Poster

Routine pre-treatment genotyping of four well-characterized toxicity risk variants in the dihydropyrimidine dehydrogenase (DPYD) gene have been widely implemented in Europe, to prevent adverse drug reactions in cancer patients treated with chemotherapeutic fluoropyrimidines.  However, current screening practices primarily focus on variants common in the European populations and other rarer risk variants or variants that are more frequent in other populations are not detected with conventionally used methods. DPYD is an exceptionally large gene consisting of 23 exons, which makes it a challenging candidate for efficient full-gene sequencing in a diagnostic setting. To address this methodological gap, we developed a time- and cost-efficient long-read sequencing method based on Oxford Nanopore Technologies (ONT), targeting the complete coding region of DPYD using full-length cDNA PCR-amplicons. The presented method is capable of capturing all variants in the entire coding region of DPYD, and providing completely phased genotypes. The latter is of particular importance for carriers of more than one risk variants, where a cis- or a trans- configuration of the risk variants would translate into different toxicity risks and dosing recommendations. We applied our method to 21 samples including two cancer patients carrying two DPYD risk-variants. We found complete concordance in base-calling of variant positions with alternative genotyping methods and found that the method is robust with regard to well-known technical issues of sequencing long-range PCR-amplified templates, such as reference alignment bias and PCR-chimera formation.

Aim

Alzheimer’s disease (AD) is the leading cause of dementia in elderly adults. The role of apolipoprotein E (apoE) in AD is well recognized but the underlying mechanisms remain unexplained. It is known that genetic variations of APOE affect the likelihood of AD development and the age of onset, with APOEε4 allele being detrimental, APOEε3 neutral, and APOEε2 protective. In the central nervous system, apoE particles are secreted by various cell types, including astrocytes, pericytes, and microglia. However, it is unknown if the compositions and functions of apoE particles are cell-type dependent. We hypothesize that protein and lipid compositions of apoE particles depend on the cell type they originate from. These lipidome and proteome signatures then influence apoE functions. Particles further differ depending on the APOEε genotype.

Methods

To test our hypothesis, we are differentiating astrocytes, pericytes, and microglia from human induced pluripotent stem cells (iPSC). We will investigate the protein and lipid compositions of apoE particles secreted by each cell type using liquid chromatography–mass spectrometry (LC-MS) and compare them with the composition of apoE in human cerebrospinal fluid (CSF).

We plan to further test the AD-related functions of cell-type-specific apoE particles using 2D cell culture and a 3D model of the human blood-brain barrier. To investigate the composition of apoE particles in a genotype-dependent manner, we will differentiate astrocytes, pericytes, and microglia from isogenic iPSC with different APOEε genotypes. We will investigate the composition and functions of apoE secreted by these cells as described above.

Results

We have differentiated human brain pericytes, microglia, and neural progenitor cells (astrocyte precursors) from iPSCs. We have confirmed that iPSC-derived cells express cell-type-specific markers. In addition, they secrete apoE. Undifferentiated human iPSCs also secrete apoE and express APOE mRNA.

Conclusion

Our project investigates the relationship between the composition and functions of cell-type-specific apoE particles.

At the end of the project, we aim to answer how the composition of apoE is linked to the cell type of origin and APOEε genotype, and how these particles affect cerebrovascular health in the context of AD.

Plasma levels of both Low and High Density Lipoprotein-Cholesterol (LDL-C and HDL-C, respectively) are mainly determined by the catabolism of these lipoproteins. While the limiting effect of the LDL receptor (LDLR) for the hepatic removal of LDL is well understood and exploited for anti-atherogenic drug therapy, the catabolism of HDL is only partially resolved. Scavenger Receptor
Class B Type I (SR-BI) mediates the selective uptake of lipids but the HDL holoparticle receptor is unknown. To identify novel regulators of hepatic LDL and HDL uptake, we performed a genome wide siRNA screen in Huh-7 hepatocarcinoma cells. We validated the candidate genes by in vitro experiments and exploring associations of their genetic variants with plasma lipoprotein levels in humans and genetically modified mice. The genome-wide siRNA screen identified six and three COPI genes that limit the uptake of HDL and LDL uptake, respectively, into Huh-7 cells. In targeted replication experiments, silencing of the six indispensable components of the COPI coatomer but not the three dispensable or paralogous COPI genes resulted in decreased uptake of labeled LDL as well as both lipid- and protein-labeled HDL and decreased cell surface expression of both LDLR and SR-BI due to aberrant glycosylation and subcellular trafficking. In the 1.65 million individuals of the Global Lipid Gene Consortium, single nucleotide polymorphisms of ARCN1 were associated with higher levels of both LDL-C and HDL-C. Rare missense mutations of COPA and COPG1 were found at increased prevalences in patients with hypercholesterolemia. Patients suffering from COPA or COPG1 syndrome had low plasma levels of HDL-C. Genetic mouse models with these mutations also had lower levels of HDL-C and triglycerides but higher levels of LDL-C and apoB than control mice. Liver specific knock-out of Copg1 led to lower plasma levels of HDL-C and triglycerides as well as higher plasma levels of nonHDL-C and apoB. Our data indicate that the COPI coatomer regulates the activity of several pivotal proteins in lipoprotein metabolism and thereby influences plasma levels of lipoproteins. The elevated levels of apoB containing lipoproteins in humans and mice with COPI mutants corroborate the limiting effect of the COPI coatomer on LDLR activity. The finding of low HDL-C despite reduced SR-BI activity and HDL holoparticle uptake indicates more complex effects of the COPI coatomer on multiple proteins regulating HDL metabolism.

Carboxylesterase 1 (CES1) is abundant in liver and metabolically active tissues and catalyses the hydrolysis of many ester-containing therapeutic agents, toxins and endogenous lipids. Genetic variation of CES1 can modify enzyme activity of this pharmacogene resulting in altered clinical response to CES1 substrates. Genetic analysis of CES1 locus has been challenging because of the presence of adjacent homologous pseudogenes and large structural variants. Short-read sequencing platforms often fail to discriminate between homologous regions and this can lead to read misalignment to a reference genome, followed by incorrect variant calls. Long read sequencing has provided powerful tools to tackle this problem and explore complex genomic regions. Here, we apply an optimized PCR-free method for targeted Oxford Nanopore Technology (ONT) sequencing of CES1 locus based on Cas9-directed cleavage of genomic DNA. The ONT Cas9-targeted sequencing (nCATS) method generates reads spanning the complete targeted region of up to 76 kb at sufficient coverage to identify CES1 structural variants and haplotype-resolved single nucleotide polymorphisms (SNPs). Moreover, we report a set of submitted CES1 SNPs in public databases that are likely to be erroneous due to short read sequencing misalignments. Additionally, we show a moderate increase in on-target reads when nCATS is combined with a software-controlled enrichment method named as adaptive sampling. With this method, we obtained more accurate reference sequences of the structurally complex genetic region of CES1 providing an important basis for future pharmacogenetic studies to promote personalized therapy of patients treated with CES1-metabolized compounds. 

The ER located protein serine palmitoyl-transferase (SPT) plays a central role in the onset of neurological disorders such as amyotrophic lateral sclerosis (ALS) or hereditary sensory and autonomic neuropathy type 1 (HSAN 1). SPT catalyzes the first reaction of the sphingolipid metabolism, which is the condensation of the amino acid serine with a fatty acyl-CoA of various chain lengths. Human SPT is active as a complex comprised of the subunit SPTLC1 and either SPTLC2 or 3, the regulatory subunits ORMDL1, 2, or 3, and the small subunits a or b (ssSPTa/b). So far, mutations in subunit 1 and 2 have been reported in both conditions. Here, we investigate a novel mutation G79R located in the transmembrane domain of SPTLC2, close to the ORMDL and ssSPT binding site.

The patient is a 16 years-old girl displaying slowly progressive spasticity of the lower limbs, a low amplitude tremor in hands, lingual fasciculation and crystalline retinopathy, a symptom previously not reported to be linked to a SPT mutation.

The sphingolipid profile in plasma and patient-derived fibroblasts was analyzed using liquid chromatography linked to tandem mass spectrometry (LC-MS). In parallel, we performed a complementation assay and subsequent isotope labeling to assess the de novo synthesis of sphingolipids in this new variant. For that, we expressed the G79R mutation in an SPTLC2 knockout HEK293 cell line. Cells were supplemented for 24h with stable isotope-labeled serine and incorporation into de novo formed sphingolipids analyzed by LC-MS.

We observed unique changes in the sphingolipid profile in patient plasma compared to the brother and unrelated healthy controls. Certain sphingolipid species seem to be under-represented in the patient plasma. This indicates a putatively altered substrate specificity of the G79R variant for its acyl-CoA substrate.

Our results show that the SPTLC2-G79R mutant is associated with a complex change in the sphingolipid profile, which is likely causally linked to the clinical phenotype.

Keywords: crystalline retinopathy, juvenile amyotrophic lateral sclerosis, sphingolipids, serine palmitoyl-transferase.

Sphingolipids (SLs) play a crucial role not only as structural components of cellular membranes but also as signaling molecules involved in diverse cellular processes such as proliferation, differentiation, inflammation, apoptosis, and autophagy. Aberrant SL metabolism predominantly affects the nervous system, which is highly enriched in SLs. Recent studies have identified Δ4-dihydroceramide desaturase 1 (DES1) deficiency as a novel cause of leukodystrophy in humans, with similar pathologies observed in DES1-deficient animal models. While the association between reduced DES1 activity and leukodystrophy is well established, the underlying pathomechanism remains unclear. DES1 deficiency leads to several metabolic alterations, including the accumulation of dihydrosphingolipids (dhSLs), a concomitant reduction in canonical SLs (Δ4E), and the emergence of a new atypical and potentially toxic SL isomer (Δ14Z).

Pharmacological (Fenretinide or GT11) or siRNA-mediated DES1 inhibition in a mammalian myelinating co-culture model, resulted in a dose-dependent increase in dhSLs, a decrease in canonical SLs, and a concomitant reduction in myelination, while cell and axon density remained unaffected. Although cell culture models indicated that the newly identified SL isomer (Δ14Z) is cytotoxic, this isomer was not formed in our myelinating co-culture due to the low expression of fatty acid desaturase 3 (FADS3), which is responsible for its formation. Blocking de novo SL synthesis with myriocin, thereby reducing SLs, did not affect myelination or axon density. Interestingly, preventing the accumulation of dhSLs in DES1 deficiency with myriocin restored the SL profile but did not prevent myelin loss. In contrast, reducing DES1 deficiency-induced reactive oxygen species (ROS) formation with Vitamin C rescued myelin loss.

In summary, our scalable mammalian cell culture model successfully recapitulated the biochemical and neuropathological phenotype observed in human patients and animal models with DES1 deficiency. Our findings indicate that DES1 deficiency not only perturbs the SL profile but also increases ROS production, which underlies hypomyelination. Reducing ROS with Vitamin C effectively prevented myelin loss, highlighting a potential therapeutic approach for treating DES1-dependent leukodystrophy.

Mutations in the genes JAK2V617F have been identified as drivers of Myeloproliferative Neoplasms (MPNs), including Polycythemia Vera (PV), Essential Thrombocytosis (ET), and Primary Myelofibrosis (PMF). JAK2V617F is included as major diagnostic criteria for MPN in the 2008 WHO diagnostic criteria. Therefore, we aimed to report the JAK2V617F mutation frequency and disease phenotype in northeast Thailand patients from January 2017 to January 2021. A total of 418 peripheral blood and bone marrow samples were analyzed by qRT-PCR. In this group, 101 (24.17%) of the patients were positive for the JAK2V617F mutation, whereas 317 (75.83%) patients were negative for the JAK2V617F mutation. The JAK2V617F mutation-positive group (101 patients) included 46 (45.54%) patients with PV, 39 (38.61%) patients with ET, 11 (10.90%) patients with primary myelofibrosis (PMF), and 5 (4.95%) patients with unclassified MPNs. The mean age of JAK2V617F-positive male patients was 59.86 years (range: 21-88), which is higher than that of female patients, whose mean age was 57.14 years (range: 23-85). Laboratory findings showed that the mean hemoglobin level (17.8 g/dL, range 14.1-21.6 g/dL) and WBC level (10.2 x 10^9/L, range 5.8-43 x 10^9/L) in PV patients were higher than those in ET patients. PV patients had a mean platelet count of 512.5 x 10^9/L (range 151-1028 x 10^9/L), which was lower than the mean platelet count of 803 x 10^9/L (range 355-2571 x 10^9/L) in ET patients. The mean values of the JAK2V617F allele burden in patients with PV and ET were 62% and 31%, respectively. In conclusion, the JAK2V617F mutation allele burden is higher in Thai patients with PV than in those with ET. The JAK2V617F mutation burden influences WBC counts and clinical characteristics of ET patients, such as WBC counts and hemoglobin levels. A higher JAK2V617F allele burden is associated with increased age, suggesting its potential role in prognostication and treatment protocol development.

Alzheimer’s disease (AD) is the leading cause of senile dementia with over 50 million affected individuals worldwide. Its well-known neuropathological hallmarks, beta-amyloid and tangles, are preceded by cerebrovascular damage. High-density lipoprotein (HDL) possesses vasoprotective functions and epidemiological studies showed associations of very low and very high HDL-cholesterol levels with the risk of AD. Studies suggest that HDL reduces AD risk by decreasing both beta-amyloid deposition within the vasculature and vascular inflammation. We previously found that HDL enriched in apolipoprotein (apo)E (HDLE⁺) are more effective in removing vascular beta-amyloid deposits than those lacking apoE (HDLE^-). However, HDL circulating in the blood must first interact with brain endothelial cells (ECs) to display its anti-AD properties, a process that is poorly understood.

HDL was isolated from plasma of healthy donors by ultracentrifugation before being further fractionated into HDLE⁺ and HDLE^- using apoE immunoaffinity chromatography. Fluorescent or ¹²⁵I-radio labeled HDL were used to measure binding (4^oC), association, internalization or transport (37^oC) through primary human brain EC or the cell line hCMEC/D3. siRNA was used to understand the role of different receptors in the trafficking of HDL by the brain endothelial cells (ECs). Using size exclusion chromatography we determined changes in the size of the HDL particles after being transported through the brain ECs.

Our in vitro data show that HDL, and both HDLE+ and HDLE- subpopulations bind to, enter in and are transported through brain ECs as intact particles. HDLE+ interacted more with the ECs and were more transported across the brain endothelium. We identified the low-density lipoprotein receptor (LDLR) as a protein limiting HDL transport through brain ECs. Moreover, we found that the uptake of the different subpopulations is mediated by different receptors. LDLR limits the trafficking of HDLE+ while the trafficking of HDLE- is mediated by LIPG and SRBI. Moreover, the different subpopulations, HDLE+ and HDLE-, only partially co-localized in brain ECs suggesting independent trafficking pathways.

Together our findings suggest distinct trafficking pathways for HDLE+ and HDLE- through brain ECs and might imply different cerebrovascular functions relevant to AD. Moreover, rHDL containing ApoE could prove to be a beneficial tool for specific drug transport across the BBB while promoting cerebrovascular health.

Background

In the process of understanding the course of infections, a key question is whether antibiotics reach sufficient concentrations at the infected site. Deep-seated musculoskeletal infections often require both surgery and antibiotic therapy for successful treatment. However, only few data about antibiotic tissue penetration is available for these patients. At the University Hospital Basel, amoxicillin in combination with clavulanic acid is the most frequently administered antibiotic before musculoskeletal surgery. Aim of this work was to measure and evaluate the amoxicillin concentrations in infected tissue samples from patients with osteomyelitis, joint infections, cellulitis, or abscess.

Methods

From 06/2021 until 03/2023, tissue samples of patients undergoing surgery for musculoskeletal infections were collected and immediately frozen. Protein precipitation and manual homogenization with micro pestles were used for preparation of the thawed samples, followed by an online solid-phase extraction for further matrix clean-up. Analysis and quantification were performed with high performance liquid chromatography and tandem mass spectrometry. Information on infection type, as well as antibiotic and surgical treatment was retrieved from the hospital clinical database.

Results

Until now, we analysed 37 tissue samples from patients receiving amoxicillin/clavulanic acid between 0.0 until 7.7 hours before surgery. Measured amoxicillin concentrations ranged from undetectable to 87 mg/kg. Only 3 samples showed concentrations below the limit of detection (0.5 mg/kg). We observed a significant correlation between the measured concentration and documented time since the last dose. Amoxicillin concentrations in abscesses were lower than in the other types of infection.

Conclusions

Our results indicate a dynamic amoxicillin penetration into the infected deep-seated tissue. For further interpretation, the results will be combined with additional clinical and diagnostic data such as renal and inflammation parameters, clinical outcome, and bacterial culture results.

Acknowledgement

This project was supported as part of NCCR AntiResist, a National Center of Competence in Research, funded by the Swiss National Science Foundation (grant number 180541).

Background: Metabolic dysfunction-associated fatty liver disease (MASLD) is a common liver condition associated with increased cardiovascular disease (CVD) risk. Cytokeratin 18 (CK-18) is indicative of liver injury and used as a surrogate biomarker covering MASLD to cirrhosis phenotypes. Autoantibodies against apolipoprotein A-1 (AAA-1) are known CVD risk factors inducing MASLD in vitro, but their role in modulating steatohepatitis and fibrosis in vivo is still elusive. We investigated AAA-1's contribution to systemic low-grade inflammation, liver steatosis, and fibrosis using a MASLD mouse model and a validated passive immunization protocol (PIP).

Methods: 10 weeks-old male C57BL/6J mice were fed a choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) and immunized with AAA-1 or control antibodies for ten days. At sacrifice, plasma samples were collected, and CK-18 levels, and proinflammatory cytokines were measured using the Mesoscale Discovery platform. Hematoxylin and Eosin and Sirius Red Fast Green staining were used to reveal liver steatosis and fibrosis, respectively. mRNA from liver tissue was then analyzed using NanoString Technologies.

Results: Compared to control IgG recipients, CDAHFD mice injected with AAA-1 exhibited significantly elevated plasma levels of CK-18 (5.3 vs 2.1, p=0.031), IL-6 (13 vs 6.9, p=0.035), IL-10 (27.3 vs 9.8, p=0.007) and TNF-α (32.1 vs 24.2, p=0.032), and liver steatosis (93.4% vs 73.8%, p=0.007). Nanostring technology experiments revealed upregulation of several pro-fibrotic m-RNAs in liver tissue from AAA-1 immunized mice compared to the IgG immunized control group. These included Serum Amyloid A (Fold Change (FC) AAA-1 vs IgG : 2.5, p=0.02) , G-protein coupled receptors 65 (FC: 2.7, p=0.01), Periostin (FC: 2.6, p=0.001), Phospholipid transfer protein (FC: 2.2, p=0.0004), Vascular Cell Adhesion Molecule 1 (FC: 2.5, p=0.02) and Angiopoietin-like 4 (FC: 2.8, p=0.01). Histologically, the amount of liver fibrosis remained unaffected, probably due to the short time of the study.

Conclusions: Short AAA-1 PIP induced enhanced liver steatosis and inflammation accompanied by an elevation of the expression of several pro-fibrotic genes in CDAHFD mice, suggesting that AAA-1 may contribute to the transition from MASLD to MASH. Knowing whether increased exposure time to AAA-1 could lead to end stage disease (including cirrhosis and HCC), and if this could apply to humans as well warrant further investigations.

Objectives. To validate the prognostic accuracy of anti-apolipoprotein A-1 IgG (AAA1) for incident major adverse cardiovascular (CV) events (MACE) in rheumatoid arthritis (RA) and study their associations with the lipid paradox at a multicentric scale. Methods. Baseline AAA1 -, lipid profile, atherogenic indexes, and cardiac biomarkers were measured on the serum of 1472 RA patients included in the prospective Swiss Clinical Quality Management registry with a median follow-up duration of 4.4 years. MACE was the primary endpoint defined as CV death, incident fatal or nonfatal stroke, or myocardial infarction (MI), while elective coronary revascularisation (ECR) was the secondary endpoint. Discriminant accuracy, and incidence rate ratios (IRR) were respectively assessed by C-statistics and Poisson regression models. Results. During follow-up, 2.4% (35/1472) patients experienced MACE, consisting in 6 CV deaths, 11 MI and 18 strokes. ECR occurred in 2.1% (31/1472) of patients. C-statistics indicated that AAA1 had a significant discriminant accuracy for incident MACE (C-statistics: 0.60, 95% confidence intervals [95%CI]: 0.57-0.98, p=0.03), mostly driven by CV deaths (C-statistics: 0.77; 95%CI: 0.57-0.98, p=0.01) and without predicting incident ECR. IRR indicated that for every unit increase in AAA1, incident CV death rate increased by 5-fold (95%CI: 3.6-7.3), independently of various models’ adjustments. At the predefined and validated cut-off, AAA1 displayed negative predictive values above 97% for the MACE components. AAA1 inversely correlated with total, and HDL cholesterol, but not with atherogenic indexes.

Conclusions. AAA1 independently predict CV deaths in RA. Whether AAA1 could improve CV risk stratification by identifying in particular low CV risk patients remains to be demonstrated.

Objectives: Efficient and timely transportation of clinical samples is pivotal to ensure accurate diagnoses and effective patient care. During the transportation process, preservation of sample integrity is crucial to avoid pre-analytical aberrations on laboratory results. Here, we present a comparative analysis between a two-step Tempus600 hub solution single-tube and a one-step, container-based pneumatic transport system (PTS) from Airco, for the in-house transportation of blood samples.

Methods: Ten blood samples from healthy volunteers were split in 10 mL collection tubes filled at full or half capacity for transportation with the two PTS (about 250 m). To compare the impact of transportation, markers of hemolysis such as lactate dehydrogenase (LDH), potassium (K+), and the hemolysis index (HI), were determined. Additionally, differences in HI in routine samples and repeated transportation was investigated. To assess and compare the mechanistic impact profiles, we recorded the acceleration profiles of the two PTS using a shock data logger.

Results: Transportation using the Tempus600 hub solution resulted in 49 and 46 % higher HI with samples filled to total or half capacity, respectively. Routine samples transported with the Tempus600 hub solution showed a higher median HI by 23 and 33 %. Additionally, shock logger analysis showed an elevated amount of shocks (6.5 fold) and shock intensities (1.8 fold).

Conclusions: The Tempus600 hub solution caused an increased number of unreportable LDH or K+ results based on the hemolysis index. However, it was only statistically significant for LDH (p<0.01 and p<0.08) – while the comparisons for K+ were not statistically significant (p<0.28 and p<0.56).

Aims: Heavy metals (HM) are combustion-related environmental factors predisposing to cardiovascular (CVD), autoimmune and liver diseases, even below regulatory concentrations. Pro-atherogenic and pro-steatotic auto-antibodies against apolipoprotein A1 (AAA1) are prevalent in the general population for unclear reasons. We explored a putative interplay between HM exposure, AAA1, CVD risk factors and a non-alcoholic fatty liver disease (NAFLD) risk score, using a geospatial clustering approach at the general population level.

Methods: Getis-Ord, Moran I² clustering and geographically weighted regression (GWR) geosptatial analyses were carried out on 6361 individuals recruited in the CoLaus/PsyCoLaus general population cohort. Geospatial dependences between serum AAA1, urinary HM (cadmium, arsenic, cobalt) concentrations, SCORE2 CVD risk, and fatty liver index (FLI) were assessed.

Results: Cadmium and cobalt were found to be associated with higher SCORE2 risk (p<0.04). AAA1 were associated with higher cadmium, cobalt, and cigarette consumption, independently of SCORE2 (p<0.04). AAA1 hotspots overlapped significantly with those of cadmium and FLI, independently of SCORE2. According to GWR, correlations between cadmium and AAA1 were the highest in railways proximity regions, increasing up to 10-fold compared to global associations. AAA1 were not associated with other HM.

Conclusions: These geospatial footprints highlight independent associations between Cadmium, AAA1, and NAFLD risk score in the general population. We hypothesize that environmental cadmium exposure may facilitate the occurrence of AAA1 in humans. Ongoing experimental studies will confirm/refute a possible causal link. Whether AAA1 could represent an early biological signature of cadmium exposure and subsequent health hazards remains to be shown.

Up to know the diagnosis of sphingolipidoses has been relying on time-consuming measurements of specific enzymatic activities and/or genetic analysis. In the recent years, lysosphingolipids in plasma have emerged as potential biomarkers of sphingolipidoses as well as Niemann-Pick disease type C. We have recently established and validated a novel method which allows to measure Globotriaosylsphingosine (LysoGb3), Glucosylsphingosine (LysoGb1), Sphingosylphosphorylcholine (LysoSM), N-Palmitoyl-O-Phosphocholineserine (LysoSM-509), 3β,5α,6β-Trihydroxycholanic acid, Lyso-monosialoganglioside-GM1 and Lyso-monosialoganglioside-GM2 for the diagnosis of Fabry and Gaucher diseases, Niemann-Pick-disease types ABC, GM1-Gangliosidosis and GM2-Gangliosidosis.

The final method, whit a total run time of just 7 min, is linear over a broad concentration range (r² > 0.99), sensitive (LLOQ = 0.012 – 2.119 nM), precise (inter-assay CV = 2.4 – 24.2%) and has satisfactory recoveries for all tested analytes (77.0 – 112.2%). Stability experiments showed that all metabolites are stable in plasma at room temperature for at least 48 hrs, enabling to ship samples at room temperature.

Several known pathological samples were measured during the validation of the method, which always led to the correct diagnosis using the established laboratory-based reference ranges.

Background: The determination of I and T subunits of cardiac troponin isoforms are the biochemical gold standard for the detection of myocardial injury. The advent of so-called highly sensitive (HS) immunoassays has optimized the diagnosis of acute coronary syndromes at the cost of increasing sensitivity to analytical interferences. The aim of the present work is to present the workflow allowing the identification of a macrotroponin, characterized by circulating IgG-troponin T immunocomplexes leading to false positive results. As different orthogonal approaches are required to adequately distinguish between the different causes of such interferences, we propose a sequential approach.  

Methods: The suspicion of interference was based on persistently elevated hs-cTnT in a ER patient hospitalized for prolonged thoracic pain, but all the diagnostic work-up allowed for the exclusion of myocardial injury.  Hs-cTnT was measured on Roche Diagnostics Cobas 8000 platforms. Various analyses were conducted. As the first step, serial sample dilution was performed to detect interference. Additionally, the serum was incubated with Scantibodies Heterophilic Blocking Tube to block heterophilic antibodies.  To rule out macro complexes in the sample, serum was precipitated using polyethylene glycol 25% (PEG). Finally, we depleted IgG from patient's serum using affinity chromatography by treating serum with a protein G.

Results: First, interference was indicated as the hs-cTnT concentration did not dilute linearly after sample dilution. Heterophile antibody interference was excluded by pre-treatment of the sample with specific heterophilic antibody blocking tubes, which did not yield a diminished recovery. PEG precipitation and protein G affinity resin enabled the retention of macrotroponin resulting in lower cTnT recovery. After both procedures, we obtained recoveries of 5.3% and 1.7% respectively, confirming the presence of a macrotroponin complex in patient's serum.

Conclusion: The laboratory investigation results are consistent with the presence of macrotroponin T. Macrotroponin complexes appear to be a rare phenomenon but responsible of unexplained persistent cTnT elevation. Recognizing the rare occurrence of macrotroponins can help reduce the need for additional tests, particularly in cases involving repeated consultations for the same issue. Laboratories and clinicians should be aware of this type of assay interference, along with the required workflow to detect them.

Post partum hemorrhage (PPH) is defined by a blood loss of 500 ml or more within 24 hours after delivery, while a blood loss of more than 1000 ml is considered as major or severe PPH. PPH is a major cause of maternal death worldwide. With the aim to better understand the PPH phenomena a prospective, monocentric cohort study was conducted at the Department of Obstetrics, University Hospital Zurich (USZ), Switzerland. Details on the study can be found elsewhere (1). We report here the measurements of the concentrations of ferritin and hemoglobin before and after delivery in women who delivered vaginally. The blood (citrate plasma) of 627 patients was analyzed within 36 hours before and within 24 hours after delivery. The blood loss of the parturient was measured according to a decribed procedure (2). According to the measured blood loss (MBL), the patients were divided in three groups: a) no PPH: women who within 24 hour after delivery lost less than 500 ml blood; b) MBL, >500 ml: women who within 24 hour after delivery lost more than 500 ml and less than 1000 ml blood; c) MBL, >1000 ml: women who within 24 hour after delivery lost more than 1000 ml blood.

The level of ferritin before partum is 12-19 µg/l for all three groups. After delivery the ferritin’s concentration increases by 20-30% for all three groups.

The level of hemoglobin before partum is 119-133 µg/l for all the three groups. After delivery the women without PPH show a decrease of about 9%, the women with >500 ml MBL the hemoglobin level decreases by about 20% and the women with >1000 ml MBL the value decreases by about 37%.

As conclusion we can state that the prepartum ferritin concentration is not predictive for PPH.

References

1. Haslinger C, Korte W, Hothorn T, Brun R, Greenberg C, Zimmermann R. The impact of prepartum factor XIII activity on postpartum blood loss. J Thromb Haemost. 2020;00:1–10. https://doi.org/10.1111/jth.1479
2. Kahr MK, Brun R, Zimmermann R, Franke D, Haslinger C. Validation of a quantitative system for real-time measurement of postpartum blood loss. Arch Gynecol Obstet. 2018;298(6):1071-1077.

Neurofilaments (Nf) belong to intermediate filaments and are part of the neuronal cytoskeleton. Among many different roles they are important for structural stability and for the radial expansion of large myelinated axons. Neurofilaments come under three forms:  heavy weight (NfH), medium weight (NfM) and light weight (NfL). Under neuronal damage or death, Nf are released into the extracellular space. Their lighter form, NfL, can then reach CSF and even peripheral blood, where it can be measured.

NfL has thus been recently identified as a sensitive marker of neurodegeneration in several diseases (Alzheimer’s, Parkinson’s, Lewy body, FTD, Huntington’s and Multiple sclerosis) for which it can be used to follow the progression of the disease. Several studies have demonstrated that CSF and serum NfL levels are highly correlated. Serum NfL detection allows the monitoring of disease progression in a much less invasive way than with CSF. However with NfL levels in blood being around 40 times lower than in CSF, more sensitive methods of detection must be employed.

The recently available Single Molecular Array (SiMoA®, Quanterix) enables the detection of proteins down to femtomolar concentrations. We describe here the method verification and implementation of serum NfL measurement within our laboratory using the SiMoA® Quanterix system.

Introduction

Fluoropyrimidines-based chemotherapy is one of the major treatments of many cancers. However, in clinical practice, 25 to 30% of patients present severe fluoropyrimidine-related toxicity, mainly due to a dihydropyrimidine dehydrogenase (DPD) deficiency.

To decrease the risk of toxicities associated with these treatments, systematic screening for DPD deficiency is becoming a strong recommendation by health authorities in Europe. Indirect phenotyping of DPD, by measuring plasma concentrations of uracil and dihydrouracil by LC-MS/MS, is the method of choice to assess potential DPD deficiency. However, due to the chemical properties of the target compounds, setting up and standardizing such a method can be difficult. Therefore, Alsachim, a Shimadzu Group Company, has developed the first reagent set for indirect phenotyping of DPD: DOSIURA^TM.

Validation of this reagent kit analytical performances has been conducted and, as requested by the European In Vitro Diagnostic Regulation (IVDR), evaluation of its clinical performances has also been carried out.

Objectives

To comply with IVDR, scientific validity, performance validation and clinical validation of the reagent set shall be demonstrated.

The objective of this presentation is to focus on the results obtained during the clinical evaluation of DOSIURA^TM.

Methods

Clinical evaluation was conducted at Limoges CHU site in France, already routinely performing the indirect phenotyping of DPD with their own laboratory-developed test.

A total of 229 samples obtained from leftovers and covering the calibration range were collected.

Validation was assessed by comparing obtained results on a same sample using DOSIURA^TM reagent solution and the current method in place, on the same LC-MS/MS instrument.

Results

A good correlation between methods was obtained with a limited bias for both uracil and dihydrouracil.

Moreover, feedback from users highlighted a good usability of the reagent set. The rapid and efficient sample preparation was highly appreciated as well as the robustness of the analytical method.

Conclusion

Evaluation of clinical performances of DOSIURA^TM reagent set was conducted and presented a good correlation between methods. Therefore, DOSIURA^TM was clinically validated to provide reliable diagnostic of DPD deficiency prior to chemotherapy with fluoropyrimidines and will be submitted for IVDR certification.

In this evaluation the Centre of Laboratory Medicine in St.Gallen tested the diagnostic performance of the measurement of NT-pro BNP (N-terminal pro-Brain Natriuretic Peptide) on LumiraDx POCT (Point of Care Testing) –system. NT-proBNP’s concentration is a biomarker to identify the presence and the severity of haemodynamic cardiac stress and heart failure. The LumiraDx NT-pro BNP Test is a microfluidic immunofluorescence assay. Results appear after 12 minutes and are displayed quantitatively. The test is approved for fingerstick, venous whole blood or plasma (lithium-heparin). The measuring range is between 50–9000 ng/l.

The evaluation of the LumiraDX was done by comparison of the NT-pro BNP’s concentrations of 53 heparin plasma samples quantitatively determined using the Cobas Elecys analyzer (Roche, Basel, Switzerland) and the LumiraDX according to the manufacturer’s instructions.

The overall performance of the LumiraDx NT-proBNP Test was conclusive. The test is easy to perform and generates fast and reliable results with high reliability.

In the broad range between 50–9000 ng/l the Passing and Bablock regression for the comparison of both methods shows good agreement. The data indicates that both methods are not proportionally different. LumiraDX gives slightly smaller values with respect to Cobas Elecys.

In the range below 1000 ng/l, which is of clinical interest, the Passing and Bablock regression indicates an even better agreement between the two methods.

References

1. Christian Mueller, Kenneth McDonald, Rudolf A. de Boer, Alan Maisel, John G.F. Cleland, Nikola Kozhuharov, Andrew J.S. Coats, Marco Metra, Alexandre Mebazaa, Frank Ruschitzka, Mitja Lainscak, Gerasimos Filippatos, Petar M. Seferovic,Wouter C. Meijers, Antoni Bayes-Genis, Thomas Mueller, Mark Richards and James L. Januzzi Jr. Heart Failure Association of the European Society of Cardiology practical guidance on the use of natriuretic peptide concentrations. European Journal of Heart Failure (2019) 21, 715–731. doi:10.1002/ejhf.1494
2. All instructions to use and further information about the LumiraDx test device can be found at https://www.lumiradx.com

Hypertension is one of the most common chronic medical conditions worldwide and remains the leading cause of cardiovascular disease and premature death. Primary aldosteronism (PA) is an endocrine disease that can affect one or both adrenal glands in which an inappropriately high production of aldosterone (independent of the renin-angiotensin-aldosterone-system) can lead to what is termed secondary hypertension. Although initially most experts previously described PA in less than 1% of patients with mild to moderate hypertension, more recent studies have suggested the presence of PA in more than 10% of all hypertensive patients.

The aldosterone-to-renin ratio (ARR) is still currently the most reliable and available screening approach for PA, this includes the quantification of plasma aldosterone and renin activity (PRA, the measure of the production of angiotensin-I) or direct renin concentration. Although in recent years and seemingly for practical reasons, many laboratories have opted for direct renin concentration approaches using automated immunochemistry-based methods, there are some concerns regarding direct renin measurements at low levels. Additionally, immunochemistry-based methods can suffer from cross reactivity with non-target molecules. Approaches based on liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) allow for a more accurate measurement and do not suffer from issues such as cross reactivity. Here we present a validated LC-MS/MS method for the determination of PRA, developed to replace a radioimmunoassay (RIA) based approach formerly employed at our hospital.

Background: When estimating reference intervals using patient’s blood test results originating from clinical routine, test results differing significantly need to be removed a priori. Exclusion criteria defined for estimating reference intervals are often applied indifferently across all patient sex and age strata. This removes a vast majority of test results e.g. from the elderly patient strata. 

Methods: The Differential Distribution Method (DDM) leverages ICD-10 labelled laboratory routine data to approximate an underlying non-diseased age and sex stratified population from mixed clinical data. By removing test results that stem from significantly different subpopulations from the general population, reference intervals stratified by sex and age taking into account the associated health conditions of the patients as signified by the ICD-10 coding system can be generated.

Results: Reference intervals for blood plasma creatinine levels estimated using the DDM were narrower than estimates from a general population. Their standard deviation was reduced by removing large portions of test results differing significantly, grouped by either their individual ICD-10 code or clusters of ICD-10 codes. The RIs were slightly wider with advancing age in both males and females, while they retained the position and width of observed variance amongst patients’ strata. 

Conclusions: The proposed DDM data mining approach for RI inference generates appropriate RIs for clinical use by involving test results based on individual associated ICD-10 codes, thereby taking patients’ health status into account. In addition, it may facilitate the discovery of novel connections between clustered diseases, multimorbidity and their effect on patients’ creatinine levels.

Acute intermittent porphyria (AIP) is an inborn error of metabolism affecting the heme biosynthesis in the liver. Patients carrying disease causing mutations are at risk to develop potentially life-threatening acute attacks triggered by factors, such as CYP450 enzyme activating drugs, stress, fasting, and infections. Acute attacks are biochemically characterized by an increased excretion of urinary porphobilinogen (PBG), one of the precursors of heme. However, so called "asymptomatic high excreters" can have a persistently elevated urinary PBG excretion without clinical signs for an acute attack. According to the recently published international consensus guideline, an increased urinary PBG excretion >10 times the upper limit of normal (ULN) together with ≥ 2 typical clinical symptoms (as defined in the guideline) is evidence of an acute attack. We aimed to determine the proportion of  patients with a confirmed diagnosis of AIP in the Swiss cohort who, despite lacking current clinical signs for an acute attack, have an elevated urinary PBG excretion >10 times the ULN.

Method: In the Swiss reference centre for porphyrias at the Stadtspital Zurich, Triemli, PBG excretion is monitored in patients with a confirmed diagnosis of AIP during the routine follow-ups. Between January to May 2024, we prospectively analysed PBG excretion and clinical information in patients with AIP who had provided their written informed consent for participation in the ongoing porphyria biobank study approved by the Cantonal Ethics Committee in Zurich (BASEC 2018-00758).

Results: In total, n=25 patients with an established diagnosis of AIP and participating in the biobank study were seen in the outpatient clinic between January to May 2024. Clinically, all 25 patients were without an acute attack during their appointments. Of these n=13 (52%) had an increase in urinary PBG excretion of >10 times of the ULN. The urinary PBG excretion in both groups as measured in our laboratory was mean 29.2 µmol/mmol creatinine (SD=12.6, median 27.5, range: 13.7-59.1, n=13) and 4.4 µmol/mmol creatinine (SD=3.1; median: 4.2, range: 1-12.4, n=12), respectively, with >12.5 µmol/mmol creatinine being >10 ULN.

Conclusion: In our cohort, in about 50% of patients with AIP, an increased urinary PBG excretion of >10 of the ULN is not sufficient to prove an acute attack but additional clinical information is imperative for the correct interpretation of the test results, in line with the consensus Guideline.

Background: Acute intermittent porphyria (AIP) is an inborn error of metabolism affecting the heme biosynthesis in the liver. Patients carrying disease causing mutations are at risk to develop potentially life-threatening acute attacks after exposure to triggering factors, such as CYP450 enzyme activating drugs, stress, fasting, and infections. Acute attacks are biochemically characterized by an increased excretion of urinary porphobilinogen (PBG), one of the precursors of heme. However, so called "asymptomatic high excreters" can have a persistently elevated urinary PBG excretion without clinical signs for an acute attack. According to the recently published international consensus guideline, an increased urinary PBG excretion >10 times the upper limit of normal (ULN) together with ≥ 2 typical clinical symptoms (as defined in the guideline) is evidence of an acute attack. We wanted to know how many patients with a confirmed diagnosis of AIP in the Swiss cohort without current clinical signs for an acute attack have an elevated urinary PBG excretion >10 times the ULN.

Method: In the Swiss reference centre for porphyrias at the Stadtspital Zurich, Triemli, PBG excretion is monitored in patients with a confirmed diagnosis of AIP during the routine follow-ups. Between January to May 2024, we prospectively analysed PBG excretion and clinical information in patients with AIP who had provided their written informed consent for participation in the ongoing porphyria biobank study approved by the Cantonal Ethics Committee in Zurich (BASEC 2018-00758).

Result: In total, n=25 patients with an established diagnosis of AIP and participating in the biobank study were seen in the outpatient clinic between January to May 2024. Clinically, all 25 patients were without an acute attack during their appointments. Of these n=13 (52%) had an increase in urinary PBG excretion of >10 times ULN. The urinary PBG excretion in both groups as measured in our laboratory was mean 29.2 μmol/mmol creatinine (SD=12.6, median 27.5, range: 13.7-59.1, n=13) and 4.4 μmol/mmol creatinine (SD=3.1; median: 4.2, range: 1-12.4, n=12), respectively, with >12.5 μmol/mmol creatinine being >10 ULN.

Conclusion: The increased urinary PBG excretion of >10 ULN in about 50% in our cohort of patients with AIP confirms and emphasises the importance of additional clinical information for the correct interpretation of the test results, in accordance with the consensus guideline.

Introduction: The new biomarker cell-free DNA (cfDNA) is on the rise for its potential in monitoring of e.g. rejections after transplantation. Most studies focus on cfDNA in plasma, however, urinary cfDNA (ucfDNA) has shown potential clinical relevance with the benefit of non-invasive collection. Since ucfDNA is more fragmented than plasma cfDNA, the size selectivity of the extraction method is important and, extraction efficiency, which is relevant for absolute ucfDNA quantification, may vary between methods.

Objective: This study aimed to identify variability between extractions, sexes and individuals for two ucfDNA extraction methods. Additionally, we evaluated the estimation of extraction efficiency using CEREBIS (an artificial spike-in) and the variability of extraction efficiency for different ucfDNA fragment lengths.

Methods: We collected urine samples from three healthy male and female volunteers each. An aliquot was used to measure creatinine and the rest was mixed with a DNA stabilizer (Streck Urine Preserve). Samples from each donor were extracted in triplicates with both extraction methods, Zymo and Qseph (adapted from literature). UcfDNA was quantified using droplet digital PCR with RPP30 as an autosomal reference gene and CEREBIS for the detection of the spike-in. Variability analysis was performed using nested ANOVA.

Results: Overall we found lower ucfDNA quantities in samples extracted with Qseph (mean = 469 cp/mL urine) compared to Zymo (mean = 1308 cp/mL). The majority of the variability was accounted for by differences between donors (99.7% and 99.9% for Qseph and Zymo, respectively) and only a very small fraction by the extractions (0.16% vs. 0.03% for Qseph and Zymo, respectively). We did not observe a reduction in the total variability when accounting for extraction efficiency estimated by CEREBIS. However, a reduction of the overall variability was observed, when accounting for urinary concentration using creatinine.

Conclusions: Overall low intra-sample variability between extractions with a high inter-individual variability shows the potential to detect biological variation between individuals for both extraction methods. Furthermore, the adjustment of ucfDNA quantities using urinary creatinine could improve the inter-sample comparability and should be used for absolute ucfDNA quantification. To account for the size specificity in ucfDNA extraction methods, analyses using different fragment lengths of spike-ins are ongoing.

Introduction: Beside a powerful analytical technique, the pre-analytical phase is a key factor for reliable quantitation of minerals and trace elements in biological fluids. Special attention in this context requires the sample tube: As no material is completely free of elements, all components of a tube, comprising tube wall, rubber stopper, anticoagulant, separator gel, clot activator, and surfactant, are sources of possible contaminations.

In this study, 33 different sample tubes (blood and urine tubes) from 3 manufacturers (Sarstedt, Greiner Bio-One, Becton Dickinson) were tested regarding their suitability for quantitation of 26 different elements. Both, special tubes for elemental analysis as well as conventional sample tubes were included. To clarify whether interaction time with the tube has an influence, different storage durations were investigated.

Methods: The sample tubes (3 of each type) were filled with 0.1% nitric acid and elements were quantitated after 2h, 6h, 24h, and 48h of storage using inductively coupled plasma mass spectrometry (ICP-MS). Between the analyses, sample tubes were stored in upright position at 4°C and shaken on a roller mixer for 15 min each time before analysing. Contaminations were considered as relevant if the measured concentration exceeded 25% of the lowest reference value.

Results: No significant concentrations were observed for the elements magnesium, iron, palladium, silver, cadmium, tin, iodine, mercury, thallium, and bismuth. All other elements (Be, Al, Ti, V, Cr, Mn, Co, Ni, Cu, Zn, As, Se, Mo, Sb, Ba, Au) showed relevant concentrations in at least one sample tube, which leads to restrictions in the choice.

Discussion & conclusion: For serum/plasma analyses, serum tubes without additives and separator gel are best suited. Lithium heparin tubes without additives are also suitable and are preferable to EDTA tubes. For urine analyses, tubes without additives are superior to tubes with additives. In general, the fewer additives in the tubes, the better. All elements can be determined from conventional sample tubes. Special trace element tubes are not necessary but suitable for most elements. Interaction time with the tube is largely irrelevant. However, for zinc an increase over time is observed in most sample containers of the manufacturer BD. Most likely the contaminations are caused by the butyl rubber stoppers. To minimize this effect a fast transfer of the specimen to a suited secondary tube is recommended.

Measurement of selenium in biological samples serves as crucial biomarker for assessing nutritional status. Nowadays, inductively coupled plasma mass spectrometry (ICP-MS) has become the preferred technique in many laboratories due to its high sensitivity, multi-element capability, and efficient sample throughput. Selenium has six natural occurring isotopes, with ⁷⁸Se (23.8%) and ⁸⁰Se (49.6%) being the most common. Despite its high abundance, ⁸⁰Se is rarely used for quantitation due to interference with an ion formed by argon gas (⁴⁰Ar⁴⁰Ar⁺). Hence, quantitation is typically performed using ⁷⁸Se.

However, gadolinium (Gd) based contrast agents, often administered during magnetic resonance imaging procedures, interfere with ⁷⁸Se measurements. This is related to the doubly charged Gd species (Gd²⁺, atomic weight 156) which results in a mass-to-charge identification of 78, indistinguishable from ⁷⁸Se⁺, leading to falsely elevated selenium results.

To overcome this interference, we opted to utilize an alternative selenium isotope, namely ⁸²Se, for quantitation. Due to its lower natural abundance (8.7%) ⁸²Se suffers from poor analytical sensitivity. However, by using the dynamic reaction cell (DRC) mode with 7% H₂ in He as the reaction gas, sensitivity could be significantly improved. Precision and accuracy of the selenium measurement in the presence of gadolinium was successfully proven, resulting in the routine measurement of ⁸²Se.

After several month in routine operation, problems arose when changing to a new lot of internal quality control (QC) materials. A substantial positive bias was observed for both QC levels (+71.2%, +38.1%). Investigation revealed that this discrepancy does not occur if quantitation is conducted by ⁷⁸Se. On demand, the manufacturer clarified that a different pre-treatment process was used in production of the latest batch, resulting in increased bromine concentration in the materials. Bromine, in combination with hydrogen (present in the reaction gas), induces an interference (⁸¹Br¹H) on ⁸²Se, that explains the falsely elevated values. As consequence, routine measurements had to be switched back to ⁷⁸Se, with Gd being monitored accordingly.

This study highlights the ongoing challenges in ICP-MS analysis and emphasises the need for constant vigilance and validation to ensure accurate results. As with any analytical procedure, it is necessary to carefully monitor potential sources of error to ensure the integrity of clinical measurements.

High-sensitivity (hs) cardiac troponin assays play a crucial role for the triage of patients with suspected non-ST-segment elevation myocardial infarction (NSTEMI) at the emergency department. Nonetheless, only few studies address the clinical utility of recently developed point-of-care (POC) hs cardiac troponin assays. Here we aimed to compare the POC-hs-cTnI-TriageTrue assay (Quidel) with the well-validated Elecsys hs-cTnT assay (Roche). Special focus was given to the clinical use by comparing the hs-cTnT/I based triage algorithms. Interestingly, the two assays revealed a moderate agreement in terms of rapid rule-out and substantial agreement in rapid rule-in. We consider the discussion of such data indispensable for technological progress and optimization of cost-effectiveness in the diagnostical health-care system.

SPT is the first and the rate-limiting enzyme of the de novo sphingolipid (SL) biosynthesis. Mammalian SPT is dynamically composed of three subunits SPTLC1, SPTLC2 or SPTLC3 and catalyzes the formation of long chain bases (LCBs). Ubiquitously expressed SPTLC1-2 synthesize C₁₈ LCBs. SPTLC1-SPTLC3, however, produces a spectrum of short and long LCBs, notably a human specific omega-3-methyl sphingosine, meC₁₈SO. Epidemiological studies associate SPTLC3 genetic locus hepatic and coronary artery disease.

Non-alcoholic fatty liver disease (NAFLD) is characterized by an aberrant accumulation of lipids in the liver, leading to steatohepatitis (NASH), cirrhosis and ultimately hepatocellular carcinoma. An emerging therapeutic target for NAFLD is the bile acid activated, farnesoid X receptor (FXR), a transcription factor that regulates lipid and glucose metabolism as well as hepatic inflammation. We show that FXR in conjunction with retinoic acid receptor alpha (RXRa) regulates hepatic sphingolipid (SL) synthesis by controlling SPTLC3 expression. FXR binds to a cis-acting DNA motif proximal to the transcriptional start site of the gene. Furthermore, HFD induced upregulation of hepatic SPTLC3 levels. Natural and derived bile acid agonists of FXR lead to SPTLC3 transcriptional repression in vitro and in vivo. Activation of FXR by the synthetic BA agonist, obeticholic acid (OCA), suppressed SPTLC3 expression and the LCB levels in mice that correlates with attenuation of fat accumulation in liver. Interestingly, human hepatic FXR and SPTLC3 show strong correlations. NAFLD patients accumulate high plasma levels of SPTLC3 specific LCBs, especially the meC₁₈SO. Atypical LCBs are therefore biomarkers for disease progression in NAFLD and may play active function in NAFLD pathology.

Background: Following the current guidelines, immunoassays for the diagnosis of heparin-induced thrombocytopenia (HIT) are interpreted in a dichotomous manner, categorizing test results as either positive or negative. However, the extent to which test results hold diagnostic significance across the entire dynamic range remains unclear.

Methods: We utilized data from the prospective TORADI-HIT study, comprising 1393 consecutive patients with suspected HIT, to assess the diagnostic significance of three H/PF4 -immunoassay test results across their respective dynamic ranges (HemoSil Acustar HIT IgG [CLIA], Lifecodes PF4 IgG [ELISA], Diamed ID H-PF4 [PaGIA]). The diagnosis of HIT was determined by a washed-platelet heparin-induced platelet activation assay (HIPA). For each measurement point in the dataset, we computed likelihood ratios (LR), sensitivities, and specificities.

Results: The prevalence of HIT was 8.5% (n=119). A likelihood ratio of ≥ 10 was first achieved at 0.3 % of the dynamic range (0.4 U/ml; CLIA), 16% (0.64 OD; ELISA), and 1.6 % (1:4 titre; PaGIA), respectively. A likelihood ratio of ≥ 100 was present at 9.4 % (12 U/ml; CLIA), 75.0 % (3.0 OD; ELISA), 25.0 % (1:64 titre; PaGIA), respectively. The slope of the linear regression line (LR ~ dynamic range) was 9.5 (CLIA), 0.9 (ELISA), and 4.6 (PaGIA). To provide post-test probabilities for individual test results, we calculated interval-specific likelihood ratios and integrated it into a web-based calculator (https://pcd-research.shinyapps.io/BayesianCalculator/).

Conclusion: In conclusion, despite all immunoassays demonstrating an association between diagnostic significance and results, the strength of that association varies with the assay, with the CLIA having the largest increase per measurement unit.

Background: Iron deficiency that cannot be resolved by oral supplements often requires intravenous infusions. IV iron must be administered separately either directly by slow injection into the vein or more conveniently as an infusion appropriately diluted into 0.9% NaCl or 5% glucose. In specific situations, co-administration with parenteral nutrition or other IV fluids might be an attractive alternative when IV-line access is limited. However, evaluating stability can be challenging, and there is limited research on the compatibility of these formulations with potentially highly reactive free iron. Various nano-colloidal intravenous iron products are available and commonly used for the necessary iron loading. It is essential to ensure the stability of the nanoparticulate complex to avoid release of ionic iron, which adds oxidizing potential in the parenteral products and eventually to the increased inflammation state of patients. The arising toxic products from lipid peroxidation in parenteral nutrition, such as malondialdehyde, 4-hydroxy-2-hexenal, and 4-hydroxy-2-nonenal, mainly generated by the essential poly-unsaturated fatty acid (PUFA) oxidation, would impair substantially the quality of parenteral nutrition admixtures and represent a safety issue.

Aim: The aim of this project was to establish a method for the measurement of lipoperoxidation in All-in-one (AiO)  parenteral nutrition (PN) after the admixing of nano-iron drug products.

Methods: Quantitative analyses: UHPLC-UV with Luna C8(2) column in gradient mode (H2O:AcN 95% to 5%). Qualitative analysis: 3D-field ultraviolet-visible spectrum. Pure standards were used, and two derivatization agents were tested (2,4-dinitrophenylhydrazine (DNPH) and 3-methyl-2-benzothiazolinone-hydrazone). Smofkabiven^®-EF was used as PN standard.

Results: Two promising UHPLC-UV methods for quantifying the lipid peroxidation were developed. DNPH produced better peak symmetry and linearity. Both methods displayed high analytical sensitivity (<1mg/ml for each substance) and specificity (no spectral or chromatographic interferences observed).

Conclusion: Iron plays a vital role in the treatment of severely ill and often iron-deficient patients with PN. Particularly when supplemented in parenteral nutrition products, the proper integrity of all substances is crucial to achieve the highest benefit for the patient in terms of safety and efficacy. We here demonstrated a reliable method to quantify lipid peroxidation a potentially limiting factor of PN after therapeutic iron admixture of widely used nano-iron products. In the next step, we will investigate the impact of various commonly used iron products in different concentrations on the stability of lipids in AiO parenteral nutrition products.

Background: Misperceptions of diagnostic test performance may lead to incorrect medical decisions, heightened patient anxiety, increased healthcare expenses, and misguided public health decisions. Therefore, knowledge about the bias introduced in studies evaluating diagnostic tests is essential.

Aim: Utilizing all studies assessing the diagnostic performance of SARS-CoV-2 immunoassays, we assessed the direction and extent of bias caused by study design and patient characteristics.

Methods: Following a detailed protocol (PROSPERO CRD42023343656), we conducted a large-scale systematic review and meta-analysis (MEDLINE, EMBASE, and iSearch portfolio) including all studies assessing the diagnostic performance of SARS-CoV-2 immunoassays, thus addressing a common diagnostic question and biological target. A broad range of study design characteristics and 2 x 2 tables were retrieved. We performed a three-level meta-analysis to control for multiple tests within one primary study. The relative effect of suboptimal design characteristics on the reported diagnostic odds ratios (DOR) was assessed by a meta-regression controlling for the type of immunoassay, target epitope, target immunoglobulin, and time from start of symptoms.

Results: Overall, 18’092 articles were identified and 1’766 were assessed in full text. In the current analysis, we eventually included 641 primary studies comprising 3’124 study groups and 1’384’556 test results. Suboptimal design characteristics were associated with a two- to five-fold increase in DOR in most domains, e.g. case-control design (55% of studies; relative DOR [RDOR] 4.4; 95% confidence interval [CI] 3.1, 6.2), unclear patient selection (65%; RDOR 3.7, 95% CI 2.5, 5.5), industry funding (4 %; RDOR 4.6; 2.3, 9.2), unclear blinding of index test interpretation (70%; RDOR 2.8; 1.9, 4.1), and different reference standard for controls (73%; RDOR 3.6; 2.7, 4.7). Few characteristics were associated with a decrease in DOR, e.g. inappropriate early timing of index test (RDOR 0.2; 0.1, 0.3), and symptoms as a reference test for COVID-19 positive patients (RDOR 0.1; 0.0, 0.6).

Conclusions: Widely used study designs resulted in a substantial overestimation of SARS-CoV-2 immunoassay test performance. Our data suggest that overestimation is a common problem and carefully designed studies are needed to establish unbiased performance measures.