Advanced technologies , including new biomarker discovery

M001

Advanced technologies , including new biomarker discovery

COMPARISON OF REFERENCE VALUES FOR SMALL EXTRACELLULAR PARTICLES IN A HEALTHY STUDY COHORT USINGNANOPARTICLE TRACKING ANALYSIS (NTA) BEFORE AND AFTER PARTICLE ISOLATION BY DIFFERENT ISOLATIONMETHODS

I. Erina 1, B. Betz 11Department of Clinical Chemistry and Laboratory Diagnostics, Jena University Hospital, 07747 Jena

BACKGROUND-AIM

The quantification of small extracellular particles (45-1000nm) in plasma is a challenging task due to the limitedresolution of many technical devices and due to the high background noise in plasma. In this study, we calculate andcompare reference values for small extracellular particles in isolated and non-isolated plasma samples from a healthystudy cohort using Nanoparticle Tracking Analysis (NTA).

METHODS

A polymer-based precipitation kit (ExoQuick, System Biosciences) and a size-exclusion chromatography (SEC) kit(qEVoriginal, Izon) were used to isolate frozen anonymized non-fasting plasma samples from healthy volunteers(n=73). ZetaView device (Particle Metrix) was used for NTA quantification of small extracellular particles.

RESULTS

The median concentration of small extracellular particles in non-isolated plasma samples was 4.19E+11/µl (2.5-97.5%Interval: 8.12E+10 - 2.02E+12/µl) with a median ratio of smaller particles (45-165nm) to larger particles (165-1000nm)of 1.7 (2.5-97.5% Interval: 0.57 - 7.50). The concentration of small extracellular particles isolated with the precipitationkit or the SEC kit were 1.10E+11/µl (2.5-97.5% Interval: 1.63E+10 - 8.86E+11/µl) and 1.04E+09/µl (2.5-97.5% Interval:3.14E+08 - 7.05E+09/µl) respectively with a smaller/larger particle ratio of 0.90 (2.5-97.5% Interval: 0.16 – 9.02) and0.93 (2.5-97.5% Interval: 0.33 - 2.83).There was no correlation between the small extracellular particle concentrations of non-isolated plasma samples andthe corresponding isolates. There was a weak positive correlation (r=0.27, p<0.05) between particle concentrations ofSEC and polymer-based isolates. In addition, the particle concentrations of SEC isolates weakly correlated with theplasma concentrations of high-sensitive C-reactive protein (r=0.28, p<0.05) and total protein (r=0.33, p<0.05).

CONCLUSIONS

The combination of an isolation method (SEC or precipitation) and NTA represents a time-effective way for thequantification of small extracellular particles in human plasma samples. The established reference values weredifferent dependent on the method of isolation and will be helpful for interpretation of future studies.

Poster Abstracts – EuroMedLab Munich 2021 – Munich, Nov 28-Dec 02, 2021 • DOI 10.1515/cclm-2021-5003 Clin Chem Lab Med 2021; 59, Special Suppl, pp S94 – S835, Nov/Dec 2021 • Copyright © by Walter de Gruyter • Berlin • Boston S94

M002


FOLLOW-UP OF NOVEL KL-6 AND ROUTINE BIOMARKERS IN COVID-19 SURVIVORS 1 YEAR POST-ICU

N. De Vos 1, C. Duterme 1, Z. Ouanani 1, D. Dirickx 1, M. Bruyneel 4, A. Roman 2, S. Alard 5, F. Ponthieux 1, M. Lauwers 1, E.

Mathieu 1, C. Goudji 1, S. André 3, D. Barglazan 1, C. Dusart 4, L. Truffaut 4, F. Cotton 11Department of Clinical Chemistry, Laboratoire Hospitalier Universitaire Bruxelles – Universitair Laboratorium Brussel(LHUB-ULB), Université Libre de Bruxelles, Brussels, Belgium2Department of Intensive Care Medicine, CHU Saint-Pierre, Brussels, Belgium and Université Libre de Bruxelles, Brussels,Belgium3Department of Pneumology, CHU Brugmann, Brussels, Belgium and Université Libre de Bruxelles, Brussels, Belgium4Department of Pneumology, CHU Saint-Pierre, Brussels, Belgium and Université Libre de Bruxelles, Brussels, Belgium5Department of Radiology, CHU Saint-Pierre, Brussels, Belgium and Université Libre de Bruxelles, Brussels, Belgium

BACKGROUND-AIM

After SARS-CoV-2 infection, a local cytopathic effect on alveolar epithelial cells upregulates Krebs von den Lungen 6(KL-6) expression. The aim of this study is to follow-up KL-6 and routine biomarkers in critical COVID-19 survivors upto 1 year post intensive care unit (ICU).

METHODS

Samples were prospectively drawn from 22 critical COVID-19 patients during their pulmonology follow-up (FU) at 3, 6and 12 months post-ICU. 10 patients lacking one of the 3 measuring points were excluded. KL-6 and procalcitonin (PCT)were analyzed on Lumipulse® G1200 (Fujirebio, Japan). C-reactive protein (CRP), ferritin, cardiac troponin T (cTnT)and lactate dehydrogenase (LDH) were measured on Cobas 8000 (Roche, Germany). Data are expressed as [median(IQR)]. Non-parametric tests were performed using GraphPad Prism 5.0 and p-values <0.05 were considered statisticallysignificant. KL-6 cut-off to distinguish between critical COVID-19 patients and healthy subjects was obtained withreceiver operating curve (Analyse-it Excel, USA).

RESULTS

Patient demographic characteristics were: 73% men, mean age of 55 years, 50% obese, 45% HTA, 27% diabetes, 100%ARDS, a mean length of stay of 21d at ICU, 64% with endotracheal intubation and 9% ECMO during the acute SARS-CoV-2 infection. At ICU, the patients showed significantly higher biomarkers KL-6 [665 U/mL (441-900)], PCT [0.30 µg/L (0.06-1.20)], CRP [97 mg/L (32-132)], ferritin [539 µg/L (133-1504)], cTnT [20.15 ng/L (10.93-34.38)] and LDH [503U/L (404-747)] compared to healthy controls (p<0.05). KL-6 and CRP marked the severity, as they were higher in acutecritical COVID-19 patients compared to asymptomatic/mild COVID-19 cases (p<0.05). Three months after ICU PCT, CRP,ferritin, cTnT and LDH turned normal in ≥90 % of patients. After 1 year, KL-6 normalized (<275 U/mL) in only 25% ofcritical COVID-19 patients. Between 3,6 and 12 months of FU, the mean KL-6 value did not significantly change (p=0.63).

CONCLUSIONS

After 1 year of post-COVID-19 follow-up, the biomarker KL-6 persisted in 75% of critical COVID-19 survivors, whilePCT, CRP, ferritin, cTnT and LDH already normalized after 3 months. Whether pulmonary sequelae are related to thepersistence of KL-6 will further be assessed with FU of pulmonary function tests and chest CT-scan.


M003


ARTIFICIAL INTELLIGENCE TECHNIQUES IN ALZHEIMER DETECTION

G. Alfonso Perez 1, J. Caballero Villarraso 11Universidad de Cordoba, Spain

BACKGROUND-AIM

DNA methylation is an epigenetic process in which a methyl group is added to the DNA in the C5 position. Methylationis involved in many biological process ranging from aging to several diseases such as cancer. The level of methylationchanges with age. Using this principle researchers have been able to build rather accurate biological clocks using DNACpGs methylation in blood as well as in tissues. The application of DNA CpGs methylation for medical purposes is anactive field of research as technology has enabled for the quick and importantly, relatively inexpensive, analysis ofDNA methylation in thousands of CpGs per sample.Currently there are public databases, such as for instance GEO database, containing a large amount of methylationdata. This data is free and easy to access. The substantial increases in data availability has been possible due to thecollaborative spirit of researchers as well as technological developments such as the Illumina methylation machines.The first generation of these machines were able to analyze approximately 27,000 CpGs. The second generation, whichis likely the most frequently used, can analyze approximately 450,000 while the last generation can analyze in excessof 800,000. Clearly, the amount of data per patient is substantially increasing. This represents both an opportunityand a challenge as appropriate techniques to analyze this ever increasing amount of data are required. Analyzingthis information can be a complex task. From a statistical point of view there are some clear challenges such as thedimensionality of the data. The proportion between the amount of data per patient (for instance 450,000 using secondgeneration machines) and the number of patients which order of magnitude varies but it is typically between 10 tono more than 10,000 individuals is not ideal for statistical analysis with the number of potential factors larger thanthe number of patients. Ideally, the number of patients and factors analyzed should be at least of the same order ofmagnitude.The large amount of data available suggest that an artificial intelligence modeling approach might be suitable. Thereare several of those techniques such as neural networks, k-nearest neighbors or support vector machines. Thesetechniques are commonly used in supervised learning in which the data is divided into two blocks i.e., the training andthe testing block. The algorithm is trained using the training data and then tested using the testing data.AlzheimerAlzheimer disease is a neurological disease with high prevalence. It is a major cause of mortality and disability inmany countries. The disease affects mostly population of advanced age (65 an above) but there are also cases ofearly onset Alzheimer disease. While the causes of the disease are not well understood yet there is clearly a geneticcomponent. Therefore, it seemed reasonable to use DNA methylation data and artificial intelligence techniques tocreate an algorithm in order to be able to differentiate between healthy individuals and patients suffering fromAlzheimer disease.

METHODS

DNA methylation data was obtained from the GEO database with a total of 190 cases including control and individualssuffering from Alzheimer disease. Approximately 450,000 CpGs methylation data were obtained from each patient. Asa first step a linear regression was run for each of the CpGs. The CpGs that were not found to be statistically significantwere excluded from the analysis. In this way, 41,784 CpGs (with a –value of less than 5%) were selected. In a secondstep a k-nearest neighbor approach was selected to differentiate between control cases and patients with Alzheimerdisease. One of the main considerations to take into account using this type of approach is selecting the number ofneighbors (k) to be used. The algorithm was trained iteratively changing the value of k from 1 to 50. After trainingthe accuracy of the models was tested with the testing data. The age of the patient was also used to account for thenormal changes in methylation due to aging.

RESULTS

The misclassification error obtained when controlling for the age of the patient was 8.4%. There was a considerableimprovement when controlling for the age of the patient. When the age of the patient is not included in the analysisthe misclassification rate reaches 16.8%. It was also observed that increasing the number of neighbors (k) did nottranslate into higher accuracy. In fact, the best results were obtained when using k=1.

CONCLUSIONS

In this article we have shown that is possible to use artificial intelligence techniques, such as k-nearest neighbors,to distinguish between control individuals and patients suffering from Alzheimer disease, using as an input DNAmethylation data. It was also shown that using this algorithm it is important to control for the age of the patient withthe accuracy of the prediction substantially increasing when age is included in the analysis.


M004


RATIONALE AND DESIGN OF A STUDY EVALUATING THE PERFORMANCES OF CARDIORENAL POINT OF CARE DEVICE TOMEASURE BLOOD POTASSIUM.

D. Gruson 2, M. Jadoul 2, M. Deltombe 2, A. Nevraumont 2, M. Berenger 1, D. Lefebvre 1, G. Henrion 1, J. Papillon 11Cardiorenal, France2Cliniques Universitaires Saint Luc

BACKGROUND-AIM

Chronic kidney disease (CKD) is a major public health problem, affecting around 10% of the global population. CKD isthe main cause of hyperkalemia, that may cause life-threatening cardiac arrythmias or sudden death. Hyperkalemiais also the main reason of discontinuation or down-titration of life-saving drugs (renin angiotensin aldosteronesystem inhibitors). Monitoring and maintenance of potassium levels within the normal range is thus crucial to avoidcardiovascular complications, as both hypokalemia (also frequent in CKD) and hyperkalemia were repeatedly foundassociated with all-cause mortality in CKD and other populations at high cardiovascular risk (e.g. in heart failure).Despite this alarming risk, serum potassium levels are currently poorly monitored. The potassium monitoring is alwaysperformed at hospitals or in clinical laboratories with invasive venous punctures. In a disruptive manner and to facesome unmet clinical needs, a fast and user-friendly in-vitro diagnostic device to monitor potassium level at the pointof care of the patient and/or by the patient him/herself is under development by CardioRenal SAS

METHODS

Our main objective is to evaluate the feasibility of potassium measurements with the CardioRenal TENOR prototypein capillary blood. ALPHA (NCT04907773) is an ongoing open, prospective, single-arm monocentric trial in a tertiaryreferral centre. The agreement of capillary potassium concentrations between the CardioRenal TENOR prototype andthe standard of care venous potassium concentrations (Roche Cobas 8000) will be assessed.

RESULTS

Study population consists in healthy volunteers (to test the normal potassium range) and in hemodialyzed patients toextend the range to abnormal concentrations (by pre and post dialysis measurements).Blood collection involves simultaneous sampling of venous blood and capillary blood. The used capillary samplingmethod limits hemolysis therefore preventing pseudo-hyperkalemia. A total of 70 subjects will be enrolled to generate100 paired capillary/venous blood samples to compare the candidate device to comparative methods (gold standard orpredicate). The sample size has been chosen from the sample size recommended in performance evaluation guidelines(CLSI, Westagard). This will allow to give a good estimate of the bias between the two measurement procedures andthe estimates for bias, at any concentration. Passing-Bablock regression and Bland Altman analysis will be used tocompare the methods and present the results.

CONCLUSIONS

The innovative capillary potassium measurement with CardioRenal TENOR device is expected to allow thedetermination of potassium value within acceptable analytical performance defined by CLIA requirement or Europeanrequirement. Such a positive outcome will be a first and huge step toward homecare for CKD and heart disease patients.


M005


HIGH PLASMA SARCOSINE LEVELS IN HOSPITALIZED PATIENTS: ANALYSIS OF A RETROSPECTIVE COHORT.

T. Ramon 3, M. Joncquel 3, M. Bout 3, M. Defevre 3, I. Kim 3, A. Kerckhove 3, L. George 3, A. Dessein 3, M. Gilleron 3, D.

Dobbelaere 1, C. Douillard 2, G. Grzych 3, P. Pigny 31CHU Lille, Centre de Référence Maladies Héréditaires du Métabolisme de l'Enfant et de l'Adulte, Jeanne de Flandre Hospital,RADEME EA 7364, F-59000 Lille, France2CHU Lille, Service d'Endocrinologie et Métabolismes, Hôpital Claude Huriez, Centre de Référence des Maladies Héréditairesdu métabolisme, F-59000 Lille, France3CHU Lille, Service d’Hormonologie, Métabolisme, Nutrition, Oncologie, F-59000 Lille, France

BACKGROUND-AIM

In literature, high plasma sarcosine levels are found in a specific inherited metabolic disease, the SARDH deficiency,but only 30 cases have been described/reported in the last 30 years. Recent papers suggest that plasma sarcosinecould also be a marker of prostate cancer. Accordingly, we sometimes found elevated plasma sarcosine levels in ourroutine analysis without SARDH deficiency. Hence, we aim to determine the physiological and pathological contextsin which plasma sarcosine is increased.

METHODS

We retrospectively extract laboratory data from patients visiting the Lille University Hospital from 2019 to 2020 whohad a disturbed aminoacidogram. Amino acids, including sarcosine, were measured by liquid chromatography coupledto mass spectrometry using the aTRAQ™ method.

RESULTS

We identified 49 patients with a plasma sarcosine concentration > 7µmol/L. Our results showed that the patients withthe highest levels of sarcosine presents hyperhomocystinuria related to cystathionine beta synthase (CBS) deficiency.A significant number patients with CBS deficiency are treated with betaine (a precursor of sarcosine). Increased levelsof sarcosine are also found in patients with liver or kidney failure. Moreover, patients with hematological diseases alsoshow high levels of sarcosine, some of these patients were treated with methotrexate. Finally, some patients with highsarcosine were chronic nitrous oxide users.

CONCLUSIONS

Sarcosine elevations have been found in numerous contexts in addition to prostate cancer. The causes of the elevationof its increase are still poorly understood and require further investigation. We suggest that alterations of one carbonmetabolism, and particularly homocysteine remethylation and folate metabolism, might be involved. In conclusion,we show here that sarcosine is not a specific biomarker of SARDH and that results should be interpreted with caution.


M006


TARGETED METABOLOMIC ANALYSIS SUGGESTS THAT TACROLIMUS ALTERS PIPECOLIC ACID AND SARCOSINEMETABOLISMS.

J. Labasque 4, M. Bout 4, A. Hamroun 2, B. Hennart 3, J. Demaret 1, M. Defevre 4, I. Kim 4, A. Kerckhove 4, L. George 4, A.

Dessein 4, M. Gilleron 4, M. Joncquel 4, G. Grzych 41CHU Lille, Institut d’Immunologie, F-59000 Lille, France2CHU Lille, Service de Néphrologie, Dialyse, Transplantations rénales, F-59000 Lille, France3CHU Lille, Service de Toxicologie et Génopathies, Métabolisme, Nutrition, Oncologie, F-59000 Lille, France4CHU Lille, Service d’Hormonologie, Métabolisme, Nutrition, Oncologie, F-59000 Lille, France

BACKGROUND-AIM

Tacrolimus (FK506), is an immunosuppressor that inhibit calcineurin. One of this described side effects isnephrotoxicity. Pipecolic acid (PA) is a metabolite derived from lysine whose concentration may increase in the eventof hepatic dysfunction but also in metabolic diseases such as peroxisomal damage or antiquitin deficiency. SignificantPA signals were routinely detected in patients treated with FK506. It is important to note that the structure of pipecolicacid is found in that of the molecule of FK506. Hence, an interferent signal could not be excluded. Aims of this studyare both to verify this possible analytical interference and to evaluate the possible link between the use of FK506 andthe increase in plasma PA concentration.

METHODS

Analysis of 30 amino acids (including PA) was performed in selected plasma samples.To confirm the link between FK506 intake and the increase of plasma PA concentration, amino acids measurementswere performed in plasma from control patients without FK506, plasma from patients under FK506 (in vivo FK506)and plasma from control patients with FK506 added in vitro. To study the link between the metabolic impact of FK506on PA and calcineurin inhibition, another anti-calcineurin immunosuppressive therapy, ciclosporin (CSA), was alsoinvestigated.

RESULTS

Increased plasma concentration of PA was observed in patients under FK506 compared to control samples (5.29+/-5.09nmol/ml Vs 1.38+/-0.46 nmol/ml, p = 0.005). In vitro additions of FK506 to control samples do not increase PA. Patientsunder CSA do not show increase in plasma PA compared to control samples (1.97+/-0.86 nmol/ml Vs 1.38+/-0.46 nmol/ml, p = 0.18), which does not support a metabolic link between the calcineurin and PA. However, in vivo FK506 wasassociated with higher plasma sarcosine (another AA derived) concentrations and a decrease in the glycine/sarcosineratio, as well as a tend to increased plasma lysine.

CONCLUSIONS

These results do not suggest any interference between FK506 and amino acids assay, nor a metabolic impact ofcalcineurin inhibition on PA. However, the plasma amino acid changes observed in patients under FK506 (increased PAand lysine and decreased glycine/sarcosine ratio) highlight a possible link between FK506 and the action of an enzymeinvolved in both PA and sarcosine catabolism, the Peroxisomal sarcosine oxidase (PIPOX). This hypothesis needs tobe confirmed by further mechanistic studies. Moreover, PA could be investigated as potential a biomarker of earlynephrotoxicity in the follow-up of patients under FK506.


M007


SERUM METABOLOME ANALYSIS OF IRON DEFICIENCY ANEMIA PATIENTS USING NUCLEAR MAGNETIC RESONANCE(QUANTITATIVE APPROACH)

A.Z. Gul 1, S. Selek 1, A.C. Goren 1, M. Demirel 1, F. Koktasoglu 1, H. Agac 1, U. Sarıkaya 11Bezmialem Foundation University

BACKGROUND-AIM

Iron deficiency anemia (IDA) is seen of high prevalence as a cause of anemia and imposes serious economical-socialburden upon societies negatively affecting child development and work productivity. Even iron replacement therapyis commonly available and beneficial, overload of exceeding amounts may unbalance oxidative processes in cell levelleading to metabolic disturbances. Setting off to gain more insights on iron metabolism and expand IDA metabolomics,we chose 40 patients and healthy controls to evaluate serum metabolites using quantitative NMR technique (targetedapproach).

METHODS

The analysis was done with 500 MHz 1D 1H NMR spectrometer (Bruker) and NOESY sequence experiment was employed.

RESULTS

According to results, patient serums significantly differed from controls regarding several metabolite levels in bothiron-associated and distinct pathways. Significant metabolites in patients included increased serum levels of N-acetylglucosamine (24 µg (c: 15); p=0.015), gluconate (37 µg (c: 27); p=0.029), arginine (41 µg (c: 30); p=0.020), glycine(45 µg (c: 34); p=0.042), allantoin (40 µg (c: 28); p=0.018) and decreased levels of 5-aminolevulinic acid (21 µg (c: 32);p=0.012), taurine (17 µg (c: 25);p =0.041), choline (24 µg (c: 33); p=0.026), phosphocholine (27 µg (c: 36 µg); p=0.042),erythritol (20 µg (c: 30); p=0.012), pantothenic acid (33 µg (c: 44); p=0.029), pyroglutamate (29 µg(c: 38); p=0.051). Eachof these metabolites represents a specific pathway and in total look, provides insights on how the metabolic status ofiron deficient individuals is affected and in which steps.

CONCLUSIONS

We believe that metabolomic profiling of IDA patients may contribute to elicit disease related symptom mechanismsand holds promise to track patients’ metabolic status to reach more rational decisions on iron replacement therapy.With current study, the quantitative serum NMR analysis in IDA patients defined novel metabolic cross talks betweenintermediates and revealed differences compared to healthy subjects. The preliminary work also posed the need offurther studies to solve emerging inquires and to validate current results.


M008


QUANTIFICATION OF CANCER-PREVENTIVE FALCARINOL FROM CARROTS IN HUMAN SERUM BY LC-MS

U. Jakobsen 1, M. Kobæk-Larsen 2, K. Domela Kjøller 2, G. Baatrup 2, M. Beck Trelle 11Department of Biochemistry, Odense University Hospital, Svendborg Hospital, Svendborg2Department of Clinical Research and Department of Surgery, Odense University Hospital, Svendborg Hospital, Svendborg

BACKGROUND-AIM

Falcarinol (FaOH) is found in carrots and other plants of the Apiaceae family, where the compound can act as a fungicide.It is also known to have biological activity in cells and mammals, and show a cancer-preventive effect in a rat modelfor colorectal cancer. To study the effect of FaOH on human health, it is advantageous to know the bioavailabilityof the compound. The analysis of FaOH in human plasma by liquid chromatography mass spectrometry (LC-MS) waspreviously described in the literature, but without sufficient detail to be readily reproduced. We therefore developed anovel method for analysis of FaOH in serum by LC-MS and used it to evaluate the bioavailability of falcarinol in humans.

METHODS

18 volunteers injested a slurry of freeze dried carrot powder in water after having their blood drawn (sample at t = 0min.). Thereafter, blood was sampled at intervals of 1-2 hours for 12 hours.Serum samples were precipitated with acetonitrile, and the supernatant was analyzed by reversed-phase liquidchromatography coupled to a triple-quadrupole mass spectrometer using electrospray ionization. Falcarinol wasquantified using single reaction monitoring of the mass transition m/z 268 → m/z 182 in the positive ion mode.Separation was achieved with a C18 column and a gradient solvent system with formic acid in acetonitrile and water.As calibrations standards and controls are not commercially available, these were produced by spiking of serum poolswith a falcarinol solution.

RESULTS

The method was found to be linear in the examined range of 0.2 to 20 ng/mL. The limit of detection was determinedto be 0.2 ng/mL and the inter- and intraday variation to be 11% and 4%, respectively.Analysis of the serum samples showed rapid increase in falcarinol levels at the earliest samplings with peak values inserum one hour after intake and declining below detection levels after 5-10 hours. The maximum values of falcarinolobtained varied in the range 0.7-4 ng/mL.

CONCLUSIONS

A new method for detection and quantification of falcarinol in human serum was developed.Falcarinol showed a rapid uptake from the digestive tract resulting in peak values measured in serum approximately 1hour after bulk intake of carrot powder in water. Subsequent clearance of falcarinol from serum was also fast.


M009


PRE-ANALYTICAL IMPACTS ON NEUROLOGICAL BLOOD-BASED BIOMARKERS

A. Ladang 1, L. Rigaud 1, G. Sqalli 1, E. Cavalier 11Clinical chemistry department CHU de Liège

BACKGROUND-AIM

New technologies allowing the measurement of neurological biomarkers in the blood are emerging. Pre-analyticalconditions are known to have an impact on the dosage of amyloid-β (Aβ) and total Tau (t-Tau) when measured incerebrospinal fluid (CSF). Therefore, pre-analytical studies on blood-based measurement of Aβ and t-Tau are required.In this study, we are assessing the impact of homogenization, delayed centrifugation and tube handling on plasmacollected with three different EDTA tubes for Aβ1-40, Aβ1-42, ratio Aβ1-42/ Aβ1-40 and t-Tau.

METHODS

Aβ1-42, Aβ1-40 and t-Tau were measured on plasma with the SiMoA° technology following various pre-analyticaltreatment. Relative and absolute concentrations as well as Aβ1-42/ Aβ1-40 ratio were calculated and an analyticalcoefficient of variation was defined.

RESULTS

The intra-run coefficient of variation were 7.1 %, 5.3 % and 7.4 % for Aβ1-42, Aβ1-40 and t-Tau, respectively.Unexpectedly, we found an increased Aβ concentration in polyethylene (PET) collection tubes when samples wereprocessed right away. Yet, these higher levels in PET collection tubes rapidly disappeared after 1 hour at roomtemperature. The observed effects were corrected by the use of Aβ1-42/ Aβ1-40 ratio. When polypropylene (PP)collection tubes were used, the homogenization, delayed centrifugation or tube handling had no impact on Aβ and t-Tau concentrations.

CONCLUSIONS

This study highlights the importance of a standardized pre-analytical procedure. The use of an EDTA polypropylenetube seems the most appropriate for Aβ blood dosage. In case the use of PP collection tubes cannot be ensured, Aβ1-42/Aβ1-40 ratio should be favored.


M010


STABILITY OF MORE THAN 400 LIPIDS IN WHOLE BLOOD AFTER SAMPLE COLLECTION

Q. Wang 1, C. Hu 1, M. Hoene 2, L. Fritsche 2, X. Liu 1, X. Zhao 1, A. Peter 2, G. Xu 1, R. Lehmann 21CAS Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academyof Sciences (CAS), Dalian 116023, China2Institute for Clinical Chemistry and Pathobiochemistry, Department for Diagnostic Laboratory Medicine, UniversityHospital Tübingen, 72076 Tübingen, Germany

BACKGROUND-AIM

Lipidomics investigations of plasma and serum are increasingly applied to profile and detect novel diagnosticbiomarkers. While the analytical part is usually highly standardized, the pre-analytical part may be not. But the timespan from blood drawing until centrifugation and sample preparation, particularly in the clinical surrounding, may belong and could be highly variable. Therefore, valid pre-analytical recommendations and, with respect to distinct lipidsas potential diagnostic biomarkers, knowledge about the stability of lipid species in whole blood are highly relevant.We aimed in our study to elucidate stable and instable lipids in whole blood in a profile of more than 400 lipid species.

METHODS

We applied UHPLC-MS to perform lipidomics of EDTA plasma originating from whole blood of > 40 individuals eitherprepared at once after blood drawing, or after exposure at 4°C or 21°C (room temperature) or 30°C (summer timeconditions) for various time spans between 30 min. and 24h. Significant alteration of a lipid was defined as changes>10% in the lipid level (p<0.05, FDR < 0.05).

RESULTS

Cooling whole blood directly after sample collection allows a transportation of up to 4h with less than 0.5% significantalterations of all lipids in our profile covering more than 400 lipids. Even after 24h at 4°C in whole blood only 6% of alllipids show significant changes. In contrast, summer time conditions, i.e. sample handling or transportation at 30°C,lead to significant changes in >6% of lipid species within 60 min. After 4h at 30° 64 lipids had significantly changed.At the same time span at room temperature 44 lipids were significantly altered. Lipid classes with the lowest stabilityunder all studied conditions are lysophosphatidylcholines, (O-acyl)-hydroxy fatty acids and free fatty acids.

CONCLUSIONS

Studying a lipid profile of more than 400 lipid species out of 15 lipid classes we revealed a time span of 4h at 4°C inwhole blood cooled at once after collection suitable for sample handling and transportation to achieve still valid andreproducible lipid pattern (the quicker the better). Several lipid classes are very robust, i.e. stable up to 24h at roomtemperature or even at 30°C. However, the stability of lipids during sample handling and transportation of whole bloodshow a high variability between and within distinct lipid classes and therefore careful consideration of the preanalyticalphase in diagnostic biomarker research is recommended.


M011


MULTI-SITE VERIFICATION OF THE AUTOMATED EXENT® MALDI-TOF-MS SYSTEM AND IMMUNOGLOBULIN ISOTYPESASSAY FOR THE IDENTIFICATION AND QUANTIFICATION OF MONOCLONAL IMMUNOGLOBULINS

N. Marrot 1, S.J. North 1, D. Mcentee 1, R. Stanton 1, D. Matters 1, D. Sakrikar 2, L. Ouverson 2, O. Berlanga 1, H. Montgomery1, B. David 2, G. Wallis 1, M. Perkins 1, S. Harding 1, J. Ashby 11The Binding Site, Birmingham, UK2The Binding Site, Rochester, MN, USA

BACKGROUND-AIM

MALDI-TOF-MS methods for the identification and quantification of monoclonal immunoglobulins have been thesubject of recent IMWG guidelines. We report on the first multi-site verification of this technology using theEXENT system. Standardisation of monoclonal peak quantification was performed using immunoglobulin assay datacalibrated to DA470k.

METHODS

EXENT (The Binding Site Group Ltd., UK) is the commercial name for the technology previously described as QIP-MS.Modified polyclonal antibodies (anti-IgG, -IgA, -IgM, -total-κ and -total-λ) were covalently attached to paramagneticmicroparticles. Patient samples were incubated with the microparticles, washed, eluted (20mM TCEP in 5%(v/v) aceticacid) and spotted onto MALDI plates with HCCA matrix. Spectra were generated using the MALDI-TOF-MS system.Quantitative IgG, IgA and IgM values were obtained using the Optilite® analyser.Light chain spectra were obtained using EXENT software to yield immunoglobulin isotype, mass-to-charge ratio (m/z) and quantity (g/L).Analytical performance was based upon CLSI guidelines: LLMI (EP17-A2), reference interval verification (EP28-A3c),precision (EP05-A2), linearity (EP06-A), interference (EP07-A2), and stability (EP25-A).Qualitative comparisons were carried out using 60 IFE-positive clinical samples. For quantitative comparisons, Passing-Bablok regression analyses were performed on 49 SPEP-positive samples and separately for those >10g/L and <10g/L.

RESULTS

LLMI was 0.015g/L for all specificities. Published Hevylite reference intervals were verified. 20-day total precisionfor polyclonal samples (<7%) and for two monoclonal samples (~1g/L and 30g/L; <15% and <10%, respectively) wasacceptable, with between laboratory precision <8% for all samples.The assay was linear for IgG, IgA and IgM across a 0.015 to 100g/L range using a 10% non-linearity acceptance limit.No significant assay interference was observed. On-board stability of reagents was 5-24h, and open-vial stability was2.6 months.The assay showed 100% concordance with IFE for the monoclonal type. Quantitative agreement with SPEP wasacceptable (y=0.91x-1.14). However, there was a substantial discordance <10g/L (y=0.75x-0.87) compared to >10g/L(y=0.95x-2.59), suggesting over-estimation by SPEP at lower levels.

CONCLUSIONS

EXENT demonstrated acceptable performance for precision, interference, and agreement with existing assays. The roleof the EXENT software in the identification and integration of peaks will ensure reliable and reproducible results acrossdifferent laboratories, an important step in standardising this methodology.


M012


UTILITY OF IMMUNOGLOBULINS G1 AND G2 SUBCLASS-SPECIFIC GLYCOSYLATION IN DIAGNOSIS AND MONITORINGTREATMENT RESPONSE OF EPITHELIAL OVARIAN CANCER PATIENTS

F. Mugeni-Wanyama 2, A. Mokomba 1, C. Nyongesa 3, V. Blanchard 21Department of Obstetrics and Gynecology , Kenyatta National Hospital, Ngong road, P.O Box 20723 - 00202, Nairobi, Kenya2Institut für Laboratoriumsmedizin, Klinische Chemie und Pathobiochemie, Charité – Universitätsmedizin Berlin, corporatemember of Freie Universität Berlin, Humboldt-Universität zu Berlin, Berlin Institute of Health: Augustenburger Platz 1,D-13353 Berlin3Texas Cancer Center, Keri road off Mbagathi way, P.O Box 13 – 00202, Nairobi, Kenya

BACKGROUND-AIM

Ovarian cancer (OC) is a common cause of cancer death among women. The mortalities are largely associated with a lackof sensitive and specific early diagnostic biomarkers to allow timely intervention. Currently, CA125 is the biomarkerroutinely used for OC detection, but it is limited due to low sensitivity and specificity as it also appears to be elevatedin benign gynecological conditions. Hence, the need to identify and validate new less invasive biomarkers that addressthe question of sensitivity and specificity to complement CA125 is urgent.Protein glycosylation is an important source of biomarkers. Previously, immunoglobulin glycopeptide (IgG) showedsignificant alterations in various malignancies including OC. We profiled changes of IgG1 and IgG2 subclass-specificglycosylations of a cohort of epithelial OC (EOC) patients of African ethnicity by comparing them with benign ovariandisease (BOD) subjects. The aim was to identify individual glycopeptides and glycosylation traits with the potential todetect EOC and monitor the treatment response of patients.

METHODS

We recruited a Kenyan cohort comprising of 52 adult EOC patients, 19 primary and 33 on various chemotherapycycles, and 46 adult women with BOD (control group). Blood samples were collected from participants in Kenya andsubsequently shipped to Charité-Universitätsmedizin Berlin. The analysis of IgG1 and IgG2 tryptic glycopeptides wasperformed by MALDI-TOF mass spectrometry and statistical analysis using SPSS.

RESULTS

Five and six glycoforms of IgG1 and IgG2, respectively, were identified as the best discriminators of EOC from BOD(p<0.001). IgG1 had 2 agalactosylated (m/z 2632.04; 2689.06) and 3 galactosylated glycoforms (m/z 2810.08; 2956.14;3159.22). While IgG2 had 3 agalactosylated (m/z 2453.98; 2600.04; 2657.06) and 3 galactosylated structures (m/z 2762.09; 2924.15; 2965.17). The glycosylation traits of IgG1 and IgG2 that were able to discriminate EOC fromBOD were agalactosylation, galactosylation and sialic acid. Improved performance was achieved by combining theglycoforms of IgG1 and IgG2 when compared with CA125 alone.

CONCLUSIONS

Specific IgG1 and IgG2 glycoforms and glycosylation traits (agalactosylation, galactosylation and sialylation) have thepotential to discriminate EOC from BOD.


M013


HYBRID IGG4 K/λ, NEW BIOMARKERS IN MYASTHENIA GRAVIS

C. Napodano 6, U. Basile 4, F. Gulli 5, K. Pocino 7, R. Di Santo 3, V. Basile 1, L. Todi 2, C. Provenzano 2, G. Ciasca 3, M. Marino 21Clinical Pathology Unit and Cancer Biobank, Department of Research, Advanced Diagnostics and Techno-logical Innovation,IFO-Regina Elena National Cancer Institute, 00128 Rome, Italy2Dipartimento di Medicina e Chirurgia Traslazionale, Sezione di Patologia Generale, Fondazione Policlinico Universitario “A.Gemelli” IRCCS, Università Cattolica del Sacro Cuore, 00168 Rome, Italy;3Dipartimento di Neuroscienze, Sezione di Fisica, Fondazione Policlinico “A. Gemelli” IRCCS, Università Cattolica del SacroCuore, 00168 Rome, Italy4Dipartimento di Scienze di Laboratorio e Infettivologiche, Fondazione Policlinico Universitario “A. Gemelli” IRCCS,Università Cattolica del Sacro Cuore, 00168 Rome, Italy5Laboratorio di Patologia Clinica, Ospedale Madre Giuseppina Vannini, 00177 Rome, Italy6Synlab Data Medica, 35133 Padova, Italy7Unità Operativa Complessa Patologia Clinica, Ospedale Generale di Zona, San Pietro Fatebenefratelli, 00189 Rome, Italy

BACKGROUND-AIM

Myasthenia gravis with antibodies (Abs) against the muscle-specific tyrosine kinase (MuSK) is a rare autoimmunedisorder (AD) of the neuromuscular junction (NMJ) and represents a prototype of AD with proven IgG4-mediatedpathogenicity. Thanks to the mechanism of Fab-arm exchange (FAE) occurring in vivo, resulting MuSK IgG4 k/λ Absincrease their interference on NMJ and pathogenicity. The characterization of hybrid MuSK IgG4 as a biomarker for MGmanagement is poorly investigated.

METHODS

We evaluated total IgG4, hybrid IgG4 k/λ, and the hybrid/total ratio in 14 MuSK-MG sera in comparison with 24 fromMG with Abs against acetylcholine receptor (AChR) that represents the not IgG4-mediated MG form.

RESULTS

In both subtypes of MG, we found that the hybrid/total ratio reflects distribution reported in normal individuals;instead, when we correlated the hybrid/total ratio with specific im-mune-reactivity we found a positive correlationonly with anti-MuSK titer, with a progressive increase of hybrid/total mean values with increasing disease severity,indirectly confirming that most part of hybrid IgG4 molecules are engaged in the anti-MuSK pathogenetic im-mune-reactivity.

CONCLUSIONS

Further analysis is necessary to strengthen the significance of this less unknown biomarker, but we retain it is full ofa diagnostic-prognostic powerful potential for the management of MuSK-MG.


M014


GROWTH DIFFERENTIATION FACTOR 15 (GDF-15): NEW BIOMARKER IN SARS-COV-2 DISEASE (COVID-19)

N.M. García Simón 1, J. Vega Benjumea 1, A.M. Roldán Cabanillas 1, F.A. Bernabeu Andreu 1, R.A. Silvestre 11Clinical Biochemistry, Hospital Puerta de Hierro, Universidad Autónoma de Madrid, Spain

BACKGROUND-AIM

Growth differentiation factor 15 (GDF-15) is a cytokine that has been associated with macrophage activation processesand might play a role in the regulation of inflammation and apoptosis. Therefore, it is considered a marker of responseto oxidative stress and inflammation. As coronavirus disease (COVID-19) pandemic rages on, there is urgent need foridentification of clinical and laboratory predictors for progression towards severe and fatal forms of this illness. In thisstudy we aimed to evaluate the discriminative ability of GDF-15 as a biomarker in patients with and without severeor fatal forms of COVID-19.

METHODS

Subjects:Controls: healthy subjects (n=52; 53 ± 1.5 years-old; 50% female). Blood Bank donors who signed the correspondinginformed consent. Negative SARS-CoV-2 PCR.• COVID-19 patients: 75 patients (62.4 ± 1.3 years-old; 24% female) from our Hospital with positive PCR for SARS-CoV-2(patients’ samples were collected as part of their clinical management):1. Mild COVID-19 (n=37): mild symptoms that do not require hospitalisation or patients with admission <10 days (notICU).2. Severe COVID-19 (n=38): admitted to ICU or Exitus.• Post-COVID-19 patients (n=23; 51.1 ± 3.1 years-old; 30% female): negative PCR for SARS-CoV-2 and/or IgG SARS-Cov2positive (with previous positive PCR).The study was approved by the Ethic Committee of our Hospital.Serum GDF-15 was measured by electrochemiluminescence immunoassay (Cobas e411, regents were generouslysupplied by Roche Diagnostics). Data are expressed as Mean±SEM and statistical analysis (unpaired t-test and ROCcurves) was performed by MedCalc® v11.4.2.0.

RESULTS

GDF-15 levels in female and male controls were comparable (621.6±32.6 vs. 622.6±31.3 pg/ml, P=0.85). GDF-15 levelswere markedly elevated in COVID-19 patients (mild+severe: 4675.6±582.4 vs. 622.1±22.3 pg/ml in controls; P<0.01).ROC curve showed AUC=0.995; p<0.01, 92.9% sensitivity (S) and 97.6% specificity (SP) for a value >878.7 pg/ml.The increase in GDF-15 levels was significantly higher in severe COVID-19 patients (8060.3823.7 in severe COVID-19vs 1566.6±10 2.6 pg/ml in mild COVID-19; P<0.01). ROC curve for severity showed AUC=0.965, 91.9% S and 94.1% SPfor a value >2411 pg/ml GDF-15. Conversely, GDF-15 decreased in post-COVID-19 subjects as compared to COVID-19patients (788.6±87.8 vs 4675.6±582.4 pg/ml, respectively; P<0.01).

CONCLUSIONS

Circulating GDF-15 concentrations increased in COVID-19 patients, especially in severe cases. GDF-15 levels decreasedin post-COVID19 subjects as compared to COVID-19 patients. Thus, GDF-15 could be considered as a new prognosticlaboratory biomarker of COVID-19 progression towards serious and fatal forms.


M015


USE OF THE VALUE OF MONOCYTE DISTRIBUTION WIDTH (MDW) AND THE NEUTROPHIL TO LYMPHOCYTE RATIO (NLR)AS SEPSIS MARKERS IN PEDIATRIC COVID-19 PATIENTS

D. Navarro Calderón 1, V.d.A. Moral Ortiz 1, L. Marques De Brito 1, S. Sánchez Berdial 1, C. Puertas López 1, M. García

Gámiz 1, M.M. González Estecha 11Department of Clinical Biochemistry, General University Hospital Gregorio Marañon, Madrid

BACKGROUND-AIM

A small percentage of SARS-CoV-2 infections can induce sepsis, there are prognostic markers to assess severity andmortality in this patients but, due to high cost or difficulty of implementation, it is necessary to develop easier and low-cost alternatives. Activated monocytes change morphology and increase in size in patients with sepsis, thus increasingthe value of MDW, which can be crucial for early sepsis detection. On the other hand, the NLR has been pointed as agood predictor of severe SARS-CoV-2 disease, enhancing its usefulness as a prognostic marker.

METHODS

A study has been carried, which included 28 patients aged ≤18 years old, who were admitted to the pediatric emergencyservice and confirmed of COVID-19 by RT- PCR. At the same time, a control group of 36 patients aged ≤18 years old wasselected. They were admitted to the pediatric emergency service for other reasons and underwent an RT-PCR protocolto exclude them from COVID- 19 disease. Peripheral blood samples were collected in containers with dipotassiumEDTA anticoagulant and subsequently analysed on a Beckman Coulter® DXH 900 haematology analyser, obtaining theMDW values and calculating the NLR value from the absolute values obtained from neutrophils and lymphocytes. Thevalues MDW and NLR results of both groups were compared using the nonparametric Mann-Whitney U test. Continuousvariables were presented as median and interquartile range (IQR) and the statistical program used was SPSS v.26.0.

RESULTS

Statistically significant differences (p <0.05) were observed when comparing the MDW and NLR values between theCOVID-19 infected group and the control group (other pathologies). In the 36 negative COVID-19 patients, the MDWvalue was 16,47 (IQR: 15,70-17,61) and the NLR value was 1,95 (IQR: 1,42-2,95), while of the 28 positive COVID-19patients, higher values were obtained for MDW and NLR, being 22,93 (IQR: 19,15-28,03) and 4,03 (IQR: 3,04-15,14),respectively.

CONCLUSIONS

Both markers increase significantly in COVID-19 positive patients, establishing them as important prognostic markersof the disease and being useful for making decisions in a short term. By not requiring additional samples, bothparameters could be seen as a quick, inexpensive and easy way to obtain more information from the patient.


M016


SAPIENIC ACID METABOLISM SUSTAINS MEMBRANE PLASTICITY AND PROTEIN SIGNALING IN BREAST CANCER

C. Ferreri 5, A. Sansone 5, C. Chatgilialoglu 5, E. Kucuksayan 2, G. Talibova 3, D. Tekeli 4, T. Ozben 11Department of Biochemistry, Faculty of Medicine, Akdeniz University, Antalya, Turkey.2Department of Biochemistry, Faculty of Medicine, Alanya Alaaddin Keykubat University, Antalya, Turkey.3Department of Histology and Embryology, Faculty of Medicine, Akdeniz University, Antalya, Turkey4Department of Pediatric Hematology-Oncology, Faculty of Medicine, Akdeniz University, Antalya, Turkey5Istituto per la Sintesi Organica e la Fotoreattività, Consiglio Nazionale delle Ricerche, Via Piero Gobetti 101, 40129 Bologna,Italy

BACKGROUND-AIM

Alternative fatty acid metabolism in cancer cells is crucial to induce faster growth and invasiveness. Sapienic acid,a monounsaturated fatty acid formed from palmitic acid by delta-6 desaturase, and its metabolic transformation to8cis-18:1 and sebaleic acid, the only PUFA pre-pared de novo, have been recently associated with cancer invasivenessand metabolic plasticity due to the incorporation of these n-10 fatty acids as membrane phospholipid constituents.However, the coupling of membrane fatty acid remodeling due to n-10 fatty acids with signaling cascades has not yetbeen explored, as well as the difference among cancer cell types. The scope of this study is to demonstrate that SAand its metabolism to n-10 MUFA and PUFA can have both structural and functional roles in breast cancer cell lines,influencing not only membrane plasticity but also membrane-based signaling in a distinctive manner for each cell type,thus providing with synergies to sustain cancer cell metabolism and resistance.

METHODS

Cultured MCF-7, BT-20, and MDA-MB-231 cells were treated with 50 and 300 µM sapienic acid for 24 h at 37˚C,analyzing in the first 3 hours the expression and activation of EGFR, m-TOR and Akt as oncogenic signaling cascade, incombination with the analysis of the membrane fatty acid remodeling.

RESULTS

We present herein the results of 50 and 300 µM sapienic acid supplementation, with parallel evaluation of membranefatty acid remodeling and expression/phosphorylation of EGFR, m-TOR and AKT as oncogenic signaling cascade, usingMDA-MB-231 and BT-20, as triple negative breast cancer cell (TNBC), as well as MCF-7, compared to untreated cells.

CONCLUSIONS

The n-10 fatty acid family displayed structural and functional effects in each of the breast cancer cell lines, influencingnot only membrane plasticity but also membrane-based signaling, which can act synergically to sustain cancer cellmetabolism and resistance.Funding:This study was supported and funded by TUBITAK (Project number: 217S253).The authors acknowledge the networking opportunity created by the COST Action NutRedOx-CA16112.


M017


PREVALENCE OF METABOLIC SYNDROME IN STUDENTS OF THE MEDICAL FACULTY IN FOčA

D. Puhalo Sladoje 1, D. Bokonjic 1, A. Mandic 1, O. Cancar 1, V. Cancar 11Faculty of Medicine Foca, University of East Sarajevo, Foca, Republic of Srpska, B&H

BACKGROUND-AIM

In the modern world, obesity is a major health problem, both among adults and among children and adolescents.Obesity is a disease characterized by abnormal or increased accumulation of fat in adipose tissue to the extent thatit damages health and leads to the development of numerous complications. The appearance of obesity at a youngerage increases the risk of early onset of these complications, which requires its timely diagnosis and treatment. Theaim of this study was to determine the presence of metabolic syndrome in students of the Medical Faculty in Foča.

METHODS

In accordance with the set goals, the research was conducted according to the type of cross - sectional study. In thefirst part of the examination, a systematic examination of first and second year students of the Medical Faculty in Foča,University of East Sarajevo was performed. The study included 175 subjects, both sexes, aged 19 to 21 years.As part of the systematic examination, each subject underwent measurement of systolic and diastolic blood pressure(mmHg), measurement of body weight (kg), body height (cm), waist circumference (cm), hip circumference (cm) andbased on the obtained results was calculated body mass index (BMI, kg / m2) and waist circumference (cm) / hipcircumference (cm) (OS / OK). Laboratory testing included: determination of total cholesterol concentration (mmol /L), cholesterol concentration in HDL (mmol / L), LDL (mmol / L) and VLDL (mmol / L) lipoproteins, apolipoprotein A-1and B concentration (g / L) and serum triacylglycerol concentration (mmol / L). Sugar metabolism was monitored bymeasuring blood glucose concentration (mmol / L).

RESULTS

Based on anthropometric parameters and biochemical indicators, and in accordance with the criteria of theInternational Diabetes Mellitus Association, the mentioned group of subjects was divided into 3 subgroups: the firstgroup - normally fed subjects (N 106), the second group - subjects with abdominal obesity but without metabolicsyndrome (N 37) and the third group - subjects with metabolic syndrome (N 32).Of the total number of subjects included in the study, 69 were found to have abdominal obesity, according to IDFcriteria. Among the subjects with abdominal obesity (N 69), in thirty - seven (N 37) no signs of metabolic syndromewere found, while 32 subjects had two components of metabolic syndrome. In the group with abdominal obesity, therewere significantly more female subjects (p <0.001). In female subjects, the most common components of the metabolicsyndrome were higher waist circumference and decreased HDL-cholesterol concentration, while in men, higher waistcircumference, increased triacylglycerol concentration and increased blood pressure were found. Decreased HDL-cholesterol concentration was measured in 93% of the female population of our study group, and among the malepopulation this percentage was 35%.The prevalence of arterial hypertension in the group with metabolic syndrome was 53.0%During our study, in the blood of subjects of all examined groups, fasting glycemic values were within the referencevalues of the applied methods and did not differ significantly between the examined groups.

CONCLUSIONS

Research indicates that the finding of metabolic syndrome components in the population of children and adolescentsincreases the incidence of fatal and non-fatal cardiovascular events in later years. Therefore, early identification andtreatment of children and adolescents with metabolic syndrome and taking preventive measures is crucial to reducethe risk of cardiovascular and other obesity complications.


M018


THE ROLE OF NON-CODING RNA AND ANGIO-MARKERS IN PREDICTING MATERNAL READINESS FOR EMBRYO TRANSFERIN THE IVF PROCESS

M. Rabajdova 2, S. Toporcerová 1, M. Kľoc 2, I. Špaková 2, K. Šoltýs 3, P. Urdzík 1, M. Mareková 21Department of Gynaecology and Obstetrics, Faculty of Medicine, Pavol Jozef Šafárik University in Košice and UNLP in Košice,Slovakia2Department of Medical and Clinical Biochemistry, Faculty of Medicine, Pavol Josef Šafaric Univerzity in Košice, Slovakia;position: associate professors3Department of Microbiology and Virology, FNS UK, Bratislava, Slovakia, Comenius University Science Park, Bratislava,Slovakia

BACKGROUND-AIM

The aim of the study was to identify molecules of non-coding RNAs and proteins in women's plasma as importantsupportive predictive biomarkers of women's biological readiness for embryo transfer. Over the last 5 years, theimplantation failure is almost unchanged, it is below 30% of successfully implanted embryos. Current studies ofimproper endometrial susceptibility have concluded that improper expression of certain biomolecules leads toperceptual disorders. There is a shift in the implantation window, or in the worst case, its extinction and thus themother's unpreparedness for embryo transfer.

METHODS

Plasma was collected from 263 women, who have been included in the IVF cycle and used for ELISA and ncRNAanalysis. From which in 49% of patient’s, IVF failed with biochemically confirmed hCG serum negativity (day 10 afterblastocyst transfer or day 11 after compacted morula transfer*). The IVF success was recorded in 51% from themas hCG level over 30 IU/L*. In these patients’ differences in expression of 15 proteins were detected on the day ofembryo transfer. For determination of proteins (e.g. Angiopoietin 1, Endoglin, PLGF, PGR, TGFβ1, VEGF, NR2F2, NOTCH3)was used commercial ELISA kits, which were examined using Synergy H4 reader platform. Isolation, detection, andsequencing libraries of ncRNA molecules were prepared too.Plasma libraries of miRNAs were examined using massively parallel sequencing on the Illumina platform. Obtaineddata was processed with freely available bioinformatic tools and machine learning.

RESULTS

Was determined by models the minimum of 18 miRNA as prediction biomarkers. The developed model can predictmaternal readiness for embryo transfer in the IVF process over the 80%. Precision, recall, F1 score indicators were usedto determine the accuracy of each model. The KNN classifier confirmed the predictive function of selected 4 proteinsin relation to the prediction of IVF success and thus the readiness of women with an accuracy of 71%.

CONCLUSIONS

Studied plasma proteins and miRNA molecules can be used as diagnostic panel important for assessment women'sbiological readiness of embryo transfer.The project was supported by the grants VEGA 1/0873/18 and VEGA 1/0620/19.


M019


ASSOCIATION OF TRIACYLGCEROL-GLUCOSE INDEX WITH LOW-DENSITY LIPOPROTEIN PARTICLE NUMBER AND SIZEMEASURED BY PROTON NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY

O. Rácz 5, M. Brenišin 4, D. Pella 3, E. Bilá 1, M. Lukačko 1, R. Gaško 21Allmedical Ltd, Bratislava, Slovakia2Biochemical Laboratory, Psychiatric Hospital, Michalovce, Slovakia3East-Slovak Institute of Cardiovascular Diseases, Inc. & Medical School, Safarik University, Kosice, Slovakia4Institute of Pathophysiology, Medical School, Safarik University, Košice, Slovakia5Institute of Pathophysiology, Medical School, Safarik University, Košice, Slovakia & Miskolc University, Faculty ofHealthcare, Hungary

BACKGROUND-AIM

The aim of this study was the comparison of triglyceride-glucose index (TYG), a marker of insulin resistance with resultsof low-density lipoprotein particle number and size measured by proton nuclear magnetic resonance spectroscopy(PNMR) in a randomly selected outpatient sample without manifest coronary artery disease (CAD) and diabetesmellitus.

METHODS

Probands: Data of 75 outpatients (56 men, 19 women, age 31-78 years) were evaluated.Methods: Basic lipid panel including triacylglycerol (TAG) concentration, and fasting blood glucose (FPG) were analyzedby standard methods, PNMR lipoprotein particle size and number assay by NIFAI NMR spectroscopic assay (Germany).In this study evaluation of total, small and large LDL particle number and LDL particle size was included. Triacylglycerol-glucose index equation: TYG = ln(TAG [mmol/L]xFPG [mmol/L]/2).

RESULTS

Evaluated data: FPG: 5,46 ± 0,45 mmol/L; TAG: 1,58 ± 0,59 mmol/L; TYG: 1,39 ± 0,40 units. Total, large, and smallLDL particle number: 1564 ± 431; 909 ± 251 and 677 ± 325 µmol/L respectively, LDL particle size 21,05 ± 0,50 nm.Associations: There were strong associations between TYG and small and total LDL particle number (r = 0,76 and 0,60,p < 0,001), but no association was found between TYG and large LDL particle number. The association between TYGand LDL size was indirect, r = -0,76, p < 0,001.

CONCLUSIONS

Assessment of CAD risk according to basic lipid markers is widely used but is not sufficient to asses the risk with highaccuracy. PNMR lipoprotein assay provides an insight into the real number of particle number and size but its use ingeneral practice is hindered by the need of specific equipment and high price. TYG is a simple integrated marker ofinsulin resistance connecting basic information on saccharide and lipid metabolism. The strong association betweenTYG and number of small LDL particles as well as the indirect association between TYG and LDL size raises the possibilityto use TYG in general practice and PNMR lipoprotein assay in research and clinical specific conditions to obtain a betterprediction of CAD risk. To prove the validity of this hypothesis further studies on sufficient number of participants arenecessary.


M020


CLINICAL ASSESSMENT OF HEPATITIS B VIRUS SURFACE ANTIGEN (HBSAG) ON THE ATELLICA AND CENTAUR XPTANALYSERS

D. Rodriguez Cano 3, M. López Melchor 2, I. Pérez De Algaba Fuentes 1, F. Rodríguez Cantalejo 31Hospital de Montilla, Montilla2Hospital San Juan de la Cruz, Úbeda3Hospital Universitario Reina Sofía, Córdoba

BACKGROUND-AIM

The change of technology in HBsAg determination has led to an increase in false positives (FP) in our laboratory results.With the installation of Atellica analysers, with chemiluminescent technology for the determination of HBsAg, we hopeto reduce these FPs.The aim of the present study is to estimate that the Atellica platform will decrease the FPs in our results, with respectto the previous Centaur XPT platform.

METHODS

To determine the clinical performance of each platform, patients with HBsAg analytical request were included. HBsAgdeterminations were performed in parallel on the Atellica and Advia Centaur XPT platforms. Patient records werereviewed to establish the clinical status of the patient. The incidence of FP and false negatives (FN) was estimated forboth methods. Sensitivity, specificity and percentage of well-classified patients from both platforms were calculatedfor the manufacturer's recommended cut-off for the serological diagnosis of hepatitis B (1 URL).Positive predictivevalue (PPV), negative predictive value (NPV), positive (PLR) and negative likelihood ratios (NLR), together with their95% CIs, were assessed.

RESULTS

The presence of FP in Centaur XPT was 5.3% and in Atellica 11.3%.FN was 1.5% in both platforms.The Centaur XPT assay had a sensitivity of 88.2% (62.3%-95.3%), specificity of 94% (87.5%-97.3%), PPV of 68.2%(45.1%-85.3%) and NPV of 98.2% (93%-99.3%). The PLR was 14.6 (6.9-30.6) and the NLR 0.12 (0.03-0.46). The CentauroXPT HBsAg assay correctly classified 93.2% of patients.The Atellica assay had a sensitivity of 88.2% (62.3%-95.3%) and specificity of 87.1% (79.3%-92.3%), PPV of 50%(31.7%-68.3%) and NPV of 98.1% (92.5%-99.2%). The PLR was 6.8 (4.1-11.3) and the NLR 0.13 (0.03-0.49). Atellicacorrectly classified 87.2% of patients.

CONCLUSIONS

Therefore, the implementation of the Atellica assay for HBsAg determination does not solve the previous issue theCentaur XPT platform, as it classifies patients less well and has lower specificity, which represents a costly This requiresan effort, both financially (extra reflexive tests) and in terms of our own resources in our management unit.


M021


MICROARRAY FOR BIOLOGICAL DMARDS THERAPY ASSESSMENT

A. Tikhonov 1, O. Smoldovskaya 1, G. Feyzkhanova 1, E. Savvateeva 1, S. Voloshin 1, E. Aleksandrova 3, A. Novikov 3, L.

Pavlushkina 2, A. Rubina 11Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia2Filatov Moscow City Pediatric Clinic No. 13, Moscow, Russia3Moscow Clinical Scientific Center n.a. A.S. Loginov, Moscow, Russia

BACKGROUND-AIM

Anti-drug antibodies (ADAs) are expressed in some rheumatoid arthritis (RA) patients during the therapy withbiological disease-modifying anti-rheumatic drugs (DMARDs) and can reduce their therapeutic effects or triggerserious side effects.

METHODS

In order to detect the ADAs, microarray that contains immobilized drugs (TNFα inhibitors (infliximab, adalimumab,etanercept), monoclonal antibodies to the IL-6 receptor (tocilizumab), CD20 (rituximab), as well as CD80, and CD86(abatacept)) and the corresponding targets has been constructed. Immobilized DMARDs can be used for the evaluationof binding effectiveness between a DMARD and a target and immobilized targets – for the characterization of theDMARD immunogenicity leading to the production of ADAs.

RESULTS

Incubation of the biotinylated DMARDs on the microarrays followed by the Streptavidin-Cy5 visualization has been heldto verify the working capacity of immobilized targets. The presence of the necessary fluorescent signals was shown forTNFα, IL-6 receptor, and CD80. The functionality of immobilized DMARDs was evaluated via the sandwich immunoassayof the targets and the following development with biotinylated DMARDs and Streptavidin-Cy5. This time, fluorescentsignals were visualized as in the cells with immobilized DMARDs (which corresponds to the triple complex “DMARD-target-DMARD”, if applicable), and in the cells with immobilized targets (double complex “DMARD-target”). Therefore,immobilization does not inactivate DMARDs and they will be able to bind with targets from the sera.

CONCLUSIONS

In the future, the microarray is planned to be used for the analysis of the sera samples of RA patients treated withbiological DMARDs in the format of the classical sandwich immunoassay or competitive immunoassay.This work was supported by the Russian Science Foundation (http://rscf.ru/en), Grant No. 19-15-00283.


M022


NU.Q® CAPTURE-MS ALLOWS THE EPIGENETIC PROFILE ANALYSIS OF CIRCULATING NUCLEOSOMES IN NON-HODGKINLYMPHOMA PATIENTS

P. Van Den Ackerveken 1, A. Lobbens 1, J. Turatsinze 1, V. Solis-Mezarino 2, M. Völker-Albert 2, A. Imhof 3, M. Herzog 11Belgian Volition SRL, 22 Rue Phocas Lejeune, Parc Scientifique Crealys, 5032, Isnes, Belgium2EpiQMAx GmbH, Am Klopferspitz 19, 82152, Planegg-Martinsried, Germany3EpiQMAx GmbH; BioMedical Center, Division of Molecular Biology, Faculty of Medicine, Ludwig-Maximilians UniversitätMünchen

BACKGROUND-AIM

There are over 544,000 new cases of non-Hodgkin lymphoma (NHL) diagnosed worldwide each year and approximately260,000 deaths. The non-specific symptoms of lymphoma often delay diagnosis. Alterations of epigeneticmodifications have been demonstrated as an important player in human cancer including lymphoma. However, theepigenetic profile of histone post-translational modifications (PTMs) on circulating nucleosomes is still not welldescribed. In this context, we investigated the histone PTMs present on circulating nucleosomes in NHL patients andcontrols by mass spectrometry (MS).

METHODS

We have developed a fast and robust enrichment method based on nucleosome immunoprecipitation toisolate intact circulating nucleosomes from blood sample. Nu.Q® Capture immunoprecipitation protocol allowsimmunoprecipitation of nucleosomes from plasma samples in a quantitative manner. 900µl of plasma samples wereincubated with anti-H3.1 magnetic coated beads. Then, the immunoprecipitated nucleosomes were subject to aliquid chromatography coupled with tandem mass spectrometry (MS) analysis. The quantification of the peptides wasachieved by using isotope labelled spike-in standards, that are introduced to the processed clinical plasma sample andare subjected to the same processing steps, to control experimental variability and increase quantitation accuracy. Weimplemented this innovative method in a pilot study composed by plasma samples from NHL cancer patients (n=9)and healthy controls (n=6)

RESULTS

We were able to isolate 92.7% ± 5.4% of the nucleosomes present in the blood sample. By comparing NHLs vs healthyplasma samples after Nu.Q® Capture-MS protocol, we identified 56 histone proteoforms. Core histone proteins suchas histone H3 and H4 were identified in all immunoprecipitated samples. Among the histone peptides identified, wefound 7 histone PTMs, located at 6 different sites, differentially represented in plasma from NHLs patients or healthydonors (p < 0.05).

CONCLUSIONS

Altogether these data suggest that the Nu.Q® Capture protocol is able to isolate circulating nucleosomes from plasmasamples of NHL patients thereby allowing their subsequent analysis by mass spectrometry, and the discovery ofpotential biomarkers for NHL cancer diagnosis.


M023


DESCRIPTIVE ANALYSIS OF MDW VALUES, A NEW MARKER OF SEPSIS OBTAINED FROM THE HAEMOGRAM.

G. Velasco De Cos 1, S. Fernandez García 2, M.A. Ainhoa 1, M. Iturralde Ros 1, A. Moyano Martinez 1, N.H. Cahuana

Santamaría 1, J.Á. Calvo Sánchez 21Clinical Analysis Service, Marqués de Valdecilla University Hospital, Spain2Haematology Department, Marqués de Valdecilla University Hospital, Spain

BACKGROUND-AIM

According to the latest definition of sepsis (2016), sepsis is defined as life-threatening organ dysfunction caused bya dysregulated response to infection. Early diagnosis is key and early initiation of therapeutic measures exponentiallyincreases survival.Clinical scales such as qSOFA and analytical parameters such as C-reactive protein (CRP), procalcitonin and lactate areavailable to identify it. In this context, a new parameter, monocytic distribution width (MDW), has been identified. Thisis an in vitro diagnostic parameter obtained through haemogram analysis that reflects the volume of monocytes inresponse to proinflammatory signals from infectious organisms.In our centre, this parameter is obtained on the Beckman Coulter® DxH900 analyser. According to the manufacturer'sliterature, values above 21.5 in EDTAK3 samples help to identify patients with sepsis or at risk of developing sepsisin the first 12 hours of admission.

METHODS

MDW determinations during the month of March were analysed and a total of 6865 patients were obtained from thelaboratory database. Together with the MDW value, CRP and leukocyte results were obtained.The medical records were searched for patients with a result >21.5 (N=530) to see if a clinical diagnosis of sepsis wasfinally made. We excluded 191 samples from oncology, COVID, hepatopathic and pregnant patients in whom the MDWvalue is not validated (N=339).Statistical analysis was performed using SPSS V21, Kolmogorov-Smirnov and Mann-Whitney test.

RESULTS

Of the total of 339 patients with MDW>21.5, 207 were admitted. We compared the MDW outcome between theseadmitted septic and non-septic patients. Statistically significant differences were obtained (p=0.01), with the medianMDW being 24.1 [21.5-37.5] for non-septics and 25.5 [22.0-42.5] for septics.Within the total number of patients analysed, we looked for differences in MDW values according to CRP and leukocytes:- Statistically significant differences (p=0.000) were found between MDW and leucocyte values <4000 and >12000 (SIRSCriterion) 17.9[11.4 -39.1] vs 18.6[11.2-42.5].- A CRP determination was performed on 3858 patients, establishing a cut-off point of CRP=8, statistically significantdifferences p=0.000 were found between MDW values (19.3 [14.2-36.8] vs 22.4 [16.9-38.5]).

CONCLUSIONS

MDW is a useful parameter in the assessment of the septic patient, both on arrival at the emergency department andon admission. There is a relationship between its value and some of the variables normally used in the assessment ofthe septic patient, so it adds value to the haemogram when diagnosing the patient.


Documents

Advanced technologies , including new biomarker discovery