Individual Plasma Samples Research Articles

Use of Affimer Technology for Inhibition of α2-antiplasmin and Enhancement of Fibrinolysis

Hypofibrinolysis is a documented abnormality in conditions with high risk of vascular occlusion. A key inhibitor of fibrinolysis is α2-antiplasmin (α2AP) and we hypothesise that the Affimer technology, comprising small conformational proteins with two nine amino acid variable regions, can be used to modulate α2AP activity and facilitate fibrinolysis.Using a phage display system, a library of Affimers was screened against α2AP. A total of 28 α2AP-specific Affimers were isolated of which one, termed Affimer A11, inhibited protein function and enhanced fibrinolysis. Affimer A11 displayed a monomeric form and consistently reduced lysis time of clots made from plasma samples of individuals with type 2 diabetes mellitus (n=15; from 150.8±100.9 to 109.8±104.8 mins) and those with cardiovascular disease (n=15; 117.6±40.6 to 79.7±33.3 mins); p<0.01 for both groups. The effects of A11 on fibrinolysis were maintained when clots were made from whole blood samples.Mechanistic studies demonstrated that A11 did not affect clot structure or interfere with incorporation of α2AP into fibrin networks but significantly enhanced plasmin activity and accelerated plasmin generation. Affimer A11 reduced α2AP binding to plasmin(ogen), while molecular modelling demonstrated interactions with α2AP in an area responsible for binding to plasminogen, explaining the effects on both plasmin activity and generation. Affimer A11, at 0.15-0.60 mg/ml, had the ability to bind 70-90% of plasma α2AP.In conclusion, we demonstrate that Affimers are viable tools for inhibiting α2AP function and facilitating fibrinolysis, making them potential future therapeutic agents to reduce thrombosis risk.

Leishmania Infection among HIV-Infected Patients in a Southern Province of Thailand: A Cross-Sectional Study.

Leishmaniasis, a neglected tropical disease, imposes a notable health burden, especially on immunocompromised individuals such as HIV patients. Recognizing its prevalence and risk factors in specific populations is vital for effective prevention. This study in Satun Province, southern Thailand, aimed to ascertain leishmaniasis prevalence and identify associated risks among HIV-infected patients. A cross-sectional study was conducted among 650 HIV-infected individuals at a tertiary care hospital. Data on demographic characteristics, clinical parameters, and potential risk factors were collected. Individual plasma, buffy coat, and saliva samples were collected. Leishmania infection was determined using the direct agglutination test and nested polymerase chain reaction (nPCR) of nPCR-buffy coat and nPCR-saliva. The association between risk factors and Leishmania infection was assessed with logistic regression analysis. The prevalence of Leishmania infection was 8.61% (56/650). Species was identified among 20 HIV-infected patients as follows: Leishmania orientalis (N = 14), Leishmania martiniquensis (N = 4), and Leishmania donovani complex (N = 2). The factors associated with Leishmania infection included age (adjusted odds ratio [OR] = 1.03), intravenous drug use (adjusted OR = 2.39), CD4 cell count <500 cells/mm3 (adjusted OR = 2.40), and a viral load ≥50 copies/mL (adjusted OR = 5.16). The prevalence of Leishmania infection among HIV-infected patients in Satun Province was considerable. These findings underscore the need for integrated care and targeted interventions to address this infection and improve public health outcomes. Further research and collaborative efforts are warranted to develop effective prevention and control strategies for Leishmania infection in the HIV-infected Thai population.

Human plasma can modulate micronucleus frequency in TK6 and OE33 cells in vitro

In this paper, we studied the potential genotoxic effects of human plasma from healthy volunteers, as well as patients with gastro-oesophageal reflux disease, Barrett’s oesophagus (BO) and oesophageal adenocarcinoma (OAC) using the oesophageal adenocarcinoma cell line (OE33) and the lymphoblastoid cell line (TK6). Both TK6 and OE33 cells were treated with plasma (10 % volume, replacing foetal bovine serum (FBS) or horse serum (HS)) at different time points of 4 h (for the micronucleus (Mn) assay and the invasion assay) and 24 h (for the cell cycle studies). Plasma-induced effects on DNA damage levels, cell viability and the cell cycle were studied by the micronucleus assay, cytokinesis block proliferation index (CBPI) and flow cytometry respectively. The expression of IL-8 in supernatants of TK6 cells and IFN-β in OE33 cells was also analysed by enzyme-linked immunosorbent assay (ELISA). Finally, we carried out an assessment of cellular invasion of OE33 cells following plasma treatment.The results of the micronucleus assay confirmed the genotoxicity of direct plasma treatment from some participants through the increase in DNA damage in TK6 cells. Conversely, some individual patient plasma samples reduced background levels of TK6 cell Mn frequency, in an anti-genotoxic fashion. In TK6 cells, (on average) plasma samples from patients with Barrett’s oesophagus induced higher micronucleus levels than healthy volunteers (p= 0.0019). There was little difference in Mn induction when using plasma versus serum to treat the cells in vitro. Cell cycle results showed that direct plasma treatment had a marked impact on OE33 cells at 24 h (p=0.0182 for BO and p=0.0320 for OAC) by decreasing the proportion of cells in the S phase, while plasma exposure was less impactful on the cell cycle of TK6 cells. Invasion of OE33 cells was also seen to be non-significantly affected by plasma treatment of OE33 cells.The addition of N-acetyl cysteine NAC in a dose-dependent matter did not alter the formation of Mn in TK6 cells, suggesting that reactive oxygen species (ROS) are not the root cause of plasma’s genotoxicity. The concentration of IL-8 in TK6 cells and IFN-β in OE33 cells was significantly higher in cells treated with OAC-derived plasma than in the untreated negative control. Collectively, our results demonstrate that plasma-specific effects are detectable which helps us better understand some important aspects of the biology of blood-based biomarkers under development.

Further evidence supporting the role of GTDC1 in glycine metabolism and neurodevelopmental disorders

Copy number variants (CNVs) represent the genetic cause of about 15–20% of neurodevelopmental disorders (NDDs). We identified a ~67 kb de novo intragenic deletion on chromosome 2q22.3 in a female individual showing a developmental encephalopathy characterised by epilepsy, severe intellectual disability, speech delay, microcephaly, and thin corpus callosum with facial dysmorphisms. The microdeletion involved exons 5-6 of GTDC1, encoding a putative glycosyltransferase, whose expression is particularly enriched in the nervous system. In a previous study, a balanced de novo translocation encompassing GTDC1 was reported in a male child with global developmental delay and delayed speech and language development. Based on these premises, we explored the transcriptomic profile of our proband to evaluate the functional consequences of the novel GTDC1 de novo intragenic deletion in relation to the observed neurodevelopmental phenotype. RNA-seq on the proband’s lymphoblastoid cell line (LCL) showed expression changes of glycine/serine and cytokine/chemokine signalling pathways, which are related to neurodevelopment and epileptogenesis. Subsequent analysis by ELISA (enzyme-linked immunosorbent assay) and HPLC (high-performance liquid chromatography) revealed increased levels of glycine in the proband’s LCL and serum compared to matched controls. Given that an increased level of glycine has been observed in the plasma samples of individuals with Rett syndrome, a condition sharing epilepsy, microcephaly, and intellectual disability with our proband, we proposed that the GTDC1 downregulation is implicated in neurodevelopmental impairment by altering glycine metabolism. Furthermore, our findings expanded the phenotypic spectrum of the novel GTDC1-related condition, including microcephaly and epilepsy among relevant clinical features.

Determination of Δ9-tetrahydrocannabinol, 11-nor-carboxy-Δ9-tetrahydrocannabinol and cannabidiol in human plasma and urine after a commercial cannabidiol oil product intake

PurposeCannabidiol (CBD) products are widely used for pain relief, sleep improvement, management of seizures etc. Although the concentrations of Δ9-tetrahydrocannabinol (Δ9-THC) in these products are low (≤0.3% w/w), it is important to investigate if its presence and/or that of its metabolite 11-nor-carboxy-Δ9-THC, is traceable in plasma and urine samples of individuals who take CBD oil products.MethodsA sensitive GC/MS method for the determination of Δ9-THC, 11-nor-carboxy-Δ9-THC and CBD in plasma and urine samples was developed and validated. The sample preparation procedure included protein precipitation for plasma samples and hydrolysis for urine samples, solid-phase extraction and finally derivatization with N,O-bis(trimethylsilyl)trifluoroacetamide) with 1% trimethylchlorosilane.ResultsFor all analytes, the LOD and LOQ were 0.06 and 0.20 ng/mL, respectively. The calibration curves were linear (R2 ≥ 0.992), and absolute recoveries were ≥91.7%. Accuracy and precision were within the accepted range. From the analysis of biologic samples of 10 human participants who were taking CBD oil, it was realized that Δ9-THC was not detected in urine, while 11-nor-carboxy-Δ9-THC (0.69–23.06 ng/mL) and CBD (0.29–96.78 ng/mL) were found in all urine samples. Regarding plasma samples, Δ9-THC (0.21–0.62 ng/mL) was detected in 10, 11-nor-carboxy-Δ9-THC (0.20–2.44 ng/mL) in 35, while CBD (0.20–1.58 ng/mL) in 25 out of 38 samples, respectively.ConclusionThe results showed that Δ9-THC is likely to be found in plasma although at low concentrations. In addition, the detection of 11-nor-carboxy-Δ9-THC in both urine and plasma samples raises questions and concerns for the proper interpretation of toxicological results, especially considering Greece’s zero tolerance law applied in DUID and workplace cases.

Abstract 6094: Detection of MRD assessment with the Personalis NeXT Personal assay using MATRIX plasma-in-plasma contrived samples

Abstract Detection of circulating tumor DNA (ctDNA) has been shown to correlate with clinical outcome of oncology patients. Molecular residual disease (MRD) profiling by ctDNA is being rapidly deployed. Current tumor-informed MRD tests assess a relatively small number of personalized variants, placing limits on their detection sensitivity and ultimately leading to false-negative results due to insufficient assay limit of detection (LOD). Here we report a performance evaluation of the Personalis NeXT Personal tumor-informed MRD assay, using a novel design for contrived samples that are highly commutable to clinical samples, enabling robust assessment of these emerging MRD assays. Novel samples were built from commercially acquired matched tumor and plasma samples, across different indications. The cancer patient plasma was diluted into a background of healthy donor plasma to create the 72 plasma-in-plasma samples in each study (total of 144). Personalis performed WGS on the matched solid tumor and normal samples from each patient. ~1800 somatic variants were selected for each patient. A personalized panel was designed and used to enrich for the selected targets in the individual plasma samples. An aggregated signal across MRD targets was evaluated to determine the presence of ctDNA in each plasma sample. MATRIX 1 panels averaged 1863 variants (range: 1818 to 1888) selected for MRD tracking. With 152 clinically relevant variants and additional assay components, an average of 2206 variants were assessed per patient. MATRIX 2 panels averaged 1836 variants (range: 1819 to 1867) selected for MRD tracking. In the MATRIX 1 study, tumor signal was detected in all analyzed samples, including 9 samples at 0.002% tumor plasma dilution, with an assay sensitivity of 100%. The MATRIX 2 study showed tumor signal detected in 14 of 16 samples at 0.001% tumor plasma dilution. The 2 false negatives were reported at the 0.001% tumor plasma dilution. Thus, the assay sensitivity was 87.5% at the lowest dilution (0.001%) and 96.8% overall. The MATRIX plasma-in-plasma approach is a robust option for assessing the analytical sensitivity of MRD assays to low levels of ctDNA. This approach addresses all parts of an MRD assay for both the plasma and tumor/normal tissues, utilizing real clinical samples while allowing direct interrogation of sensitivity. The Personalis NeXT Personal assay shows ultra-high sensitivity with reproducible data down to the 1-3 PPM range. Citation Format: Paul Labrousse, Hugh Russell, Sobia Hamid, Gabor Bartha, John Lyle, Dan Norton, Josette Northcott, Sean Michael Boyle, Richard Chen, Dan Stetson, James Hadfield. Detection of MRD assessment with the Personalis NeXT Personal assay using MATRIX plasma-in-plasma contrived samples [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 6094.

Metabolomics profiling for diagnosis of acute renal failure after cardiopulmonary bypass.

Acute renal failure (ARF) is one of the most serious complications of cardiopulmonary bypass (CPB) surgery. Serum creatinine level is a key compound examined to understand whether renal function is normal. However, its level may vary based on age, gender, race, muscle mass, nutrition, and drugs taken by an individual. In addition, it may not be detected without a 50% reduction in renal function and may lead to delays in treatment. New markers are needed for early diagnosis of ARF. They were determined for early diagnosis of ARF after CPB. Metabolic differences in plasma samples of individuals who developed and did not develop ARF after cardiopulmonary bypass were determined. This study was the first to perform an untargeted metabolomics analysis for early diagnosis of ARF after CPB surgery. Plasma samples were taken from 105 patients (9 ARF patients) at five time points to identify the time at which a more accurate ARF diagnosis can be made. A total of 687 samples, including quality control samples, were analyzed. Two hundred twenty-six metabolites were identified using retention index libraries. Based on the statistical evaluations, tryptophan, threonine, and methionine were found in lower concentrations in patients with ARF compared to the control group at all time points. Whereas gluconic acid, hypoxanthine, and lactic acid showed a decreasing trend over time, longitudinal analysis showed that cysteine, hippuric acid, and uric acid levels increased over time in the ARF group. These metabolites are candidate biomarkers for early diagnosis of ARF as well as biomarkers for tracking the recovery of ARF patients.

Pesticides in small volume plasma samples: Method development and application to smallmouth bass (Micropterus dolomieu) from the Chesapeake Bay watershed, USA

Nontarget organisms are exposed to pesticides following applications in agricultural and urban settings, potentially resulting in deleterious effects. Direct measurements of pesticides in biological tissues may aid in characterizing exposure, accumulation, and potential toxicity versus analyses in environmental media alone (e.g., water, soil, and air). Plasma represents a nonlethal sampling medium that can be used to assess recent exposures to contaminants. Herein, a method was developed to test the extraction of 210 pesticides and their transformation products in small volume plasma samples (100 μL). Plasma samples were protein precipitated with 0.5 % formic acid in acetonitrile added to the sample (ratio of 3.5:1). Pass-through solid phase extraction was used for sample matrix and lipid removal and samples were analyzed by liquid chromatography and gas chromatography with tandem mass spectrometry. Recoveries of 70.0–129.8 % were achieved for 182 pesticides and degradates across the low (25 ng mL−1), medium (100 ng mL−1), and high (250 ng mL−1) spike levels. Method detection levels ranged 0.4–13.0 ng mL−1. Following development, the method was applied to smallmouth bass (Micropterus dolomieu) plasma samples (n = 10) collected from adults in the Chesapeake Bay watershed. Individual plasma samples resulted in four to seven analytes detected with summed concentrations ranging 16.4–95.0 ng mL−1. Biological multiresidue pesticide methods help elucidate recent exposures of bioactive compounds to nontarget organisms.

SCUBE1 promotes pulmonary artery smooth muscle cell proliferation and migration in acute pulmonary embolism by modulating BMP7.

After an episode of acute pulmonary embolism (APE), activated platelets have the ability to release various bioactive factors that can stimulate both proliferation and migration of pulmonary artery smooth muscle cells (PASMCs). SCUBE1 has been previously reported to engage in platelet-platelet interactions, potentially contributing to the activation of platelets in early onset thrombi. The purpose of this study was to examine the alterations in SCUBE1 expression in PASMCs after APE, as well as understand the mechanism behind these changes. The platelet-rich plasma samples of both APE patients and healthy individuals were collected. A hyperproliferative model of PASMCs was established by using platelet-derived growth factor (PDGF) as a stimulator and various assays were used to investigate how SCUBE1-mediated BMP7 can regulate PDGF-induced PASMC proliferation and migration. Elevated level of SCUBE1 were observed in platelet-rich plasma from patients with APE and in PASMCs induced by PDGF. SCUBE1 interference ameliorated PDGF-driven cell proliferation and migration, and also downregulated PCNA expression. Additionally, mechanistic studies demonstrated that SCUBE1 could directly bind to bone morphogenetic protein 7 (BMP7) and enhance BMP7 expression, which completely abolished the impact of SCUBE1 silencing on proliferation and migration ability of PASMCs after PDGF treatment. In the PDGF-induced proliferation of PASMCs, the expression of SCUBE1 and BMP7 was upregulated. Silencing of SCUBE1 impeded PDGF-induced proliferation and migration of PASMCs by restraining BMP7.

Development of tandem antigen capture ELISAs measuring QSOX1 isoforms in plasma and serum

QSOX1 is a sulfhydryl oxidase that has been identified as a potential biomarker in multiple cancer types as well as acute decompensated heart failure. Three anti-QSOX1 monoclonal antibodies (mAbs) were generated: 2F1, 3A10, and 56-3. MAbs 2F1 and 3A10 were generated against the short isoform of recombinant QSOX1 (rQSOX1-S), and mAb 56-3 was generated against a peptide (NEQEQPLGQWHLS) from the long isoform of QSOX1 (QSOX1-L). Using these mAbs, tandem antigen capture ELISAs were developed to quantify both short and long isoforms of QSOX1 (Total QSOX1 ELISA) and QSOX1-L (QSOX1-L ELISA) in serum and plasma samples. The Total QSOX1 ELISA pairs mAbs 2F1 and 3A10 and has a limit of detection of 109.5 pM, while the QSOX1-L ELISA pairs mAbs 2F1 and 56–3 and has a limit of detection of 10 pM. The levels of total QSOX1 and QSOX1-L were measured in a cohort of paired sera and plasma from 61 donors ≥40 years old and 15 donors <40 years old. No difference in QSOX1 levels was detected between QSOX1-L and QSOX1-S in serum, but the mean concentration of QSOX1-L was found to be 3.21 nM in serum and 5.63 nM in plasma (**p = 0.006). Our tandem ELISAs demonstrate the wide range of concentrations of QSOX1-L and QSOX1-S among individual serum and plasma samples. Since the epitope of mAb 2F1 was mapped to the first CxxC motif at residues C70 and C73 and mAb 56-3 was generated against NEQEQPLGQWHLS in QSOX1-L, our findings support previous research which suggested that QSOX1-L is secreted from cells despite a putative transmembrane domain. The ELISAs reported here may be a useful tool for investigating QSOX1 isoforms as potential biomarkers in cancer and/or heart failure.

Acoramidis produces near-complete TTR stabilization in blood samples from patients with variant transthyretin amyloidosis that is greater than that achieved with tafamidis

Abstract Introduction Transthyretin (TTR) amyloidosis (ATTR) is a progressive, fatal disease caused by destabilizing TTR variants (TTRv) and age-related factors. Dissociation of tetrameric TTR initiates protein misfolding, aggregation, and tissue deposition which constitutes the mechanism of disease. More destabilizing variants drive more severe clinical phenotypes. TTR stabilizers have demonstrated clinical benefits for neuropathic and cardiovascular outcomes correlated with the extent of TTR stabilization. Acoramidis (AG10) is a novel TTR stabilizer in development for the treatment of TTR amyloid cardiomyopathy. Hypothesis: Acoramidis achieves near-complete in vitro TTR stabilization, exceeding levels achieved with clinically relevant concentrations of tafamidis (a TTR stabilizer in clinical use), when added to blood samples from ATTRv patients across a spectrum of destabilizing TTR variants. Methods Two established assays assessed TTR stabilization: fluorescent probe exclusion (FPE; measures binding site occupancy), and Western blot (WB; quantifies tetrameric TTR persistence under conditions of accelerated dissociation). Over 60 individual patient samples representing 18 unique TTRv across a spectrum of intrinsic instability and clinical phenotypes were assayed. Acoramidis was added at its target clinical trough concentration (10 µM) and compared to tafamidis added at its clinical peak (26 µM) and trough (16 µM) concentrations reported for its maximal approved dose. Results Acoramidis bound serum TTR to a greater extent (103 ± 13%) than either peak (87 ± 14%) or trough (71 ± 14%) concentrations of tafamidis. WB assays showed that addition of acoramidis resulted in significantly greater and more durable TTR stabilization (93 ± 14%) than adding tafamidis (peak: 49 ± 14%, trough: 36 ± 13%) in all paired individual patient plasma samples tested (Figure 1, p &amp;lt; 0.0001). Rare variants (A97S, D38A, F64L, L58H, P24S, Y114C) also demonstrated near complete stabilization upon in vitro addition of acoramidis. Conclusions At its target therapeutic trough concentration, acoramidis achieved near-complete TTR stabilization across 18 unique genotypes; in the subset of paired samples, the stabilization was significantly greater for acoramidis than for tafamidis even at its peak clinical concentration. This observation held across a range of destabilizing mutations, including a∼2-fold greater stabilization than tafamidis for the prevalent cardiomyopathic V122I variant. Based on the mechanism of disease and the association between TTR destabilization and severity of clinical outcomes, these data suggest that acoramidis has the potential to be a clinically differentiated and efficacious treatment option for patients with ATTRv, independent of variant genotype.

Alterations in glutamate, arginine, and energy metabolism characterize cerebrospinal fluid and plasma metabolome of persons with HIV-associated dementia.

HIV-associated dementia (HAD) is the most severe clinical expression of HIV-mediated neuropathology, and the processes underlying its development remain poorly understood. We aimed to exploit high-dimensional metabolic profiling to gain insights into the pathological mechanisms associated to HAD. In this cross-sectional study, we utilized metabolomics to profile matched cerebrospinal fluid (CSF) and plasma samples of HAD individuals ( n = 20) compared with neurologically asymptomatic people with HIV (ASYM, n = 20) and healthy controls (NEG, n = 20). Identification of plasma and CSF metabolites was performed by liquid-chromatography or gas-chromatography following a validated experimental pipeline. The resulting metabolic profiles were analyzed by machine-learning algorithms, and altered pathways were identified by comparison with KEGG pathway database. In CSF, HAD patients displayed an imbalance in glutamine/glutamate ratio, decreased levels of isocitrate and arginine, and increased oxidative stress when compared with ASYM or NEG. These changes were confirmed in matched plasma samples, which in addition revealed an accumulation of eicosanoids and unsaturated fatty acids in HAD individuals. Pathway analysis in both biological fluids suggested that alterations in several metabolic processes, including protein biosynthesis, glutamate and arginine metabolism, and energy metabolism, in association to a perturbed eicosanoid metabolism in plasma, may represent the metabolic signature associated to HAD. These findings show that HAD may be associated with metabolic modifications in CSF and plasma. These preliminary data may be useful to identify novel metabolic biomarkers and therapeutic targets in HIV-associated neurological impairment.

Diagnostic value of FOXF1 gene promoter-methylated DNA in the plasma samples of patients with colorectal cancer.

Epigenetic modifications such as DNA methylation in the CpG islands of genes occur at a high rate. In this study, we measured the methylation level of the promoter region of the FOXF1 gene as a new blood biomarker for the detection of colorectal cancer in the early stages. The methylation level of the promoter region of the FOXF1 gene was measured in the plasma samples of 50 colorectal cancer patients and 50 normal individuals. DNA was extracted after exposure to sodium bisulfite by the MethyLight polymerase chain reaction (PCR) method. The percentage of promoter region was measured in all samples, and statistical analysis was done using SPSS v24 software. The average promoter region between the plasma samples of colorectal cancer patients and healthy individuals had a significant difference (P < 0.001). The average promoter region of the FOXF1 gene in tumor plasma samples was 7.1 and in the control samples was 0.48. The sensitivity and specificity of the sample plasma levels were 78% and 89.5%, respectively. The promoter region value of the FOXF1 gene in plasma samples using the MethyLight PCR method had high sensitivity and specificity as a non-invasive method for colorectal cancer diagnosis. This research is the first report that has been presented regarding the investigation of FOXF1 gene methylation in plasma samples in colorectal cancer. Therefore, it is necessary to conduct more studies with larger size samples to evaluate the efficiency of the gene under investigation.

A-023 Performance Evaluation of the Atellica IM High-Sensitivity Troponin I Assay on the Atellica CI 1900 Analyzer

Abstract Background The Atellica® CI 1900 Analyzer is an automated, high-throughput, integrated chemistry and immunoassay analyzer employing both Atellica CH and Atellica IM assays. This study was designed to evaluate the analytical performance of the Atellica IM High Sensitivity Troponin I (TnIH) assay* on the Atellica CI 1900 Analyzer*. Methods Precision and method comparison were used as performance indicators for the Atellica CI 1900 Analyzer. Precision studies were performed according to CLSI EP05-A3 and method comparison according to CLSI EP09-A3. Precision studies used a panel of four native and one contrived serum/lithium-heparin plasma samples. One aliquot of each sample pool was tested in duplicate in two runs per day &amp;gt;2 h apart on each analyzer for &amp;gt;20 days with two reagent lots on two systems. For the method comparison, individual native human lithium-heparin plasma samples were analyzed using the Atellica IM TnIH assay on both the Atellica IM and Atellica CI 1900 Analyzers. Detection capability studies including limit of blank (LoB), limit of detection (LoD), and 20-day functional sensitivity (LoQ) were performed according to CLSI EP17-A2 guidelines. Expected values of normal healthy individuals and hook effect were also verified on the Atellica CI 1900 Analyzer. Results Repeatability and within-lab %CVs ranged from 0.7% to 4.4% and 1.3% to 5.1%, respectively, at TnIH concentrations ranging from 13.73 to 23 197.12 ng/L. Method comparison between the Atellica IM TnIH assay on the Atellica CI 1900 Analyzer vs the Atellica IM Analyzer showed a regression slope of 1.00 and an intercept of 0.04 ng/L (n = 170). In detection capability studies, four lots of the Atellica IM TnIH assay on two Atellica CI 1900 analyzers showed a LoB of 0.35 ng/L and an LoD of 0.76 ng/L. LoQ, defined as the lowest amount of analyte in a sample at which within-lab %CV = 20%, was 1.10 ng/L; %CV was approximately 10% at a TNIH concentration of 2.57 ng/L. In addition, &amp;gt;50% of healthy individuals tested had TnIH values &amp;gt;LoD. The 99th percentile for the Atellica IM TnIH assay (45.20 ng/L) was verified on Atellica CI 1900 Analyzer and was consistent with the Atellica IM Analyzer. No interference from biotin was observed up to a concentration of 3500 ng/mL, and no hook effect was observed up to a concentration of 500 000 ng/L. Conclusion Evaluation of the Atellica IM TnIH assay using the Atellica CI 1900 Analyzer demonstrated reproducible assay precision, sensitive detection capabilities, and equivalent performance compared to the same assay on the Atellica IM Analyzer. This evaluation shows that the Atellica IM TnIH assay on Atellica CI 1900 Analyzer meets the definition of a true high-sensitivity troponin I assay as defined by the International Federation of Clinical Chemistry (IFCC) Task Force on Clinical Applications of Cardiac Biomarkers. *The products/features mentioned here are not commercially available in all countries. Their future availability cannot be guaranteed.

B-256 Genotype Distribution of the Hepatitis C Virus: 10 Years Data of a Clinical Laboratory in Brazil

Abstract Background Hepatitis C virus (HCV) infection is a significant health problem worldwide and is a major cause of severe liver disease including chronic hepatitis, cirrhosis, and hepatocellular carcinoma. HCV is divided into six genotypes with several sub-genotypes depending on the geographic region, some genotypes are endemic. The purpose of this study was to describe the frequency of the HCV genotype (G) profile by gender and age using a database of a large Brazilian clinical laboratory. Methods This was a retrospective analysis from January 2012 to December 2022 based on molecular data of 3.566 isolated from clinical samples. The automated extraction and purification of nucleic acids were conducted from EDTA plasma samples of individuals suspected of HCV infection. Then, nucleic acid amplification was performed using oligonucleotide probes labeled with fluorescent dyes specific for the qualitative identification of HCV genotypes 1 to 6 and subtypes A and B of genotype 1. Results A total of 3.566 HCV genotype cases were analyzed during the period. The genotype distribution was (2.653/74.4%) G1 (48.1% G1A and 46.4% G1B), (733/ 20.5%) G3, (133/3.7%) G2, (35/0.9%) G4, (11/0.3%) G5 and (1/0.02%) G6. Twelve patients had multiple genotypes over the last ten years. At least one individual changed the genotype over a 3-year evaluation period, and 11 (0.30%) changed it twice. The genotypes G1 and G3 were predominant in all years analyzed. The prevalence of G1 and G3 was 75% and 19.6% among females and 74% and 21.2% among male patients, respectively. The analysis was conducted on individuals aged 6 to 99 years, with an overall average age of 52.6±13 years. The average age among men was 51.1 ± 12 years and among women, it was 54.7 ± 13 years. According to the age groups evaluated, genotype 1 was the most frequent, ranging from 69.3% (21 to 30 years old) to 75.8% (over 60 years old). The main subtype in the over 60 years old group was 1B. The highest frequency of genotype 2 was observed in the group over 60 years old (6.1%). The patient's symptoms, clinical history, or further diagnostic procedures were not accessible. Conclusion The study found that genotypes 1 and 3 were the most prevalent in Brazil over the ten-year period, with genotype 1 being the most frequent among all age groups. Genotype 1B was most prevalent in individuals above 60 years old. The change of genotype per individual was less than 1%, but further attention is required for potential virus tropism.

Characterization of antibodies to SARS-CoV-2 in lyophilized plasma prepared with amotosalen-UVA pathogen reduction.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)-infected patients exhibit disease ranging from asymptomatic to severe pneumonia, multi-organ failure, and death. convalescent COVID plasma (CCP) from recovered patients with high levels of neutralizing antibodies has demonstrated therapeutic efficacy to reduce the morbidity of coronavirus disease 2019 (COVID-19) in some studies. The development of assays to characterize the activity of CCP to neutralize SARS-CoV-2 infectivity offers the possibility to improve potential therapeutic efficacy. Lyophilization of CCP may increase the availability of this therapy. We hypothesized that SARS-CoV-2 antibody profiles of pooled lyophilized pathogen-reduced CCP from COVID-19-recovered blood donors retains virus-neutralizing efficacy as reported for frozen pathogen-reduced CCP. Pooled lyophilized pathogen-reduced plasma was prepared from recovered COVID plasma donors. Antibodies to SARS-CoV-2 were characterized in each donor plasma prior to pathogen reduction and lyophilization and after lyophilization of individual CCP, and in the lyophilized CCP pool. Several complimentary assays were used to characterize antibody levels, neutralizing capacity, and the spectrum of antigen reactivity. The mean values for individual plasma samples and the value in the pool were compared. The mean ratio for antibody binding to SARS-CoV-2 antigens before and after treatment was 0.95 ± 0.22 mean fluorescent intensity (MFI) units. Antibody activity to an array of influenza virus antigens demonstrated a mean activity ratio of 0.92 ± 0.12 MFI before and after treatment. The antibody activity in pooled pathogen-reduced lyophilized CCPs demonstrated minimal impact due to pathogen reduction treatment and lyophilization.

MATRIX plasma-in-plasma contrived samples assessment and detection of MRD using the Personalis NeXT Personal assay.

e15056 Background: Detection of circulating tumor DNA (ctDNA) has been shown to correlate with clinical outcome of oncology patients. Molecular residual disease (MRD) profiling by ctDNA is being rapidly deployed. Current tumor-informed MRD tests assess a relatively small number of personalised variants, placing limits on their detection sensitivity and ultimately leading to false-negative results due to insufficient assay limit of detection (LOD). Here we report a performance evaluation of the Personalis NeXT Personal tumor-informed MRD assay, using a novel design for contrived samples that are highly commutable to clinical samples, enabling robust assessment of these emerging MRD assays. Methods: Our novel samples were built from commercially acquired matched tumor and plasma samples, across different indications. The cancer plasma was diluted into a background of healthy donor plasma to create the 72 plasma-in-plasma samples in each study (total of 144). Personalis performed WGS on the matched solid tumor and normal samples from each patient. ~1800 somatic variants were selected for each patient. A personalized panel was designed and used to enrich for the selected targets in the individual plasma samples. An aggregated positive variant score was evaluated to determine the presence of ctDNA in each plasma sample. Results: MATRIX 1 panels averaged 1863 variants (range: 1818 to 1888) selected for MRD tracking. With 152 clinically relevant variants and additional assay components, an average of 2206 variants were assessed per patient. MATRIX 2 panels averaged 1836 variants (range: 1819 to 1867) selected for MRD tracking. With 2130 clinically relevant variants they averaged 4094 variants assessed per patient. In the MATRIX 1 study, tumor signal was detected in all analyzed samples, including 9 samples at 0.002% tumor plasma dilution, with an assay sensitivity of 100%. The MATRIX 2 study showed tumor signal detected in 14 of 16 samples at 0.001% tumor plasma dilution. The 2 false negatives were reported at the 0.001% tumor plasma dilution. Thus, the assay sensitivity was 87.5% at the lowest dilution (0.001%) and 96.8% overall. Conclusions: The MATRIX plasma-in-plasma approach is a robust option for assessing the sensitivity of MRD assays to low levels of ctDNA. This approach addresses all parts of an MRD assay for both the plasma and tumor/normal tissues, and is the closest thing to real clinical samples while allowing direct interrogation of sensitivity. Additionally, it allows assessment of any genomic or epigenomic biological feature. The Personalis NeXT Personal assay shows ultra-high sensitivity with reproducible data down to the 1-3 PPM range, and expansion of the personalized panel fixed clinical content allows tracking of more driver and resistance mutations with no impact to the MRD sensitivity.

Monocyte pathology in human tuberculosis is due to plasma milieu changes and aberrant STAT signalling.

Monocyte-derived macrophages contribute centrally to immune protection in Mycobacterium tuberculosis infection and changes in monocyte phenotype characterize immunopathology in tuberculosis patients. Recent studies highlighted an important role of the plasma milieu in tuberculosis immunopathology. Here, we investigated monocyte pathology in patients with acute tuberculosis and determined tuberculosis plasma milieu effects on phenotype as well as cytokine signalling of reference monocytes. Patients with tuberculosis (n = 37) and asymptomatic contacts (controls n = 35) were recruited as part of a hospital-based study in the Ashanti region of Ghana. Multiplex flow cytometry phenotyping of monocyte immunopathology was performed and effects of individual blood plasma samples on reference monocytes prior to and during treatment were characterized. Concomitantly, cell signalling pathways were analysed to elucidate underlying mechanisms of plasma effects on monocytes. Multiplex flow cytometry visualization characterized changes in monocyte subpopulations and detected higher expression of CD40, CD64 and PD-L1 in monocytes from tuberculosis patients as compared to controls. Aberrant expression normalized during anti-mycobacterial treatment and also CD33 expression decreased markedly. Notably, higher CD33, CD40 and CD64 expression was induced in reference monocytes when cultured in the presence of plasma samples from tuberculosis patients as compared to controls. STAT signalling pathways were affected by the aberrant plasma milieu and higher levels of STAT3 and STAT5 phosphorylation was found in tuberculosis plasma-treated reference monocytes. Importantly, high pSTAT3 levels were associated with high CD33 expression and pSTAT5 correlated with CD40 as well as CD64 expression. These results suggested plasma milieu effects with potential implications on monocyte phenotype and function in acute tuberculosis.

Poor comparability of plasma renin activity measurement in determining patient samples: the status quo and recommendations for harmonization.

This study aims to investigate and update the consistency and comparability of plasma renin activity (PRA) assays in measuring clinical samples. The contributions of recalibration, blank subtraction, and incubation strategies to interchangeability were also explored. Five different laboratories were evaluated using forty-six individual plasma samples, including four liquid chromatography-tandem mass spectrometry (LC‒MS/MS) assays and one chemiluminescence immunoassay (CLIA). Spearman correlation coefficient (R), Passing-Bablok regression, and Bland‒Altman plot analyses were used to evaluate the consistency among assays. Consistency before and after recalibration, blank subtraction, and incubation strategy unification was compared. A good correlation was observed among all assays (R>0.93). None of the samples measured by all assays showed coefficient variation (CV) <10 %, and 37 % of samples showed overall CVs >20 %. The 95 % confidence intervals (CIs) for slopes did not contain 1 for most assay pairs. Large relative biases (-85.1-104.2 %) were found, and 76 % (52-93 %) of samples had unacceptable biases. Recalibration reduced the calibration bias. Ignoring blank subtraction improved the comparability across all assays while unifying incubation did not. The interchangeability of PRA measurement was unsatisfying. Harmonization on calibrator and ignoring blank were recommended. Unifying incubation strategy was unnecessary.

Quantification of 782 Plasma Peptides by Multiplexed Targeted Proteomics.

Blood analysis is one of the foundations of clinical diagnostics. In recent years, the analysis of proteins in blood samples by mass spectrometry has taken a jump forward in terms of sensitivity and the number of identified proteins. The recent development of parallel reaction monitoring with parallel accumulation and serial fragmentation (prm-PASEF) combines ion mobility as an additional separation dimension. This increases the proteome coverage while allowing the use of shorter chromatographic gradients. To demonstrate the method's full potential, we used an isotope-labeled synthetic peptide mix of 782 peptides, derived from 579 plasma proteins, spiked into blood plasma samples with a prm-PASEF measurement allowing the quantification of 565 plasma proteins by targeted proteomics. As a less time-consuming alternative to the prm-PASEF method, we describe guided data independent acquisition (dia)-PASEF (g-dia-PASEF) and compare its application to prm-PASEF for measuring blood plasma. To demonstrate both methods' performance in clinical samples, 20 patient plasma samples from a colorectal cancer (CRC) cohort were analyzed. The analysis identified 14 differentially regulated proteins between the CRC patient and control individual plasma samples. This shows the technique's potential for the rapid and unbiased screening of blood proteins, abolishing the need for the preselection of potential biomarker proteins.