Serum Samples Of Patients Research Articles

Effects of Various Concentrations of Pronase on Flow Cytometric Crossmatching Patients Treated With Rituximab and Donor HLA-Specific Antibodies.

Pronase pretreatment can reduce rituximab (RTX) interference by degrading CD20 in B-cell flow cytometry crossmatch (FCXM) testing. However, it may also reduce the assay sensitivity by degrading HLA molecules. We investigated the effects of various pronase concentrations on RTX interference and the analytical sensitivity of B-cell FCXM testing. Using 59 patient serum samples and 38 donor lymphocyte samples, we designed 97 recipient-donor pairs and divided them into three groups according to RTX use and the presence of weak-to-moderate donor HLA-specific antibody (DSA) reactions: RTX+/DSA-, RTX+/DSA+, and RTX-/DSA+. FCXM was performed after pretreating lymphocytes with six different pronase concentrations (0, 0.5, 1, 2, 3, and 4 mg/mL). With B-FCXM testing, false-positive results due to RTX in the RTX+/DSA- group markedly decreased with increasing pronase concentrations. The median channel shift values in the RTX+/DSA+ and RTX-/DSA+ groups did not significantly decrease when the pronase concentration was increased from 1 mg/mL to 2 or 3 mg/mL. All eight RTX+/DSA+ cases that were positive at 1 mg/mL pronase but negative at 2 or 3 mg/mL had mean fluorescence intensity (MFI) DSA values of less than 3,000 except for DQ5 (MFI: 5,226). With T-cell FCXM, false-positive results were observed in 2.9% of 315 FCXM tests with pronase pretreatment. Higher concentrations (2 or 3 mg/mL) of pronase effectively eliminated RTX interference but still carried a risk for false negativity for weak DSA reactions in B-cell FCXM. Higher pronase concentrations can be used as an auxiliary method to detect moderate-to-strong DSA reactions in RTX-treated patients.

Zeolite Functionalized with Magnesium/Aluminum/Lanthanum Ternary Hydroxide for the Phosphometabolite Profiling of Malignant Neoplastic Serum Samples.

ATP upregulation is a significant driver of aggressive cancer cell phenotypes. Phosphometabolites participate in metabolic pathways and are overexpressed in cancer cell activity. Therefore, developing novel and accurate methods for detecting phosphometabolites in biological fluids is essential. In this research, a novel zeolite composite comprising magnesium, aluminum, and lanthanum hydroxides (MALZ) is developed and used for the first time to enrich phosphorylated metabolites via its inherent interaction with phosphate groups. SEM micrographs show a crystalline cubic structure with a small diameter of 36.62 nm. FTIR analysis confirms the phosphate adsorption and desorption using AMP and ATP as the standards. XRD analysis of MALZ provides structural information about the synthesized composite. Adsorption-desorption parameters, such as pH, shaking time, and MALZ concentration, are optimized to analyze the binding capacity of the fabricated material for phosphorylated metabolites. A kinetic study reveals the rapid and effective AMP and ATP adsorptions on MALZ. The multiple hydroxyl groups of ternary hydroxides and high affinity of lanthanum toward the phosphate group enrich 26 phosphometabolites from serum samples of malignant neoplastic patients. The LC-MS profile shows characteristic phosphometabolites that may act as signatures of cancer-related abnormal metabolic pathways. This study may provide an experimental pathway for detecting metabolites in human body fluids.

Circ_0005699 Expedites ox-LDL-Triggered Endothelial Cell Injury via Targeting miR-384/ASPH Axis.

Atherosclerosis (AS) is an inflammatory disease with multiple causes. Multiple circular RNAs (circRNAs) are known to be involved in the pathogenesis of AS. To explore the function and mechanism of circ_0005699 in oxidative low-density lipoprotein (ox-LDL)-induced human umbilical vein endothelial cells (HUVECs) injury. Ox-LDL treatment restrained HUVECs viability, cell proliferation, and angiogenesis ability, and accelerated HUVECs apoptosis, inflammatory response, and oxidative stress. Circ_0005699 was up-regulated in the serum samples of AS patients and ox-LDL-induced HUVECs. Interference of circ_0005699 effectively rescued ox-LDL-induced injury in HUVECs. Additionally, miR-384 could bind to circ_0005699, and miR-384 depletion inverted the effects of circ_0005699 deficiency on ox-LDL-mediated HUVEC injury. Moreover, ASPH was a direct target of miR-384, and the enforced expression of ASPH overturned miR-384-induced effects on ox-LDL-induced HUVECs. Importantly, circ_0005699 regulated ASPH expression via sponging miR-384. Interference of circ_0005699 protected against ox-LDL-induced injury in HUVECs at least partly by regulating ASPH expression via acting as a miR-384 sponge.

High Serum S100A12 as a Diagnostic and Prognostic Biomarker for Severity, Multidrug-Resistant Bacteria Superinfection and Herpes Simplex Virus Reactivation in COVID-19.

Neutrophils are critical immune cells in severe coronavirus disease 2019 (COVID-19). S100 calcium-binding protein A12 (S100A12) is highly expressed in neutrophils during acute inflammation. The aim of this study was to evaluate serum S100A12 levels as a diagnostic and prognostic tool in COVID-19. Serum samples of patients with moderate and severe COVID-19 were collected during 2020 to 2024. Enzyme-linked immunosorbent assay was used to measure serum S100A12 levels in 63 patients with moderate COVID-19, 60 patients with severe disease and 33 healthy controls. Serum S100A12 levels were elevated in moderate COVID-19 compared to controls and were even higher in severe cases. In moderate disease, serum S100A12 levels positively correlated with immune cell counts. While C-reactive protein and procalcitonin are established inflammation markers, they did not correlate with serum S100A12 levels in either patient cohort. Patients with severe COVID-19 and vancomycin-resistant enterococcus (VRE) infection had increased S100A12 levels. Elevated S100A12 levels were also observed in patients with herpes simplex reactivation. Fungal superinfections did not alter S100A12 levels. These data show that serum S100A12 increases in moderate and severe COVID-19 and is further elevated by VRE bloodstream infection and herpes simplex reactivation. Therefore, S100A12 may serve as a novel biomarker for severe COVID-19 and an early diagnostic indicator for bacterial and viral infections.

Serological immune biomarker for disease severity in dengue-infectedpediatric hospitalized patients.

Clinical manifestation of dengue disease ranges from asymptomatic, febrile fever without warning sign (DOS) to serious outcome dengue with warning sign (DWS) and severe disease (SD) leading to shock syndrome and death. The role of antibody response in natural dengue infection is complex and not completely understood. Here, we aimed to assess serological marker for disease severity. Antibody response of dengue-confirmed pediatric patients with acute secondary infection were evaluated against infecting virus, immature virus, and recombinant envelop protein. Immature virus antibody titers were significantly higher in DWS as compared to DOS (p = 0.0006). However, antibody titers against recombinant envelop protein were higher in DOS as compared to DWS, and antibody avidity was significantly higher against infecting virus in DOS. Serum samples of DOS patients displayed higher in vitro neutralization potential in plaque assay as compared to DWS, whereas DWS serum samples showed higher antibody-dependent enhancement in the in vitro enhancement assays. Thus, antibodies targeting immature virus can predict disease severity and could be used in early forecast of disease outcome using anenzyme-linked immunoassay assaysystem which is less laborious and cheaper than plaque assay system for correlates of protection and could help optimize medical care and resources.

Analysis of Th17 cell population and expression of microRNA and factors related to Th17 in patients with premature ovarian failure

Folliculogenesis is the process where follicles in the ovaries develop and eventually lead to ovulation. Any disruption to this process can cause premature ovarian failure. miR-326 is one of the microRNAs whose expression leads to Th17 production. Th17 activates the immune system to respond more vigorously, and by producing interlukins and cytokines causes inflammation and autoimmune disorders. Th17-induced inflammation and Th17/Treg imbalance can result in POF. This investigation took samples from 30 POF patients and 30 healthy people. The study utilized PCR to assess the expression levels of cytokines, specific transcription factor (ROR-γt), and miR-326. Additionally, ELISA was employed to analyze serum levels of IL-17, IL-21, IL-23. Furthermore, flow cytometry was utilized to determine the frequency of Th17. Compared to the control group, our results demonstrated a rise in the transcription factor RORɣt and a considerable rise in the frequency of Th17 cells in patients with POF. The level of inflammatory cytokines IL-17, IL-21, and IL-23 secreted in serum samples of patients with POF increased significantly compared to the control group. Results of investigating microRNA associated with Th17 cells also showed increased expression of miR-326 in females suffering from POF. The elevation of pro-inflammatory markers in women with POF contrary to the control group underscores the significant involvement of the immune system in pregnancy disorders pathogenesis. Consequently, immunological factors may serve as promising biomarkers for predicting POF likelihood in high-risk women in the future.

Extracellular Vesicle DNA Extraction and Sequencing in Ancient Serum Samples From Patients With Breast Cancer.

Extracellular vesicle DNA (EV-DNA) has emerged as a novel biomarker for tumor mutation detection using liquid biopsies, exhibiting biological advantages compared to cell-free DNA (cfDNA). This study assessed the feasibility of EV-DNA and cfDNA extraction and sequencing in old serum samples of patients with breast cancer (BC). A total of 28 serum samples of 27 patients with corresponding clinical information were collected between 1983 and 1991. EV-DNA was extracted using Exo-GAG kit (Nasabiotech) and cfDNA using QIAsymphony DSP Virus/Pathogen Midi Kit (Qiagen), respectively. Subsequently, 10 matched samples (EV-DNA n=5, cfDNA n=5) of five patients were subjected to sequencing using the Oncomine™ Breast cfDNA Research Assay v2 (Thermo Fisher Scientific). Samples were collected on median 1.9 years after primary diagnosis [interquartile range (IQR)=0.2-7.2]. Median follow-up was 9.5 years (IQR=5.2-14.2). Median age of serum samples was 36.1 years (IQR=34.5-37.3). EV-DNA and cfDNA were extracted from 100% (28/28) of the included samples. Both, DNA quantity and concentration were comparable between EV-DNA and cfDNA. Sequencing was successfully performed in 100% (10/10) of the included samples. Two matched analyses yielded equivalent results in EV-DNA and cfDNA (no mutations, n=1; PIK3CA mutation, n=1), whilst in two analyses, PIK3CA mutation was only found in cfDNA, and in one analysis, a TP53 mutation was only found in EV-DNA. EV-DNA extraction and sequencing in old serum samples of patients with BC is feasible and has the potential to address clinically relevant questions in longitudinal studies.

Microfluidic Immunoarray for Point-of-Care Detection of Cytokines in COVID-19 Patients.

The "cytokine storm" often induced in COVID-19 patients contributes to the onset of "acute respiratory distress syndrome" (ARDS) accompanied by lung infection and damage, multiorgan failure, and even death. This large increase in pro-inflammatory cytokines in blood may be related to severity. Rapid, on-demand cytokine analyses can thus be critical to inform treatment plans and improve survival rates. Here, we report a sensitive, low-cost, semiautomated 3D-printed microfluidic immunoarray to detect 2 cytokines and CRP simultaneously in a single 10 μL serum sample in 25 min. Accuracy was validated by analyzing 80 COVID-19 patient serum samples, with results well correlated to a commercial Meso Scale protein immunoassay. Capture antibodies immobilized in detection microwells in a flat well plate-type flow chamber facilitate the immunoassay, with a programmable syringe pump automatically delivering reagents. Chemiluminescence signals were captured in a dark box with a CCD camera integrated for 30 s. This system was optimized to detect inflammation biomarkers IL-6, IFN-γ, and CRP simultaneously in blood serum. Ultralow limits of detection (LODs) of 0.79 fg/mL for IL-6, 4.2 fg/mL for CRP, and 2.7 fg/mL for IFN-γ with dynamic ranges of up to 100 pg/mL were achieved. ROC statistical analyses showed a relatively good diagnostic value related to the samples assigned WHO COVID-19 scores for disease severity, with the best results for IL-6 and CRP. Monitoring these biomarkers for coronavirus severity may allow prediction of disease severity as a basis for critical treatment decisions and better survival rates.

Hyperkalemia presentation at a clinic during the cold season.

Familial pseudohyperkalemia (FP) is an underrecognized cause of pseudohyperkalemia, caused by the leaking of potassium from red blood cells. This extracellular shift of potassium is temperature-dependent and occurs when blood samples are stored below room temperature or for a long time, manifesting as apparent hyperkalemia. We report a suspicious case of FP, which demonstrated an apparent seasonal trajectory of serum-potassium levels at the local clinic. At first, laboratory test results did not show an increase in the serum-potassium levels in our tertiary hospital. However, by replicating the clinic's storage conditions, the patient's serum sample showed hyperkalemia only when it was stored at a temperature of 4°C or 20°C for 4-8h. Hyperkalemia was not observed in the patient's sample when it was stored at 37°C, or in the healthy control's sample at a temperature of 20°C or 37°C. When encountering hyperkalemia without an obvious cause and symptoms, physicians should consider pseudohyperkalemia in the differential diagnosis. In particular, if a seasonal trajectory of serum-potassium levels is observed, FP should be suspected as a potential cause of pseudohyperkalemia. Although a genetic test is needed to properly diagnose FP, confirming it by verifying the sample storage conditions or proving it by replicating the test using different storage conditions is easy and very important, as it can prevent unnecessary treatment.

Peroxynitrite reduces Treg cell expansion and function by mediating IL-2R nitration and aggravates multiple sclerosis pathogenesis

T-helper 17 cells and regulatory T cells (Treg) are critical regulators in the pathogenesis of multiple sclerosis (MS) but the factors affecting Treg/Th17 balance remains largely unknown. Redox balance is crucial to maintaining immune homeostasis and reducing the severity of MS but the underlying mechanisms are unclear yet. Herein, we tested the hypothesis that peroxynitrite, a representative molecule of reactive nitrogen species (RNS), could inhibit peripheral Treg cells, disrupt Treg/Th17 balance and aggravate MS pathology by inducing nitration of interleukin-2 receptor (IL-2R) and down-regulating RAS/JNK-AP-1 signalling pathway. Experimental autoimmune encephalomyelitis (EAE) mouse model and serum samples of MS patients were used in the study. We found that the increases of 3-nitrotyrosine and IL-2R nitration in Treg cells were coincided with disease severity in the active EAE mice. Mechanistically, peroxynitrite-induced IL-2R nitration down-regulated RAS/JNK signalling pathway, subsequently impairing peripheral Treg expansion and function, increasing Teff infiltration into the central nerve system (CNS), aggravating demyelination and neurological deficits in the EAE mice. Those changes were abolished by peroxynitrite decomposition catalyst (PDC) treatment. Furthermore, transplantation of the PDC-treated-autologous Treg cells from donor EAE mice significantly decreased Th17 cells in both axillary lymph nodes and lumbar spinal cord, and ameliorated the neuropathology of the recipient EAE mice. Those results suggest that peroxynitrite could disrupt peripheral Treg/Th17 balance, and aggravate neuroinflammation and neurological deficit in active EAE/MS pathogenesis. The underlying mechanisms are related to induce the nitration of IL-2R and inhibit the RAS/JNK-AP-1 signalling pathway in Treg cells. The study highlights that targeting peroxynitrite-mediated peripheral IL-2R nitration in Treg cells could be a novel therapeutic strategy to restore Treg/Th17 balance and ameliorate MS/EAE pathogenesis. The study provides valuable insights into potential role of peripheral redox balance in maintaining CNS immune homeostasis.

Ceftobiprole quantification in human serum by HPLC-UV to implement routine TDM in clinical practice

Background and aimsCeftobiprole is a recent 5th generation parenteral cephalosporin with antibacterial activity against a large range Gram+ and Gram− bacteria. Therapeutic drug monitoring (TDM) is an essential tool for maintaining plasma concentrations of antibiotics above the MIC by the end of the dosing interval, thus preventing the resistant strain diffusion. TDM is already recommended for other cephalosporins, and it is a reasonable tool contributing to the safety and efficacy of these drugs. During the treatment of patients in real-life, a number of pharmacokinetic (PK) changes not normally seen in healthy volunteers can occur which can impair the pharmacokinetic/pharmacodynamic target attainment.We aimed to develop simple and rapid HPLC-UV method for determination of ceftobiprole in human serum to implement TDM in clinical practice and support PKs and pharmacokinetic/pharmacodynamic (PK/PD) studies. Materials and methodsSamples preparation of calibration standards, QC, and anonymous patients serum samples was performed by protein precipitation by adding 0.01 ml of sulphosalicylic acid at 30 % to 0.1 ml of each sample. Then samples were vortexed and the centrifuged at 12,000 rpm for 10 min at 4 °C. Fifty microlitres of clear supernatant were diluted 1:1 with mobile phase and transferred into HPLC autosampler held at 8 °C. Chromatographic separation was carried out in a gradient mode at 35 °C on an ultra-Biphenyl column using a Thermo Scientific chromatographic system with a Diode array. Data management was performed with Chromeleon 7.4 software. ResultsThe HPLC-UV method proved to be linear over wide concentration ranges (0.5–50.0 mg/L) and was accurate and reproducible in the absence of matrix effects, allowing for robust, specific, and rapid quantification of ceftobiprole from a low amount of serum (0.1 mL). The mean steady state Ctrough and Cend values measured in the anonymous patients’ samples were 6.26 ± 3.81 mg/L and 22.56 ± 15.69 mg/L, respectively. ConclusionsWe report a broadened simple and fast HPLC with UV detection method for quantification of ceftobiprole in human serum to implement ceftobiprole TDM as clinical routine, and support future (PK/PD) studies in special patients’ population.

O-GlcNAc Modification Is a Promising Therapeutic Target for Diabetic Retinopathy.

Diabetic retinopathy (DR) is a very serious diabetes complication. Changes in the O-linked N-acetylglucosamine (O-GlcNAc) modification are associated with many diseases. However, its role in DR is not fully understood. In this research, we explored the effect of O-GlcNAc modification regulation by activating AMP-activated protein kinase (AMPK) in DR, providing some evidence for clinical DR treatment in the future. Bioinformatics was used to make predictions from the database, which were validated using the serum samples of diabetic patients. As an in vivo model, diabetic mice were induced using streptozotocin (STZ) injection with/without an AMPK agonist (metformin) or an AMPK inhibitor (compound C) treatment. Electroretinogram (ERG) and H&E staining were used to evaluate the retinal functional and morphological changes. In vitro, 661 w cells were exposed to high-glucose conditions, with or without metformin treatment. Apoptosis was evaluated using TUNEL staining. The protein expression was detected using Western blot and immunofluorescence staining. The angiogenesis ability was detected using a tube formation assay. The levels of O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) in the serum changed in the DR patients in the clinic. In the diabetic mice, the ERG wave amplitude and retinal thickness decreased. In vitro, the apoptotic cell percentage and Bax expression were increased, and Bcl2 expression was decreased in the 661 w cells under high-glucose conditions. The O-GlcNAc modification was increased in DR. In addition, the expression of GFAT/TXNIP O-GlcNAc was also increased in the 661 w cells after the high-glucose treatment. Additionally, the Co-immunoprecipitation(CO-IP) results show that TXNIP interacted with the O-GlcNAc modification. However, AMPK activation ameliorated this effect. We also found that silencing the AMPKα1 subunit reversed this process. In addition, the conditioned medium of the 661 w cells may have affected the tube formation in vitro. Taken together, O-GlcNAc modification was increased in DR with photoreceptor cell degeneration and neovascularization; however, it was reversed after activating AMPK. The underlying mechanism is linked to the GFAT/TXNIP-O-GlcNAc modification signaling axis. Therefore, the AMPKα1 subunit plays a vital role in the process.

Genetic determinants of host- and virus-derived insertions for hepatitis E virus replication

Hepatitis E virus (HEV) is a long-neglected RNA virus and the major causative agent of acute viral hepatitis in humans. Recent data suggest that HEV has a very heterogeneous hypervariable region (HVR), which can tolerate major genomic rearrangements. In this study, we identify insertions of previously undescribed sequence snippets in serum samples of a ribavirin treatment failure patient. These insertions increase viral replication while not affecting sensitivity towards ribavirin in a subgenomic replicon assay. All insertions contain a predicted nuclear localization sequence and alanine scanning mutagenesis of lysine residues in the HVR influences viral replication. Sequential replacement of lysine residues additionally alters intracellular localization in a fluorescence dye-coupled construct. Furthermore, distinct sequence patterns outside the HVR are identified as viral determinants that recapitulate the enhancing effect. In conclusion, patient-derived insertions can increase HEV replication and synergistically acting viral determinants in and outside the HVR are described. These results will help to understand the underlying principles of viral adaptation by viral- and host-sequence snatching during the clinical course of infection.

Distinct metabolomic and lipidomic profiles in serum samples of patients with primary sclerosing cholangitis.

Identification of specific metabolome and lipidome profile of patients with primary sclerosing cholangitis (PSC) is crucial for diagnosis, targeted personalized therapy, and more accurate risk stratification. Nuclear magnetic resonance (NMR) spectroscopy revealed an altered metabolome and lipidome of 33 patients with PSC [24 patients with inflammatory bowel disease (IBD) and 9 patients without IBD] compared with 40 age-, sex-, and body mass index (BMI)-matched healthy controls (HC) as well as 64 patients with IBD and other extraintestinal manifestations (EIM) but without PSC. In particular, higher concentrations of pyruvic acid and several lipoprotein subfractions were measured in PSC in comparison to HC. Of clinical relevance, a specific amino acid and lipid profile was determined in PSC compared with IBD and other EIM. These results have the potential to improve diagnosis by differentiating PSC patients from HC and those with IBD and EIM.

Development of a novel non-invasive biomarker panel for hepatic fibrosis in MASLD

Accurate non-invasive biomarkers to diagnose metabolic dysfunction-associated steatotic liver disease (MASLD)-related fibrosis are urgently needed. This study applies a translational approach to develop a blood-based biomarker panel for fibrosis detection in MASLD. A molecular gene expression signature identified from a diet-induced MASLD mouse model (LDLr−/−.Leiden) is translated into human blood-based biomarkers based on liver biopsy transcriptomic profiles and protein levels in MASLD patient serum samples. The resulting biomarker panel consists of IGFBP7, SSc5D and Sema4D. LightGBM modeling using this panel demonstrates high accuracy in predicting MASLD fibrosis stage (F0/F1: AUC = 0.82; F2: AUC = 0.89; F3/F4: AUC = 0.87), which is replicated in an independent validation cohort. The overall accuracy of the model outperforms predictions by the existing markers Fib-4, APRI and FibroScan. In conclusion, here we show a disease mechanism-related blood-based biomarker panel with three biomarkers which is able to identify MASLD patients with mild or advanced hepatic fibrosis with high accuracy.

Exosomal Serum Biomarkers as Predictors for Laryngeal Carcinoma.

The lack of screening methods for LSCC is a critical issue, as treatment options and the treatment outcome greatly depend on the stage of LSCC at initial diagnosis. Therefore, the objective of this study was to identify potential exosomal serum biomarkers that can diagnose LSCC and distinguish between early- and late-stage disease. A multiplexed proteomic array was used to identify differentially expressed proteins in exosomes isolated from the serum samples of LSCC patients compared to the control group (septorhinoplasty, SRP). The most promising proteins for diagnosis and differentiation were calculated using biostatistical methods and were validated by immunohistochemistry (IHC), Western blots (WB), and ELISA. Exosomal insulin-like growth factor binding protein 7 (IGFBP7) and Annexin A1 (ANXA1) were the most promising exosomal biomarkers for distinguishing between control and LSCC patients and also between different stages of LSCC (fold change up to 15.9, p < 0.001 for all). The identified proteins represent potentially novel non-invasive biomarkers. However, these results need to be validated in larger cohorts with a long-term follow-up. Exosomal biomarkers show a superior signal-to-noise ratio compared to whole serum and may therefore be an important tool for non-invasive biomarker profiling for laryngeal carcinoma in the future.

#2124 Accuracy and interferences in the T50 test: assessing the reliability of a novel diagnostic tool

Abstract Background and Aims A novel blood-based in vitro diagnostic test determines the calcification propensity in patients, e.g. chronic kidney disease (CKD) patients. This assay is known as the ‘T50 test’ and is based on the one-half maximal transition time (T50; minutes) of calciprotein particle 1 (CPP1) to calciprotein particle 2 (CPP2) in serum (Fig. 1). Numerous studies have associated a lower T50 with cardiovascular and all-cause mortality in chronic kidney disease (CKD) patients at stages 3, 4 and 5, kidney transplant recipients, and in the general population. To date, the T50 test has been performed at a single site (Calciscon AG, Biel, Switzerland). The aim of this study was to determine the accuracy of the T50 test. Additionally, the effect of haemolysis, hyperbilirubinemia and lipidemia interferences on the T50 was studied and compared to the manufacturer's claims. Method The T50 was measured using T50 test kits and according to the protocol of Calciscon AG using an automatic pipetting machine (EVOware Freedom, Tecan) and a nephelometer (NEPHELOstar, BMG). Patient serum samples were spiked with haemoglobin, bilirubin, or lipids (triglyceride) for the interference analysis. In total, three dilution series with interferences and four additional non-spiked patient serum samples were included (n = 32). The T50 of all samples were measured at two laboratory sites: Amsterdam UMC (Amsterdam, The Netherlands) and Calciscon AG. Data were statistically analysed for accuracy using the Passing Bablok regression and the Bland Altman plot. Interference was established with deviations &amp;gt;10% in T50 compared to the control sample. Results The measurements of all samples at both sites were similar with a mean bias of 0.24% (±4.84%) and a great agreement was found (slope = 1.012; 95% CI [0.931 to 1.107]) (Table 1). Interference was observed at higher levels of haemoglobin and triglyceride with concentrations of at least 0.1 g/dL and 1124.8 mg/dL, respectively. The triglyceride interference is in agreement with the manufacturer's claim (&amp;gt;1000 mg/dL) in contrary to haemoglobin (&amp;gt;0.3 g/dL). No interference was detected for bilirubin with concentrations up to 59.8 mg/dL. Conclusion The accuracy of the T50 test was high with nearly identical T50 results between two laboratory sites. Furthermore, no interference was observed within usual clinical ranges of haemoglobin, bilirubin, and lipid levels. Interference was present at extremely high values only.

#749 Components in blood collection tubes interfere with the spectral signatures of patients with kidney disease: analysis by infrared microspectroscopy

Abstract Background and Aims Kidney disease is a worldwide public health problem, affecting between 8%-16% of the global population. Chronic Kidney Disease (CKD) is a silent disease and its detection is often late, making the treatment difficult. Considering the emerging Fourier-Transform Infrared spectroscopy (FTIR) as a biophotonic resource for the biomedical sciences, we aimed to identify spectral signatures related to biomarkers of CKD in human blood serum using the microscopy FTIR option. Method Blood serum samples from 17 healthy in comparison to 33 CKD patients were analyzed by micro-reflectance FTIR spectroscopy. The first aspect to be addressed refers to the possible influence of components present in the container tube. Here we showed that separator gel in VACUETTE® tubes is a relevant source of spectral interference which could decrease the accuracy of discrimination from 85% to 65%. Acquiring a diluted gel signal as a background would improve the discrimination performance in spite of some gel bands still present in sample data. Results In this case, our results indicated that vibrations associated with galactose-4-sulfate, CH2 bending of the methylene chains, C=C in lipids and fatty acids, C=N stretching, CH bending vibration from the phenyl rings, N-H bending vibration coupled to C-N stretching and β-sheet of Amide II and ring base are modulated in blood serum samples of chronic kidney disease patients compared to healthy patients. Urinary trypsin inhibitors, fatty acids, phenolic derivatives, tryptophan, and plasminogen were the biomolecules related to these assignments were the biomolecules related to these assignments. Conclusion In conclusion, FTIR is a viable option for fast diagnosis of chronic kidney disease being also a powerful tool for monitoring the disease. Due to its capability of large batch analysis, the micro-FTIR would be an eligible technology for clinical laboratories in healthcare facilities. However, for direct clinical applications, ATR-FTIR would be the option of choice.

#3004 Serum miRNAs as novel biomarkers and modulators of rapidly progressing chronic kidney disease in patients with diabetes

Abstract Background and Aims Chronic kidney disease (CKD) is very common in diabetes, occurring in around 40% of patients. Without treatment, CKD can lead to end stage renal disease (ESRD), which requires costly dialysis and transplantation. Current treatments aim to prevent CKD or slow down its progression to ESRD. The speed of CKD progression is patient-specific, and at present there are no biomarkers which can predict which patient with diabetes will develop CKD; level of albuminuria is an important but variably reliable biomarker of speed of CKD progression. The current methods for rapid identification of patients prone to accelerated CKD progression are limited, as they are based on measuring estimated glomerular filtration rate (eGFR) over prolonged periods of time, as well as albuminuria. Therefore, there is a clinical need for novel biomarkers that can predict early potential rapid CKD progression in patients. The aim of this project is to determine whether serum microRNAs (miRs) can be used as biomarkers to predict the speed of CKD progression in diabetes patients, and to identify potential treatment targets associated with such dysregulated miRs. Method miRs were isolated from sera of 34 patients with type 2 diabetes classed with either slow or fast progressing CKD by their eGFR (n = 17; obtained from the Salford Kidney Study sample collection). Several miRs were found to be significantly dysregulated in fast progressing patients by using Next Generation Sequencing (NGS) of miRs in individual serum samples. An additional 49 diabetic CKD patient serum samples (slow (n = 29) or fast progressors (n = 20)) were used to validate the potential miR biomarker panel by qPCR. Results The NGS miR analysis of the initial 34 CKD patient sera produced a panel of 16 miRs that were either significantly upregulated or downregulated in the fast-progressing patients compared to slow progressors. The qPCR validation of this initial panel in 49 additional CKD patient serum samples demonstrated that 6 miRs were significantly upregulated in the fast-progressing patients. The levels of these miRs correlated with demographic and clinical parameters. ROC curve analysis revealed that the individual predictive power of each of those 6 miRs to define either a slow or fast progressing CKD patient was between 63-75%. Determination of a miR combination the strongest predictive power is ongoing. Conclusion A potential biomarker panel of 6 miRs has been identified and validated by qPCR, with each miR having a predictive power of between 63-75% for fast progression of CKD. A panel of miRs that could accurately predict progression speed of CKD patients could lead to faster and earlier diagnosis of patients to enable better treatment. Work is ongoing to explore the predictive power of this miR biomarker panel, and potential novel therapeutic targets to prevent CKD or delay its progression to ESRD.

Detection of Glutamate Decarboxylase Antibodies and Simultaneous Multi-Molecular Translocation Exploration by Glass Nanopores.

Glutamic acid decarboxylase antibody (GADAb) has emerged as a significant biomarker for clinical diagnosis and prognosis in type 1 diabetes (T1D). In this study, we investigated the potential utilization of glass capillary solid-state nanopores as a cost-effective and easily preparable platform for the detection of individual antigens, antibodies, and antigen-antibody complexes without necessitating any modifications to the nanopores. Our findings revealed notable characteristic variations in the translocation events of glutamic acid decarboxylase (GAD65) through nanopores under different voltage conditions, discovered that anomalous phenomenon of protein translocation events increasing with voltage may potentially be caused by the crowding of multiple proteins in the nanopores, and demonstrated that there are multiple components in the polyclonal antibodies (GADAb-poly). Furthermore, we achieved successful differentiation between GAD65, GADAb, and GADAb-GAD65 complexes. These results offer promising prospects for the development of a rapid and reliable GADAb detection method, which holds the potential to be applied in patient serum samples, thereby facilitating a label-free, cost-effective, and early diagnosis of type I diabetes.