) levels and total antioxidant capacity (TAC). In addition, interleukin 6 (IL6), vascular endothelial growth factor (VEGF), and caspase 3 were assayed. The VIT samples were collected to measure the expression levels of all the above‐mentioned molecules.Results: Our findings support the presence of molecules involved in OS, inflammation, angiogenesis, and apoptosis in the PLS and VIT samples from T2DM. In PLS from DRG, there was a decrease in the antioxidant load and an increase in pro‐angiogenic molecules, but an increase in pro‐oxidants and a decline in antioxidants intravitreally. In PLS from DMEG, pro‐oxidants and pro‐inflammatory molecules were augmented and the antioxidant capacity diminished, but the pro‐oxidants increased, and antioxidants decreased intravitreally. Furthermore, we found a positive correlation between the PLS‐CAT and the VIT‐SOD levels in DRG, and a negative correlation between the PSD‐4HNE and the VIT‐TAC levels in DMEG.Conclusions: We conclude that hyperglycaemia induces anomalies in biochemical pathways with crosstalk between them; MDA, 4HNE, TAC, VEGF, and IL6 are promoters of the clinical manifestations of PDR and DME. The above molecules have been selected as biomarker candidates for distinguishing T2DM patients at risk of DR, DME and vision loss.

Neurodegeneration in diabetic retinopathy: An insight on its main signalling pathways

Aims/Purpose: In the latest years neurodegeneration has been considered as one of the main pathological processes involved in proliferative diabetic retinopathy's (PDR) onset and progression. We aimed to increase the understanding on the role of the main signalling molecules that participate in this phenomenon, including apoptotic, excitotoxic and neurotrophic factor pathways.Methods: Participants of both sexes were selected and programmed for vitrectomy by its diabetes mellitus 2 (DM2)‐derived vitreoretinal complications (PDR group, PDRG; n = 20), or by suffering macular hole, epiretinal membrane or rhegmatogenous retinal detachment (as the surrogate control group, SCG; n = 26). Vitreous body (VIT) and plasma (PLS) samples were collected and processed to determine the glutamate, caspase‐3 (CAS‐3) and brain‐derived neurotrophic factor (BDNF) levels. Next‐Generation Sequencing (NGS) techniques were applied to characterize the miRNA expression pattern in both groups.Results: Significantly higher VIT glutamate (p &lt; 0.05) and CAS‐3 (p &lt; 0.05), and significantly lower BDNF (p &lt; 0.05) levels were detected in the PDRG. Significantly higher PLS glutamate (p &lt; 0.05) and CAS‐3 (p &lt; 0.05) levels were found in diabetics. However, PLS levels of BDNF showed no differences between groups (p = 0.80). NGS analysis showed a different miRNA expression pattern between both groups.Conclusions: Diabetes‐induced changes in retinal neurons/glia may be the key to discover preclinical biomarkers for better managing diabetic eyes. Glutamate, caspase‐3 and BDNF, playing important roles in the neurodegenerative processes occurring in PDR, are pivotal candidates to identify diabetics at high risk of sight‐threatening complications. Additionally, certain miRNAs could be playing an important role in these signalling pathways, being involved in PDR's neurodegenerative phenomenon.

A comparative study of plasma and dried blood spot metabolomics and its application to diabetes mellitus

Metabolomics has become a promising method for understanding pathological mechanisms. Plasma (PLS) is the most common sample type used for metabolomics studies, and dried blood spot (DBS) sampling has been regarded as a good strategy due to its unique characteristics. However, how results obtained from DBS can be correlated to results obtained from PLS remains unclear. To bridge the results and to investigate the feasibility of using DBS to study metabolomics, we performed a comparative study using 64 paired PLS and DBS samples. The number of features extracted from the two different sample types was investigated. The concentration correlations of the identified metabolites between the DBS and PLS were individually studied. Approximately 47% showed a strong correlation, 19% showed a moderate correlation, and 34% showed a low or even negligible correlation. Finally, we applied both PLS- and DBS-based metabolomics to explore the dysregulated metabolites in diabetes mellitus (DM) patients. Thirty-two non-DM subjects and 32 DM patients were enrolled, and 2 significant metabolites were found in both PLS and DBS samples. In summary, detailed correlation information between PLS and DBS metabolites was first explored in this study, and it is anticipated that these results could facilitate future applications in DBS-based metabolomics.

Influence of exposure of customized dental implant abutments to different cleaning procedures: an in vitro study using AI-assisted SEM/EDS analysis

PurposeDental implant abutments are defined as medical devices by their intended use. Surfaces of custom-made CAD/CAM two-piece abutments may become contaminated during the manufacturing process in the dental lab. Inadequate reprocessing prior to patient care may contribute to implant-associated complications. Risk-adapted hygiene management is required to meet the requirements for medical devices.MethodsA total of 49 CAD/CAM-manufactured zirconia copings were bonded to prefabricated titanium bases. One group was bonded, polished, and cleaned separately in dental laboratories throughout Germany (LA). Another group was left untreated (NC). Five groups received the following hygiene regimen: three-stage ultrasonic cleaning (CP and FP), steam (SC), argon–oxygen plasma (PL), and simple ultrasonic cleaning (UD). Contaminants were detected using scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDS) and segmented and quantified using interactive machine learning (ML) and thresholding (SW). The data were statistically analysed using non-parametric tests (Kruskal–Wallis test, Dunn’s test).ResultsSignificant differences in contamination levels with the different cleaning procedures were found (p ≤ 0.01). The FP–NC/LA groups showed the most significant difference in contamination levels for both measurement methods (ML, SW), followed by CP–LA/NC and UD–LA/NC for SW and CP–LA/NC and PL–LA/NC for ML (p ≤ 0.05). EDS revealed organic contamination in all specimens; traces of aluminum, silicon, and calcium were detected.ConclusionsChemothermal cleaning methods based on ultrasound and argon–oxygen plasma effectively removed process-related contamination from zirconia surfaces. Machine learning is a promising assessment tool for quantifying and monitoring external contamination on zirconia abutments.Graphical

Therapeutic drug monitoring of mycophenolic acid (MPA) using volumetric absorptive microsampling (VAMS) in pediatric renal transplant recipients: ultra-high-performance liquid chromatography-tandem mass spectrometry analytical method development, cross-validation, and clinical application

BackgroundMycophenolic acid (MPA) is widely used in posttransplant pharmacotherapy for pediatric patients after renal transplantation. Volumetric absorptive microsampling (VAMS) is a recent approach for sample collection, particularly during therapeutic drug monitoring (TDM). The recommended matrix for MPA determination is plasma (PL), and conversion between capillary-blood VAMS samples and PL concentrations is required for the appropriate interpretation of the results.MethodsThis study aimed to validate and develop a UHPLC-MS/MS method for MPA quantification in whole blood (WB), PL, and VAMS samples, with cross and clinical validation based on regression calculations. Methods were validated in the 0.10–15 µg/mL range for trough MPA concentration measurement according to the European Medicines Agency (EMA) guidelines. Fifty pediatric patients treated with MPA after renal transplantation were included in this study. PL and WB samples were obtained via venipuncture, whereas VAMS samples were collected after the fingerstick. The conversion from VAMSMPA to PLMPA concentration was performed using formulas based on hematocrit values and a regression model.ResultsLC–MS/MS methods were successfully developed and validated according to EMA guidelines. The cross-correlation between the methods was evaluated using Passing-Bablok regression, Bland–Altman bias plots, and predictive performance calculations. Clinical validation of the developed method was successfully performed, and the formula based on regression was successfully validated for VAMSMPA to PLMPA concentration and confirmed on an independent group of samples.ConclusionsThis study is the first development of a triple matrix-based LC–MS/MS method for MPA determination in the pediatric population after renal transplantation. For the first time, the developed methods were cross-validated with routinely used HPLC–DAD protocol.Graphical

Oxidative Stress, Inflammatory, Angiogenic, and Apoptotic molecules in Proliferative Diabetic Retinopathy and Diabetic Macular Edema Patients.

The aim of this study is to evaluate molecules involved in oxidative stress (OS), inflammation, angiogenesis, and apoptosis, and discern which of these are more likely to be implicated in proliferative diabetic retinopathy (PDR) and diabetic macular edema (DME) by investigating the correlation between them in the plasma (PLS) and vitreous body (VIT), as well as examining data obtained from ophthalmological examinations. Type 2 diabetic (T2DM) patients with PDR/DME (PDRG/DMEG; n = 112) and non-DM subjects as the surrogate controls (SCG n = 48) were selected according to the inclusion/exclusion criteria and programming for vitrectomy, either due to having PDR/DME or macular hole (MH)/epiretinal membrane (ERM)/rhegmatogenous retinal detachment. Blood samples were collected and processed to determine the glycemic profile, total cholesterol, and C reactive protein, as well as the malondialdehyde (MDA), 4-hydroxynonenal (4HNE), superoxide dismutase (SOD), and catalase (CAT) levels and total antioxidant capacity (TAC). In addition, interleukin 6 (IL6), vascular endothelial growth factor (VEGF), and caspase 3 (CAS3) were assayed. The VITs were collected and processed to measure the expression levels of all the abovementioned molecules. Statistical analyses were conducted using the R Core Team (2022) program, including group comparisons and correlation analyses. Compared with the SCG, our findings support the presence of molecules involved in OS, inflammation, angiogenesis, and apoptosis in the PLS and VIT samples from T2DM. In PLS from PDRG, there was a decrease in the antioxidant load (p < 0.001) and an increase in pro-angiogenic molecules (p < 0.001), but an increase in pro-oxidants (p < 0.001) and a decline in antioxidants (p < 0.001) intravitreally. In PLS from DMEG, pro-oxidants and pro-inflammatory molecules were augmented (p < 0.001) and the antioxidant capacity diminished (p < 0.001), but the pro-oxidants increased (p < 0.001) and antioxidants decreased (p < 0.001) intravitreally. Furthermore, we found a positive correlation between the PLS-CAT and the VIT-SOD levels (rho = 0.5; p < 0.01) in PDRG, and a negative correlation between the PSD-4HNE and the VIT-TAC levels (rho = 0.5; p < 0.01) in DMEG. Integrative data of retinal imaging variables showed a positive correlation between the central subfield foveal thickness (CSFT) and the VIT-SOD levels (rho = 0.5; p < 0.01), and a negative correlation between the CSFT and the VIT-4HNE levels (rho = 0.4; p < 0.01) in PDRG. In DMEG, the CSFT displayed a negative correlation with the VIT-CAT (rho = 0.5; p < 0.01). Exploring the relationship of the abovementioned potential biomarkers between PLS and VIT may help detecting early molecular changes in PDR/DME, which can be used to identify patients at high risk of progression, as well as to monitor therapeutic outcomes in the diabetic retina.

The variability of arsenic in blood and urine of humans

BackgroundHumans are exposed to inorganic and organic arsenic. The total arsenic (As) concentration in urine is a commonly used biomarker of exposure. However, little is known about variability of As in biological fluids and the diurnal variation of As excretion. ObjectivesMain objectives were to assess the variability of As in urine, plasma (P-As), whole blood (B-As), and the blood cell fraction (C-As), and to assess diurnal variation of As excretion. MethodsSix urine samples were collected at fixed times during 24 h on two different days around one week apart among 29 men and 31 women. Blood samples were collected when the morning urine samples were delivered. The intra-class correlation coefficient (ICC) was calculated as the ratio of the between-individuals variance to the total observed variance. ResultsGeometric mean (GM) 24 h urinary excretions of As (U-As24 h) were 41 and 39 µg/24 h on the two days of sampling. Concentrations of B-As, P-As and C-As were highly correlated with U-As24 h and As in first void morning urine. No statistically significant differences were observed for the urinary As excretion rate between the different sampling times. A high ICC was observed for As in the cellular blood fraction (0.803), while ICC for first morning urine corrected for creatine was low (0.316). ConclusionsThe study suggests that C-As is the most reliable biomarker for use in exposure assessment of individual exposure. Morning urine samples have low reliability for such use. No apparent diurnal variation was observed in the urinary As excretion rate.

Proteomic profiling of extracellular vesicles in synovial fluid and plasma from Oligoarticular Juvenile Idiopathic Arthritis patients reveals novel immunopathogenic biomarkers.

New early low-invasive biomarkers are demanded for the management of Oligoarticular Juvenile Idiopathic Arthritis (OJIA), the most common chronic pediatric rheumatic disease in Western countries and a leading cause of disability. A deeper understanding of the molecular basis of OJIA pathophysiology is essential for identifying new biomarkers for earlier disease diagnosis and patient stratification and to guide targeted therapeutic intervention. Proteomic profiling of extracellular vesicles (EVs) released in biological fluids has recently emerged as a minimally invasive approach to elucidate adult arthritis pathogenic mechanisms and identify new biomarkers. However, EV-prot expression and potential as biomarkers in OJIA have not been explored. This study represents the first detailed longitudinal characterization of the EV-proteome in OJIA patients. Fourty-five OJIA patients were recruited at disease onset and followed up for 24 months, and protein expression profiling was carried out by liquid chromatography-tandem mass spectrometry in EVs isolated from plasma (PL) and synovial fluid (SF) samples. We first compared the EV-proteome of SF vs paired PL and identified a panel of EV-prots whose expression was significantly deregulated in SF. Interaction network and GO enrichment analyses performed on deregulated EV-prots through STRING database and ShinyGO webserver revealed enrichment in processes related to cartilage/bone metabolism and inflammation, suggesting their role in OJIA pathogenesis and potential value as early molecular indicators of OJIA development. Comparative analysis of the EV-proteome in PL and SF from OJIA patients vs PL from age/gender-matched control children was then carried out. We detected altered expression of a panel of EV-prots able to differentiate new-onset OJIA patients from control children, potentially representing a disease-associated signature measurable at both the systemic and local levels with diagnostic potential. Deregulated EV-prots were significantly associated with biological processes related to innate immunity, antigen processing and presentation, and cytoskeleton organization. Finally, we ran WGCNA on the SF- and PL-derived EV-prot datasets and identified a few EV-prot modules associated with different clinical parameters stratifying OJIA patients in distinct subgroups. These data provide novel mechanistic insights into OJIA pathophysiology and an important contribution in the search of new candidate molecular biomarkers for the disease.

Plasma and urinary metanephrine and normetanephrine concentrations using liquid chromatography with tandem mass spectrometry in healthy cats and in a cat with pheochromocytoma

Pheochromocytoma (PCC) is rare in cats and plasma (PL) and urinary (U) metanephrines (metanephrine [MN]; normetanephrine [NMN]) measurement is rarely described in cats. We evaluated the utility of PL and U MNs measurement in 10 healthy cats and a cat with a confirmed diagnosis of pheochromocytoma (PheoCat), using liquid chromatography with tandem mass spectrometry (LC-MS-MS). Urine and EDTA PL samples collected from each of the 10 cats and the PheoCat were promptly stored at -80°C and remained frozen until analysis. To evaluate U MNs stability, an additional urine sample collected from the healthy cats was refrigerated for 24 hours before freezing. Urinary creatinine concentration (Creat) was assessed using the same spot urine samples to calculate U MNs-to-creatinine ratios. The PL-MN and PL-NMN median concentrations of the healthy cats were 2.73 and 7.02 nmol/L, respectively. The median U-MN/Creat and U-NMN/Creat ratios were 70 and 139 μg/g, respectively. The PheoCat had a PL-MN of 3.68 nmol/L, PL-NMN of 66.27 nmol/L, U-MN/Creat of 179 μg/g, and U-NMN/Creat of 1262 μg/g. The PheoCat had markedly increased concentrations of both PL and U MNs when compared to the healthy cats. No significant difference was found between U MNs measured in urine samples that underwent 24 hours of refrigeration in comparison to those that were frozen immediately. We report preliminary reference intervals for PL and U MNs in cats using LC-MS-MS and the potential clinical applicability of these biomarkers for the diagnosis of PCC in cats.

Matrix Metalloproteinase-9 Level in Synovial Fluid-Association with Joint Destruction in Early Rheumatoid Arthritis.

Background and objective: Matrix metalloproteinases (MMPs) are the key enzymes in the pathogenesis of cartilage and joint damage and potentially a new biomarker of the early erosive form of rheumatoid arthritis (RA). Firstly, the study aimed to compare the level of MMP-9 in plasma (PL) and synovial fluid (SF) of patients with RA and osteoarthritis (OA). Secondly, the goal was to examine the association of MMP-9 level in PL and SF with early erosive changes in RA, and finally, to determine the association of MMP-9 level with serological parameters of the disease (rheumatoid factor-RF and anti-citrulline protein antibodies-ACPA). Materials and Methods: A total of 156 subjects were involved in this study (84 patients with RA and 72 patients with OA, who were involved as a control group). MMP-9 level was measured in PL and SF of all subjects by the sandwich enzyme-linked immunosorbent assay (ELISA) method. Standard radiographs of the hands and feet were used to detect joint damage and classification into erosive or non-erosive RA. The Larsen score (LS) was used for the quantitative assessment of joint damage, and its annual change (∆ LS) was used to assess the radiographic progression of the disease. Results: MMP-9 level in PL and SF was significantly higher in RA compared to controls (PL: 19.26 ± 7.54 vs. 14.57 ± 3.11 ng/mL, p&lt; 0.01; SF: 16.17 ± 12.25 vs. 0.75 ± 0.53 ng/mL, p &lt; 0.001) as well as in SF of patients with erosive compared to non-erosive RA (18.43 ± 12.87 vs. 9.36 ± 7.72; p &lt; 0.05). Faster radiographic progression was recorded in erosive compared to non-erosive early RA (11.14 ± 4.75 vs. 6.13 ± 2.72; p &lt; 0.01). MMP-9 level in SF, but not in PL, significantly correlates with the radiographic progression in both erosive and non-erosive RA (ρ = 0.38 and ρ = 0.27). We did not find a significant association between RF and MMP-9 level in early RA, but the ACPA level significantly correlates with MMP-9 level in SF (r = 0.48). Conclusion: The level of MMP-9 in plasma and synovial fluid of patients with RA is significantly higher compared to patients with osteoarthritis. The level of MMP-9 in synovial fluid is significantly higher in erosive than non-erosive early RA. It is significantly associated with the radiographic progression of the disease and the level of anti-citrulline protein antibodies.

Maternal-fetal biodisposition of buprenorphine and its metabolites in opioid dependent pregnant individuals

To determine the concentration of buprenorphine (BUP) and its metabolites in maternal and fetal samples and investigate the relationship between BUP concentrations and neonatal opioid withdrawal syndrome (NOWS). Matched maternal and fetal samples were collected from 12 individuals maintained on BUP for opioid use disorder during pregnancy. All delivered at term (≥37 weeks gestation) via cesarean section. Samples were collected at delivery and included maternal plasma (PL), amniotic fluid (AF), and umbilical cord blood (CB). Concentrations of BUP, its biologically active metabolite norbuprenorphine (NBUP) and its inactive metabolites glucuronide forms of buprenorphine (BUP-Gluc) and norbuprenorphine (NBUP-Gluc) were determined by liquid chromatography-mass spectrometry. The concentrations of BUP and its metabolites were compared between neonates who developed NOWS, defined as withdrawal symptoms requiring pharmacologic treatment, and those who did not. Five (42%) of neonates born to mothers in the study developed NOWS. There was no difference in gestational age, birthweight or other baseline characteristics between the two groups. All individuals in the study were receiving 16 mg daily of BUP at delivery. BUP concentrations were higher in cord blood (0.65 v 0.31ng/mL, p=0.01) and amniotic fluid (0.64 v 0.0 ng/mL, p=0.04) in neonates who developed NOWS, compared to those who did not. The concentrations of NBUP were also higher in neonates who developed NOWS (2.01 v 0.0ng/mL, p=0.03). There were no differences in maternal plasma concentrations between the two groups. Despite similar maternal dosing and plasma concentrations, NBUP concentrations in AF and BUP concentrations in AF and CB were higher for infants later diagnosed with NOWS, suggesting accumulation of the drug in the fetal compartment. Further studies are needed to determine the mechanisms and whether analysis of these specimens at delivery may help predict NOWS in buprenorphine-exposed neonates.

Cerebrospinal fluid-derived circulating tumor DNA is more comprehensive than plasma in NSCLC patients with leptomeningeal metastases regardless of extracranial evolution

IntroductionMetastases to the central nervous system (CNS) are devastating neurological complications. Circulating cell-free tumor DNA (ctDNA) from cerebrospinal fluid (CSF) better represents genomic alterations in CNS tumors compared to plasma (PLA). However, the clinical value of cerebrospinal fluid (CSF) as a liquid biopsy medium in non-small cell lung cancer patients with leptomeningeal metastases (NSCLC-LM), regardless of extracranial evolution, remains unclear. Patients and methods14/48 NSCLC-BM patients and 34/48 NSCLC-LM patients were enrolled in this study. The genomic mutation profiles in CSF and matched PLA for patients with single CNS progression (cohort one, N = 22) or intracranial progression with extracranial disease progression (cohort two, N = 12) were compared. ctDNA in the CSF and simultaneously collected PLA was subjected to next-generation target sequencing (NGS) of 168 cancer-relevant genes. ResultsCSF is more comprehensive of driver genomic mutation profile than in matched PLA in patients with a single CNS progression. In addition, potential prognostic markers are much higher in CSF samples than related PLA. For example, the detection rate of EGFR-amp in CSF was more than twice of the rate in matched PLA. Moreover, CDKN2A/B, PIK3CA/G, CDK4/6, and MET were detected uniquely in CSF samples and, all of these genetic mutations were correlated with poor outcomes.Almost all genetic mutation profiles detected in PLA could be seen in matched CSF samples in cohort two. With the driver genes, such as EGFR or ALK, have a higher detection rate in CSF compared to PLA. Moreover, the potential survival maker genes CDK4/6 (6/12, 50%), CDKN2A/B (2/12, 17%), EGFR-amp (1/12, 8%), MET (1/12, 8%), and PIK3CA (1/12, 8%) were unique to the CSF samples. ConclusionFor NSCLC -LM patients, regardless of single intracranial progression or intracranial progression simultaneously with extracranial evolution, CSF is superior to matched PLA.

CNSC-04. CEREBROSPINAL FLUID-DERIVED CIRCULATING TUMOR DNA IS SUPERIOR TO PLASMA IN NSCLC PATIENTS WITH CNS PROGRESSION

Abstract Introduction Central nervous system (CNS) metastases are devastating neurologic complications. However, the clinical value of cerebrospinal fluid (CSF) as liquid biopsy medium in EGFR-mutated or ALK-rearranged patients with brain metastases (BM) or leptomeningeal metastases (LM) remains unclear. Patients and Methods We retrospectively analyzed the gene map of 14/48 NSCLC-BM patients and 34/48 NSCLC-LM patients. Compare the clinical predictive value of CSF and matched plasma (PLA) for patients with single CNS progression (cohort 1) or intracranial progression simultaneously with extracranial disease progression (cohort 2). Circulating cell-free tumor DNA (ctDNA) in CSF and matched PLA underwent next-generation sequencing targeting 168 cancer-relevant genes. Results For patients with single CNS progression, the mutation rate of major driver genes is much higher than the rate in matched PLA. Besides, CSF also demonstrated higher levels of potential prognostic markers in comparison with matched PLA samples, showing EGFR-amp presented more than two times the rate compared to matched PLA, and the detection rate of TBX3, PTEN were almost double the rate in CSF than paired PLA samples. In addition, poor outcomes associated with makers including CDKN2A/B, PIK3CA/G, CDK4/6, and MET showed exclusively in CSF samples.Almost all mutations identified in the PLA could detect in matched CSF samples in NSCLC-LM patients with both intracranial and extracranial disease progression. In addition, detection rates of driver mutations, consisting of EGFR or ALK are much higher in CSF compared to matched PLA, and these potential survival marker genes like CDK4/6, CDKN2A/B, EGFR-amp, MET, and PIK3CA identified uniquely in CSF samples. Conclusion CSF is more comprehensive in genomic mutation profile than matched PLA in NSCLC -LM regardless of single intracranial progression or intracranial progression simultaneously with extracranial evolution.