61st National Congress of the Hungarian Society of Laboratory Medicine

Department of Laboratory Medicine, University of Pécs, Medical School, Pécs, Hungary

In my presentation I would like to show how our long-term basic research could serve as a good starting point for the development of novel assays in various fields of biomarker studies. In the 80ies we could establish a model of fluorescence polarization immunoassay which later became a routine technique in the clinical laboratory. Using chemiluminescence labeling we were the first to measure procalcitonin in collaboration with the Department of Intensive Care Unit in Pécs. We tried to find novel biomarkers in systemic inflammatory diseases including sepsis and Crohn’s disease, first by electrophoresis and Western blot methods. We were focusing on serum actin and actin binding proteins and on urinary markers that might help the diagnosis and the prediction of the severity of systemic inflammation including acute kidney failure. Because there were no commercially available assays for the chosen markers [actin, gelsolin, serum and urinary Gc globulin, urinary orosomucoid (u-ORM), cystatin-C (u-CYSC), and neutrophil gelatinase-associated lipocalin (u-NGAL)] we developed automated immune turbidimetric assays and ELISA techniques for their fast and reliable measurement. Recently, we established a new test of presepsin/gelsolin ratio useful in critically ill patients. Our ROC analysis results show that the novel laboratory markers could improve diagnosis and therapeutic decision-making especially on the day of admission of the patients. The newly introduced methods may complete the classical clinical and laboratory approaches and are also useful in the follow-up of the patients. Going back to basic research again, most recently we developed a fluorescence multiplexed in situ quantitative protein expression assay (total and phosphorylated forms as well) in cultured cells to follow cell signaling processes directly in 96-well culture plates.

Department of Clinical Biochemistry and Clinical Molecular Biology, University of Padova, Padova, Italy

Sustainability in healthcare has shifted from an environmental concern towards a holistic definition that includes balancing socio-ecological and socio-technical systems, including effectiveness and cost efficiency of health services. Laboratory medicine should contribute to a sustainable healthcare system ensuring that resources can be used efficiently from ecological, social, and economical perspectives, while providing high-quality services to patients and physicians. Clinical laboratories can limit their environmental impact and provide sustainable laboratory services making reduction in 4 key areas: 1) Energy consumption; 2) Water consumption; 3) Waste production; and 4) Use of hazardous chemicals. However, the second and main contribution of clinical laboratories to a sustainable healthcare system is to promote value-based laboratory medicine. According to the seminal concept of the brain-to-brain loop (1), “Laboratory tests that matter are those that produce actionable results to bring a positive outcome benefit for the patient”. This, in turn, means that laboratorians should all be concerned not only on analytical performances, but about the effects of laboratory tests and whether the laboratory information was useful for the patient or for the public’s health. Therefore, value in medicine and in laboratory medicine should be defined as “outcomes relative to costs”. Even if the link between laboratory testing and outcomes, is usually «an indirect» relationship, thus making difficult the search for outcome measures, the integration of process and outcome measures plays a fundamental role in value-based laboratory medicine (2). Process measures are well known in laboratory medicine as internal quality control (IQC) and external quality assessment (EQA) programs have significantly improved the analytical quality in the last decades. The introduction of quality indicators for evaluating and monitoring quality in extra-analytical phases led to further improvement of quality in the total testing process (TTP) (3). It’s time to finally integrate these measures with outcome measures to improve the visibility and value of laboratory medicine.

Department of Clinical Biochemistry and Clinical Molecular Biology, University of Padova, Padova, Italy

In the last decades, the progressive laboratory automation changed the peculiarity of clinical chemistry testing, so that minutes instead of hours are needed to complete an analysis. This quickening of analytical process has markedly improved intra-laboratory TAT, so that automation has become essential in clinical laboratories striving to deliver timely results. However, to make shorter the total examination process and thus reduce therapeutic TAT, further automation of extra-analytical steps was needed, implementing IT management of all processes, and connecting several fully automated analyzers to pre- and post-analytical modules by a track system. In addition, evidence has been collected to demonstrate the improvement in analytical performances due to both automation, better training of the staff and adoption of quality control and assurance programs. However, recent technological developments, particularly point-of-care testing and examinations, patient self-testing, and patient self-collection of specimens are addressing one of the current key barriers, namely access sufficiently close to the patient to be convenient, often described as the so-called last mile problem (1). Ultimately, these developments aim to reduce the diagnostic gap (i.e., the proportion of the population with a particular condition who are underdiagnosed) (2). More recently, the proliferation of health tracking apps, wearables and home sensors have created new clinical data streams controlled by the patient, with capture granular information about lifestyle and micro-environmental exposures. In an era of big data and artificial intelligence, the main challenge for clinical laboratories is to promote the integration between centralized and decentralized laboratory testing and to promote the value of laboratory information for early diagnosis, prognosis, guide to therapy and wellness maintenance as well. Another fundamental goal is to move clinical laboratories out from the “silo model” to promote integrated diagnostics.

Department of Laboratory Medicine, Faculty of Medicine, University of Debrecen, Debrecen, Hungary

In the last decade, with the advancement in laboratory technology, the analysis of body fluids has seen a drastic increase in clinical examinations. Cell counting and classification in the body fluid software of hematology analyzers conducted by semiconductor laser flow cytometry and nucleic acid fluorescence staining techniques is a reliable method and potentially reduces the ratio of manual cell counting in chamber as well as it shortens the turnaround time (TAT). Based on several preliminary experiments (within-run and between-run reproducibility for WBC, RBC, polymorphonuclear %, mononuclear %; comparison of cell counts and differential between Sysmex-BF module and light microscopy, is presented on a poster at this meeting: Kürti-G.-Szabó E. et al) and by consultations with clinical and laboratory specialists finally we created an algorithm for the evaluation of body fluid samples. IT improvements were needed for the new algorithm (e.g., display of the BF-WDF channel image in the laboratory IT system). The result format has also been changed, in body fluid samples: the corrected and uncorrected WBC number along with the MN % and PMN % are indicated. Our study also evaluates the advantages of incorporating this algorithm in routine laboratory work. Previously we counted all CSF RBC and WBC below 20 cells/µL in Fuchs-Rosenthal chamber. After the introduction of the new algorithm the percentage of WBC manual counts during a 10-day-period decreased from 67 % to 24 % while RBC manual counts in the same period decreased drastically from 82 % to 32 % in CSF samples. The new algorithm for the evaluation of body fluid samples has optimized the number of samples that need to be counted under the microscope, however, in addition to the MN % and PMN % provided by the analyzers, microscopic examination of the cytospin preparation is essential in all cases when WBC differential was ordered.

¹SYNLAB Laboratory, Dunaújváros, ²SYNLAB Hungary Ltd., Budapest, Hungary

In 2013, SYNLAB Hungary Ltd. set a goal to improve patient safety. Patient safety risks were estimated and compared using the FMEA (Failure Mode and Effects Analysis) method. In 2016, we processed data from 360 post-post-analytical questionnaires. In 2023, we have more than doubled the number of potential patient risks as part of our preparations for accreditation. We have expanded the questionnaire and repeated the survey among respondents from clinical chemistry laboratories in the network. We received 626 responses. 72 % of the responses were from primary care. Half of the respondents have direct contact with SYNLAB’s diagnostic centre in Budapest. In the FMEA table, several potential risks related to the reference range have been identified. We wanted to know whether the respondents considered that there were any studies for which no reference range was indicated but which would be necessary for the assessment. According to the survey, 95 % of them did not think there was such a test. Also, 96 % of them said that they did not usually encounter an unclear comment on the report. 9 % of respondents said there is a group of tests where more information would be needed to assess the results. We asked the physicians’ opinions on factors (lipemia, hemolysis, icterus) that could significantly (>10 %) influence the measured parameters. The majority (72 %) considered that the result should always be included in the report with a comment on LIH. 51 % of the respondents repeat the requested test if they see contradictory results on the report, and 46 % consult the laboratory staff. A potential risk was considered if the name of the parameter/test specified by the laboratory is not clear (e.g., abbreviation or there are several names for the same test). We wondered how often this mismatch was noted by test requesters. 29 % of respondents have experienced this. In line with this, our aim is to integrate the standardized IT system and, thus, the standardization of the nomenclature as part of risk management into the network’s post-post analytical processes.

St. Imre Teaching Hospital, Central Laboratory, Budapest, Hungary

The estimated glomerular filtration rate (eGFR) is used clinically as a marker for kidney disease. The European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) recommends not implementing version 2021 of the Chronic Kidney Disease Epidemiology Consortium (CKD-EPI) equation in European laboratories and keeping the 2009 version of the CKD-EPI equation. At the same time, the European Kidney Function Consortium (EKFC) published a new equation for eGFR. On our adult patient population, which also includes significant dialysis patients, we examined the effect of the 2 new equations on eGFR results compared to the 2009 version. Creatinine was determined with enzymatic method (Diasys, Creatinine PAP FS) on Abbott Architect c8000 chemistry analyzer. eGFR based on creatinine (eGFR_cr) was calculated by three equations (2009 and 2021 CKD-EPI, EKFC). We processed the data of 11,710 men and 15,279 women. Patients were divided into CKD categories (1,2,3a,3b,4,5). The reclassification of CKD stages was calculated using the three equations in each group separately for males and females. The eGFRcr values in both groups were usually higher in each CKD stage based on the 2021 formula, while they were usually lower according to the EKFC equation comparing the 2009 formula. With the 2021 formula, the % of men/women moved into a higher CKD category: 20.9/19.2 % from 2 to 1; 10.5/11.6 % from 3a to 2; 20.5/20.4 % from 3b to 3a; 12.9/13.9 % from 4 to 3b; 8.2/7.6 % to 4 out of 5. With the EKFC equation, the % of men/women fell into a lower category: 30.2/19.2 % from 1 to 2; 10.5/11.6 % from 2 to 3a; 20.5/20.4 % from 3a to 3b; 12.9/13.9 % from 3b into 4; 4:8.2/7.6 % from 4 to 5. We must keep in mind that all GFR equations remain an estimation of GFR and patients with CKD stage 3 or higher should undergo more intense clinical evaluation and surveillance.

¹University of Debrecen, Department of Laboratory Medicine, Debrecen, Hungary,

²University of Debrecen, Department of Internal Medicine, Debrecen, Hungary,

³ESZFK Nonprofit Kft, Budapest, Hungary

A questionnaire survey was conducted to assess the practice of lipid diagnostic tests in Hungary, as several new international recommendations have been published in recent years.

A total of 39 laboratories from level II and III laboratories answered the questions. The upper limit of the reference range for triglyceride is around 1.7 mmol/L in most laboratories. Twenty-one laboratories reported a specific panic value for triglyceride. The optimal cholesterol level for adults is below 5.2 mmol/L in 30 laboratories, and it is below 5 mmol/L in only 3 laboratories. Fifteen laboratories report non-HDL concentrations, of which 2 report target values for different risk groups, while in 6 cases no target value is reported. In case of LDL cholesterol (LDL-C), 4 laboratories display the target values for each risk group on the report. The LDL-C cut-off values for the other laboratories range from 2.58 to 4.1. LDL-C levels are always determined by measurement in 12 laboratories, by calculation only in 12 others, while 14 laboratories measure or calculate LDL-C, depending on the origin of the test request and triglyceride level. For calculation the Friedewald formula is predominantly used. In total, 5 laboratories measure ApoA-I and ApoB. Lp(a) results are reported by 11 laboratories, with a variety of units and target values, the most common limits are 300 mg/L and 75 nmol/L.

In conclusion, similar to the international tendencies, Hungarian laboratory practice is not uniform in reporting of lipid parameters. Therefore, there is a need for standardization of national laboratory practice. At the University of Debrecen, we have introduced a new report format based on 2019 ESC/EAS and EAS/EFLM recommendations.

Central Hospital of Northern Pest - Military Hospital, Budapest, Hungary

LDL-C (low-density lipoprotein cholesterol) analysis plays a key role in screening, diagnosis, and management of cardiovascular disease risk estimation. The aim of this study was to look for news in lipidology focused on the LDL-C determination method, effective LDL-C lowering options, strategies for increasing compliance during treatment, target values in different cardiovascular risk groups based on current literature data and EFLM and ESC consensus guidelines.

In the field of preanalytics fasting is not routinely required for determination of total-C, TG, HDL-C, LDL-C (I/C). Beta quantification reference measurement procedure is still “the gold standard”. Estimation methods can be used knowing their strengths and limitation. Sampson and Martin/Hopkins method, in case of

LDL-C underestimation with Friedewald equation in those with low LDL-C and/or elevated TG, will help to inform treatment decisions.

Cholesterol-year-score evaluates vascular cholesterol burden. Earlier initialization of aggressive lipid lowering therapy (LLT) might contribute to stabilization and regression of coronary atherosclerotic plaques and reduction in the incidence of CV events. Guidelines were updated to lower the recommended LDL-C threshold in ASCVD. Their consensus recommendation is a comprehensive cardiovascular-renal-metabolic risk reduction management, especially for patients with CKD and diabetes. For long-term safety of combinated, effective LLT it is recommended patient education, counseling, and personalized therapy selection. By blocking HMG-CoA reductase statins not only decrease the LDL-C level but also block the coenzyme Q₁₀ biosynthesis pathway. Statin therapy needs supplementation with coenzyme Q_10. The used of fixed combinations significantly increases adherence and reduces clinical events. A short update on new approaches to LLT therapy: “the lower the better” and “the longer the better” and “the earlier the better”.

University of Szeged, Institute of Laboratory Medicine, Szeged, Hungary

Background: The COVID-19 pandemic had a huge impact on healthcare systems as well as on almost all other areas of our life. The front-line patient care system was fully saturated, and it also presented great challenges to laboratories. The pandemic highlighted the strengths and weaknesses of laboratory workflows and processes and encouraged the staff to adapt all the new situations and to better prepare for similar future events. The purpose of this study was to identify changes in laboratory request, testing and economic issues related to COVID-19 pandemic.

Method: We performed a retrospective assessment of laboratory test requests from patient care providers from the region belonging to the Institute of Laboratory Medicine. We examined the changes in the composition of senders and the proportion of urgent/routine test orders in relation to the waves of the COVID-19 pandemic. We also investigated the weekly changes in demand for seven types of laboratory tests, before and during the pandemic.

Results: Between January 1, 2020, and October 10, 2022, 1126578 laboratory request/order forms were registered, 14 % less than in the last two years before the pandemic. Surprisingly, the proportion of routine and urgent examination requests did not change during this time. There was a net decrease in overall demand across nearly all laboratory sections. Increases in testing were noted only for tests related to COVID-19 management. We also experienced a decrease in the number of submitters. During the COVID-19 pandemic, new submitters appeared, but simultaneously, the number of patient care providers engaged in rehabilitation activities decreased significantly.

Conclusion: Despite the decrease in number of test requests and in number of submitters during the COVID-19 epidemic, the laboratory’s reagent costs did not alter since the majority of diagnostic tests for diagnosing and monitoring COVID-19 patients belong to the highly expensive tests performed by the laboratory.

Semmelweis University, Institute of Laboratory Medicine, Budapest, Hungary

The ability to provide timely identification of the causative agents of lower respiratory tract infections may contribute to better outcome and support antimicrobial stewardship efforts. Because of the importance of pathogens absent in PCR panels and multifactorial mechanisms of antibiotic resistance, culture is still recommended as an essential adjunct to pneumonia-specific multiplex PCR tests. Early detection of viral infections may prevent unnecessary antibiotic treatment and may indicate antiviral therapy.

Syndrome-specific multiple PCR tests have long been introduced as an aid in identification of respiratory pathogens. We reviewed the results of pneumonia panel PCR tests (BioFire Filmarray Pneumonia panel; BioMerieux) performed in our laboratory and compared them with the results of conventional culture during two years of COVID pandemia (2021-2022; n=980 patient). In the same study period, test results (n=1600) of the upper respiratory tract panel (BioFire FilmArray® Respiratory Panel; BioMerieux) also were analyzed. We analysed the link between SARS-CoV-2 infections and other viral respiratory infections, as well as investigated the dynamics of other viral respiratory infections in this period.

The most commonly detected bacteria with PCR were Pseudomonas aeruginosa, Staphylococcus aureus and Acinetobacter baumannii. In SARS-CoV-2-positive inpatients, 25.8 % of the detected bacteria were S. aureus. 44 % of the bacterial strains detected by PCR were not verified by cultures, whereas cultures indicated several bacterial, fungal strains and antibiotic resistance mechanisms other then detected by multiplex PCR tests.

Lock-down and other restrictive measures to limit pandemia significantly reduced the incidence rate of respiratory viral pathogens other than SARS-CoV-2. Viral respiratory co-infections were apparently absent.

¹Central Hospital of Southern Pest-National Instituter of Hematology and Infectious Diseases, Budapest, Hungary,

²Healtware Consulting Ltd, Budapest, Hungary

Background: Current publications suggest that residual symptoms persisting for more than four weeks after the onset of COVID-19 are related to the severity of the disease and the state of the patient’s immune system, as well as the number and type of vaccinations.

Patients and methods: 1496 healthcare workers volunteered from our and its associate hospitals to participate in our research. In addition to filling out a questioannaire, SARS-CoV-2 S-antibody titer measurements were conducted, and in 141 volunteers the cellular immune response was also evaluated. Based on the time of the infection in relation to vaccination, we formed 5 groups of volunteers (COVID-19 before vaccination, within 90 days of vaccination, within 180 days of vaccination, after 180 days of vaccination, the last group had no SARS-CoV-2 infection).

Aims: We examined the correlation between the median values of SARS-CoV-2 S-antibody titer, the number of N and S protein responsive T cells and the severe acute and post-COVID symptoms.

Results and conclusions: 186 volunteers had residual post-COVID symptoms, comparing their data to the other participants, we managed to conclude the following: 1. Higher SARS-CoV-2 S IgG titers indicated undetected infections in some cases. 2. Higher IgG levels in similar immunized and exposed patients are associated with more symptoms generally, but it’s also influenced by comorbidities and the size of T-cell response. 3. Residual symptoms are more common at post-vaccination infections when N type T-cell response no observed. 4 Conversely, this is associated with a higher T-cell reaction among infected before vaccination. 5. Chronic symptoms are more likely to persist where severe acute symptoms appeared at infection. 6. Incidence of residual symptoms is higher in the elderly and recurrent infections. Further population-level studies needed to verify and specify these conclusions.

University of Szeged, Department of Laboratory Medicine, Szeged, Hungary

Introduction: At the end of 2019, a new type of virus strain belonging to the Coronaviridae family was described in Wuhan, Hubei Province, China. The SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) virus strain has been causing a pandemic for almost 3 years, which is the pathogenic factor of the upper respiratory disease COVID-19. Prior to beginning of obligatory vaccination of healthcare workers a longitudinal study was designed with the aim of investigating the kinetics of both vaccination and infection induced humoral immune response based on the determination of the titers of the two relevant antibodies raised against the nucleocapsid and spike proteins of the virus.

Materials and methods: Serum samples were collected from voluntary healthcare workers of University of Szeged Albert Szent-Györgyi Clinical Center prior to both vaccinations and thereafter on a monthly frequency for 24 months between December 2020 and December 2022. The measurements were performed on Roche cobas e801 immunochemistry analyzer using ECLIA method. Information and medical history about the participants were collected by using an Evasys online questionnaire. During the data analysis, we used descriptive statistics and related sample variance analysis, as well as dynamic clustering.

Results: We present the results of 546 healthcare workers, of whom 204 received vaccinations without reported infection. All authorized vaccines were represented in the examined population. Most results were attributable to Comirnaty/BNT162b (Pfizer/BioNTech).

Conclusion: We successfully described several clusters based on the kinetic changes in antibody titer of the participants. We compared the antibody responses induced by the booster vaccinations with respect to vaccinations and time. In addition, based on the reports of the participants, we also described several correlations e.g., antibody titers and clinical symptoms, habits of taking medicine and mental supplements, smoking habits; existing chronic diseases.

Department of Laboratory Medicine, Faculty of Medicine, University of Debrecen, Debrecen, Hungary

Background: Immunogenicity induced by the 3^rd (booster) dose of COVID-19 vaccines has recently got in focus to decide whether the 4^th dose needs to be administered to those without any comorbidities. Here we have assessed anti-SARS-CoV-2 T-cell responses after 1 year of the mRNA booster immunization in parallel to serological tests.

Methods: Sixty-nine volunteers aged between 24-66 years were enrolled who were tested 1 year (375-437 days) after their booster dose of BNT162b2 mRNA homologous vaccinations with a history of previous mild SARS-CoV-2 infection but no other chronic diseases. As controls, age-matched 15 individuals with different heterologous immunizations were tested. T-cell responses were studied via IFN-gamma levels using Elecsys^® IGRA SARS-CoV-2 test (Roche), while total anti-SARS-CoV-2 S1-RBD antibody (S-Ab) levels were determined on a Elecsys^® e602, respectively. Cellular immunity was estimated according to manufacturer’s algorithm for the IGRA test.

Results: Except for one person in the BNT162b2 mRNA group, T-cells were reactive showing a median (IQR) level of IFN-gamma (0.80 [0.43-1.78] IU/mL). These individuals demonstrated induced levels of anti-SARS-CoV-2 S-Ab (10226 [5198-28473] BAU/mL). In terms of the efficacy of different vaccination strategies, there was no difference in IFN-gamma (P=0.2008) between two cohorts, while significantly higher S-Ab titers were (P=0.0007) measured after mRNA vaccines.

Conclusions: The Roche IGRA test is a suitable assay to measure T-cell responses following different vaccination strategies.

¹Department of Laboratory Medicine, Faculty of Medicine, University of Debrecen, Debrecen, Hungary;

²Department of Internal Medicine and Hematology, Semmelweis University, Budapest, Hungary;

³National Institute of Hematology and Infectious Disease, Budapest, Hungary;

⁴Institute of Pediatrics, Faculty of Medicine, University of Debrecen, Debrecen, Hungary

Aims: We analyzed some selected cytokines in acute multisystem inflammatory syndrome in children (MIS-C) in association with disease severity along with routinely measured COVID-19 parameters.

Methods: Thirty-five MIS-C patients at the age of 8.41± 4.1 years were recruited. The WHO classification was used to determine the clinical score for disease severity based on the number of affected organs. Serum concentrations of IFN-γ, IL-1α, IL-1RA, IL-8, IL-10, IL-17A, IL-18, IP-10, MCP-1, and TNF-α were measured by a MILLIPLEX^® Human Cytokine/Chemokine panel (Sigma-Aldrich) using a MAGPIX^® instrument (Merck) in baseline samples obtained at admission and 26 follow-up sera before discharge. Associations were studied between these cytokines, routinely available laboratory parameters and clinical characteristics.

Results: Significantly (p<0.05) higher levels of IL-18, TNF-α and ferritin with reduced lymphocyte count were found in those with the highest clinical scores (4-5) compared to moderate cases (1-3). Furthermore, cardiac dysfunction was associated with larger ACE2 activity and IL-6 (p<0.05). Reduced cytokine values monitored IVIG treatment in all these MIS-C subjects showing remission.

Conclusions: Baseline IL-18, TNF-α and ferritin predict disease severity and are sensitive to follow treatment efficacy in MIS-C.

¹Division of Clinical Laboratory Science, Department of Laboratory Medicine,

²Department of Laboratory Medicine, Faculty of Medicine, University of Debrecen, Debrecen, Hungary

Diagnosis of platelet disorders is sometimes challenging and there are also several differential diagnostics issues. Next-generation sequencing (NGS) is a useful tool to overcome these problems. However, in this group of diseases, there are still few literature data available obtained by this methodology. The aim of our study was to analyze the value of NGS testing in platelet disorders in cases with well-characterized clinical and laboratory phenotype in a tertiary clinical center.

We selected 45 patients with probable hereditary platelet disorders based on their laboratory results. A custom designed gene panel was used including ADRA2A, FLI1, GFI1B, GNAI1, GNAQ, NBEA, NBEAL2, P2RY1, P2RY12, PLA2G4A, PLAU, PLCB2, RASGRP2, STXBP2. In whom no mutations were found by panel sequencing (n=15) clinical exome sequencing was performed. Using this platform, we were able to analyze 4,727 genes. Sanger sequencing was performed with SeqStudio Genetic Analyzer to verify the detected mutations. To assess the pathogenicity of novel genetic variations MutPred2, PolyPhen-2, SIFT, MutationTaster, VarSome and Franklin applications were used.

We were able to detect genetic variations in a total of 30 patients (66 %) by panel sequencing. Furthermore, we detected additional genetic variations in 10 cases (66.7 %) by clinical exom sequencing.

The NGS genetic analysis seems to be a valuable tool in the diagnosis of platelet function disorders, which helps in establishment of genotype-phenotype correlations.

Department of Laboratory Medicine, Faculty of Medicine, University of Debrecen, Debrecen, Hungary

Aims: COVID-19 is associated with the activation of various cell types causing thrombotic complications. Platelet activation in COVID-19 patients was investigated in association with disease outcome.

Methods: Thirteen severe COVID-19 patients (7 survivors and 6 deceased) and 12 age- and sex-matched healthy controls were included. Platelet reactivity was assessed using flow cytometry to quantify platelet P-selectin receptor expression, platelet-leukocyte heterotypic aggregates and platelet-derived microparticles (PMP). The extent of platelet aggregation induced by ADP, adrenaline, and collagen was analyzed by lumi-aggregometry, while plasma P-selectin and CD40 ligand (CD40L) levels were measured with ELISA. Total RNA was isolated from leukocyte-depleted platelets (LDP) purified by anti-CD45 antibody-coated magnetic beads. Profiling of platelet gene expressions was analyzed by NGS regarding 28-day survival (n=3/group). Altered SELP (P-selectin gene) mRNA was validated using RT-qPCR.

Results: Increased platelet activation was observed in COVID-19 patients showing elevated P-selectin positivity, more PMPs and platelet-monocyte aggregates compared to controls (P<0,05). Platelet aggregation was more pronounced, and significantly higher (P<0,001) plasma P-selectin and CD40L concentrations were measured in those with adverse outcomes. Substantially altered gene profile was detected with 319 increased and 482 reduced transcripts. Elevated SELP mRNA level was confirmed in non-survivors (P<0,05).

Conclusions: Abnormal platelet reactivity may contribute to COVID-19 disease progression and predict clinical outcomes.

Grant: NTP-HHTDK-22-0051

Department of Laboratory Medicine, Semmelweis University, Budapest, Hungary

Apixaban and rivaroxaban are non–vitamin K antagonist oral anticoagulants (NOAC). Optimization of NOAC is often a challenge; therefore, patient adherence to therapy should be monitored. An attracting way could be the collection of capillary blood microsamples that are dried and mailed by patients themselves without medical supervision. Apixaban and rivaroxaban levels were measured with liquid-chromatography tandem mass spectrometry (LC-MS/MS) method. We investigated the optimal extraction procedure of apixaban and rivaroxaban and evaluated various approaches to calibrate microsample assay and optimize its performance. Intra-day precision and accuracy, the hematocrit dependence of analyte recovery, and 1-week benchtop stability of the dried samples were assessed. Apixaban and rivaroxaban concentrations (the number of levels were 15 and 25, respectively) were measured in the dried blood and the plasma samples of patients taking drug. Extraction using acetonitrile-methanol 1:1 yielded the highest recoveries. The application of dried whole blood or dried plasma calibrators were optimal for quantitation. Measurement results were slightly hematocrit dependent. The length of storage period of calibrators dried onto the sampling devices had no impact on the quality of analysis up to one week of storage. Extremely strong linear correlation was found between the concentrations of apixaban, or rivaroxaban measured in dried blood and in plasma (r=0.9825 and r=0.9927, respectively) when dried plasma calibrators were used. Conclusions: a methodology is presented for the monitoring of apixaban or rivaroxaban concentrations from dried blood microsamples. The presented method allows the integration of capillary sampling into the monitoring of patients receiving NOAC therapy.

¹University of Debrecen, Department of Laboratory Medicine, Division of Clinical Laboratory Sciences, Debrecen, Hungary,

²University of Debrecen, Department of Gynecology and Obstetrics, Debrecen, Hungary

Background and aim: Coronavirus disease-19 (COVID-19) is associated with disturbed coagulation and fibrinolysis balance. We aimed to investigate COVID-19-associated fibrinolysis alterations in third trimester pregnancies and their associations with the clinical course and post-partum hemostasis events.

Methods: In this observational case-control study, 100 women with acute COVID-19 infection at 24-40 gestational weeks (COVID-19+ group) and 95 healthy age and gestational week matched pregnant women (COVID-19- group) were enrolled. All women were outpatients with mild/no symptoms at admission. Acute infection was confirmed/ruled out using SARS-CoV-2 RT-PCR and/or antigen test. In addition to screening tests of coagulation, a comprehensive set of fibrinolysis markers including D-dimer, plasminogen activity, α2-plasmin inhibitor (α2PI) activity, FXIII activity and FXIII-A₂B₂ antigen, plasminogen activator inhibitor-1 (PAI-1) activity and antigen levels, in vitro clot-lysis were measured. Detailed clinical parameters of pregnancy, labor and post-partum period were registered.

Results: Clot-lysis times (CLT) were significantly shorter in the COVID-19+ group as compared to controls (50 %CLT median [IQR]: 25 [21-42] min vs. 46 [41-58] min, respectively, p<0,001). A significant decrease in plasminogen activity was observed in the COVID-19+ group compared to control pregnancies (COVID-19+: 162 [IQR:143-190] % and COVID-19-: 174 [IQR:164-197] %, p= 0.002). In case of more severe COVID-19 (stage 2 disease), FXIII levels and plasminogen activity showed a significant decrease as compared to mild cases (stage 1 disease). Fibrinogen, D-dimer, PAI-1 activity, and antigen levels did not differ between the groups. In the COVID-19+ group, postpartum hemorrhage (PPH) developed in 4 cases, associated with significantly reduced plasminogen and α2PI levels as compared to those without PPH. Thrombotic events did not occur in either group.

Conclusions: In this cohort, third trimester COVID-19+ pregnancies were associated with marked hemostasis alterations and hyperfibrinolysis. In patients with PPH, reduced plasminogen and α2PI levels were observed.

Funding: NKFI TKP2021 EGA19

Central Laboratory of Szent Erzsébet Hospital, Jászberény, Hungary

I examined the respiratory tract and blood culture samples of patients suffering from COVID-19 and requiring inpatient care at Jászberény Szent Erzsébet Hospital in 2021. The results were compared to those in 2019 when SARS-CoV-2 was not yet present. The number of respiratory samples was similar, but the rate of lower respiratory tract samples among respiratory tract samples was higher in 2021. The number of blood culture tests was 148.18 % higher in 2021 than in 2019.

An increased proportion of Candida albicans and Stenotrophomonas maltophilia strains was identified in the respiratory tract samples. The number and proportion of pathogens cultured from the blood culture samples also showed a difference compared to the test year used as a control. In 2021, the rate of coagulase-negative Staphylococcus continued to rise; this pathogen accounted for 68.8 % of strains cultured in anaerobic blood culture bottles. We also isolated Candida albicans from the aerobic blood cultures bottles, which we had not experienced before. Smaller numbers of other strains appeared as novelties, but the number of other Gram-negative pathogens decreased or did not grow at all in COVID-19 patients.

¹University of Debrecen, Faculty of Medicine, Division of Clinical Laboratory Sciences, Debrecen, Hungary,

²University of Debrecen, Faculty of Medicine, Department of Pediatrics, Debrecen, Hungary

Background. Inflammatory bowel diseases (IBD; Crohn’s disease: CD and ulcerative colitis: UC) are associated with higher thrombotic risk and enhanced thrombin generation (TG) in adults. Case reports indicated that adverse events, e.g., IBD flare, might be more common in children after SARS-CoV-2 mRNA vaccination.

Aims. To find out whether the balance of hemostasis, as observed by TG, is altered in children with IBD as compared to healthy controls. To find out whether changes in TG occur in children after SARS-CoV-2 mRNA vaccination.

Methods. In this observational case-control study, 37 children with IBD (CD:16, UC: 21) aged 12-18 years and 55 healthy age-matched children were enrolled. Blood was collected before and 2-4 weeks after the second dose of BNT162b2 (Pfizer-BioNTech) vaccine dose. Whole blood count, fibrinogen, inflammatory markers (CRP, ferritin), anti-SARS-CoV-2 antibody levels were investigated, TG assay was carried-out using platelet-poor plasma. Lag time, endogen thrombin potential (ETP), peak thrombin, time-to-peak were calculated. Detailed clinical parameters including post-vaccination symptoms, COVID-19 history, disease activity scores (PUCAI, Mayo score, PCDAI) were registered.

Results. CRP was significantly elevated in children with IBD and showed a positive correlation with ETP (CD: r=0.700; p=0.003 and UC: r=0.501; p=0.020). TG parameters did not differ between patients and controls pre- or post-vaccination. TG parameters remained unaltered post-vaccination in both groups. IBD disease flare was not observed post-vaccination but reduced anti-SARS-CoV-2 antibody titers were found in 3 patients receiving immunosuppressive therapies. Previous COVID-19 infection had no effect on TG levels.

Conclusions. Although TG parameters correlated with the level of inflammation in children with IBD, the extent of TG was not significantly different from healthy controls. TG parameters and IBD disease activity scores did not increase significantly following mRNA vaccination. Our results support the safety of SARS-CoV-2 mRNA vaccination in children with IBD, highlighting observations of lower antibody titers in immunosuppressed children.

Funding: NKFI FK128582

¹Semmelweis University, Department of Laboratory Medicine, Budapest, Hungary,

²Semmelweis University, Department of Pediatrics, Budapest, Hungary

IBD is a collective term for inflammatory bowel diseases including ulcerous colitis (UC) and Crohn’s disease (CD). The complex therapy of IBD extends from lifestyle modification to medical therapy and surgery. Medical therapy includes the administration of immunosuppressants, aminosalicylates, steroid and, in an increasing number of patients, biological agents.

The therapeutic drug monitoring (TDM) services for the first-line adalimumab / infliximab therapies and second line vedolizumab / ustekinumab therapies are available at the Department of Laboratory Medicine, Semmelweis University since mid-2020 and mid-2022, respectively. Due to the immunogenicity of these therapies and related loss of efficacy each TDM tests is complemented by measurements of levels of antibodies generated against these agents. The measurment of active drug and antibody levels is offered in a regular basis with 1 week of turn-around-time. The service is QC-controlled by external QC programme CTCB (Toulouse Center for Quality Control in Clinical Biology).

During the period 2020 and 2022 we analyzed about 300 infliximab 200 adalimumab 50 vedolizumab and 50 ustekinumab samples with our CE-IVD ELISA methods respectively. In many patients had several measurement points. Our main results show that in case of infliximab there is a higher chance of low drug trough-levels (<0,3 µg/ml) and high antibody levels (>10 ng/ml) compared to the other drugs.

In my presentation, I would like to present the background and results of measuring the levels of biological therapeutic drugs used in IBD.

¹ Semmelweis University, Department of Laboratory Medicine, Budapest, Hungary,

² Uzsoki Street Hospital, Central Department of Anaesthesiology and Intensive Care, Budapest, Hungary

Piperacillin/tazobactam is the combination of a broad-spectrum beta-lactam antibiotic and a beta-lactamase inhibitor administered to critically ill pneumonia patients empirically as an intravenous infusion in the initial phase of intensive care. To facilitate successful therapy, piperacillin/tazobactam treatment would require careful pharmacokinetic-pharmacodynamic planning and the continuous monitoring of drug concentrations due to the pharmacokinetic instability of these patients. One- and two-compartment nonparametric population pharmacokinetic models were constructed by evaluating piperacillin concentrations in 72 serum samples obtained from 12 adult patients. Seventeen clinical laboratory parameters were tested as candidate model covariates. The performance of the experimentally established one- and two-compartment models was similar, but there were significant differences regarding model selection criteria. Creatinine clearance displayed strong correlation with the elimination rate constant, nevertheless, its inclusion as a covariate did not improve the performance of either model. Based on these findings, several in silico population models were generated by assigning random values to the pharmacokinetic parameters within the experimentally defined parameter ranges. The performance of the in silico models was comparable in terms of the agreement of predicted and observed piperacillin concentrations, as well as model bias and imprecision, to that of the experimentally established population models. The results demonstrate that in silico models may offer a useful alternative to population models which do not yield acceptable concentration estimates for individuals or for a certain set of patients.

Semmelweis University, Department of Laboratory Medicine, Budapest, Hungary

The value of supporting antibiotic treatment at intensive care units bytherapeutic drug monitoring (TDM) is increasingly recognized. Apart from aminoglycosides and glycopeptides, high-performance liquid chromatography-ultraviolet light absorbance detection (HPLC-UV) is currently the primary tool of analysis, which limits the availability of assays to five beta-lactams and linezolid. Our aim was to develop and validate an analytical method relying on liquid chromatography-mass spectrometry (LC-MS/MS), and to extend assay capabilities to further beta-lactams and to beta-lactamases. The presented approach allows the high-throughput quantitation of ampicillin, cefepim, ceftazidime, ceftriaxone, linezolid, meropenem, piperacillin, sulbactam and tazobactam. All validation criteria were met. The preanalytical stability of these antibiotics in the patient samples is of primary concern, therefore, multiple candidate preservative substances were tested. The cross-validation of the performance of the LC-MS/MS method with that of a CE-IVD certified reagent kit relying on HPLC-UV was accomplished for linezolid, meropenem and piperacillin. LC-MS/MS offers high throughput, flexibility in terms of the set of analytes, and high analyte selectivity. Beta-lactamases can be assayed reliably, an option not provided by the HPLC-based CE-IVD reagent kit. The handling of samples in the preanalytical phase, as well as sample logistics, are of key importance. As new beta-lactams, beta-lactamases and oxazolidinone antibiotics continue to become available, efficient TDM methods are increasingly needed for guiding the antimicrobial therapy of critically ill patients.

Division of Clinical Physiology, Department of Cardiology, Faculty of Medicine, University of Debrecen, Debrecen, Hungary

Sarcoidosis is a granulomatous inflammatory disease, which can lead to serious pulmonary complications. Determination of the usually elevated angiotensin-converting enzyme (ACE) activity is important in establishing the diagnosis and in assessing the success of treatment. However, ACE inhibitor drugs (ACEI) can significantly reduce ACE activity, influencing postanalytical evaluation, diagnostic and treatment decisions.

The aim of the study was to set up an analytical method that can detect the presence of any ACEI in the sample, and to investigate to what extent the requested ACE activity measurements were influenced by taken medication, and how this affected clinical decision making.

In this study, the results of fluorescent kinetic ACE activity assay were analyzed of patients who had diagnostic ACE activity measurements between 2014 and 2021 in Debrecen, Hungary.

A total of 1853 diagnostic measurements were performed during the study period, of which 30 patients’ results were not evaluated due to missing data. In 302 (17 %) cases ACEI effect could be observed, resulting a significant decrease in serum ACE activity compared to patients not taking ACEI (median [interquartile range]: 4.42 [2.93-6.75] U/L; 1.32 [8.79-13.92] U/L; p<0.01, respectively). 83 % of patients with results below the reference range (RR) would fall at least in the RR, while 43 % of patients with results in the RR would have a value above RR if they were not taking ACEI. Thus, sarcoidosis in at least 61 patients may not have been detected in time or at all due to ACEI treatment during the study period. Physicians associated low ACE activity with ACEI treatment in only 3 cases.

With this analytical method, the misleading presence of ACEI can be highlighted to the physician, helping to ensure proper interpretation of results and decision making. This can significantly reduce the time and cost, as well as increase the efficiency of establishing the diagnosis of sarcoidosis.

Diagnosticum Zrt., Budapest, Hungary

The future of laboratory medicine is continuous innovation. The objective of the presentation is to demonstrate the Atellica CI 1900 Analyzer of Siemens Healthineers, latest member of Atellica Solution family. The Atellica CI 1900 is designed as a cost-effective integrated analyzer for low- and mid-volume labs, which can use the instruments in a hub-and-spoke setting in which the hub laboratories are running the high-volume Atellica Solution instruments. Atellica CI 1900 Analyzer is independently integrating clinical chemistry and immunoassay testing under a single platform which operate as standalone. The system is standardized to the core laboratory platform, the Atellica Solution with the same comprehensive menu, technology, assay methodology, user interface, workflow, reagents, and consumables – so you can scale capabilities where and when you need them. Additionally, all offered in a formidable footprint of 1.9 meters squared. Atellica CI 1900 Analyzer is specifically developed to harnessing the power of Atellica Solution in a smaller footprint, bring world-class diagnostic capabilities into smaller laboratory environments and provide clinical equivalence across your network.

Emerging Markets Europe Abbott Molecular

The lecture “HPV Primary Screening – Europe Experience” gives insight into the problematic screening programs for cervical cancer elimination. The overview of the epidemiology and mortality of this disease and the prevalence of HPV infection underlying the importance of these screening programs. Because persistent infection with oncogenic HPV genotypes is responsible for more than 95 % of cervical cancers, we bring the overview of how the prevention programs established in European countries reflect this fact and implement HPV testing. There is the latest evidence and recommendations for HPV-based primary screening, including criteria and guidelines, which the HPV PCR test must fulfill to be clinically validated for primary screening. In the final part, Abbott’s fully automated solution for HPV PCR testing is discussed and the impact of this automation on the laboratory workflow is demonstrated.

¹Biological Research Centre, Institute of Genetics, Szeged, Hungary,

²Delta Bio 2000 Ltd, Szeged, Hungary

Metagenomics is continuously improving in both quality and quantity of data available. Based on over 2000 gut metagenomic samples analyzed, our laboratory established a standard sample analysis pipeline in which bioinformatics plays a key role. While several classifiers exist that apply various methods for classification, we were able to show that these tools provide incongruent results and are often inaccurate. To address this, we propose a new classifier, NABAS+, which improves species classification and reduces false positives. We evaluated the performance of our classifier by creating 30 NGS in silico runs from diverse mock microbial communities with exactly known compositions and compared them to industry-leading classifiers. We found that NABAS+ demonstrated superior classification accuracy and reduced false positives, while not requiring high computational power. Furthermore, we demonstrated substantial differences between the classifiers, showing that popular ones like DIAMOND, Kaiju, and CLARK identify a high number of false positive species, whereas classifiers utilizing smaller, filtered reference databases or species-specific marker sequences, such as MetaPhlAn3 and Kraken2, produce a lower rate of false positives. In our comparative analysis, we show how the most widely used algorithms fail to accurately identify bacteria and describe a classifier, NABAS+, which can accurately identify bacteria in low abundance even with the best accuracy. We can use this new classifier to accurately classify metagenomic samples and utilize the mock metagenomic data as a reliable benchmarking data source for microbiome studies in the future.

¹Delta Bio 2000 Kft., Szeged, Hungary,

²European Life Technologies Hungary Kft., Szeged, Hungary

³Albert Szent-Györgyi Health Centre, Department of Pathology, Szeged, Hungary,

⁴Albert Szent-Györgyi Health Centre, Department of Oncology, Szeged, Hungary

Tumour is characterised by a high degree of genetic heterogeneity already at the time of diagnosis. Subsequently, the therapy used exert selection pressure, with the consequence that therapy-resistant subclones become predominant. Liquid biopsy based Next Generation Sequencing is one of the best tools to regularly monitor the tumor for new potentional molecular targets. ESR1 mutation is a marker of aromatase resistance in ER+/HER2- advanced or metastatic breast cancer. The PADA-1 clinical trial of Elacestrant, a novel SERD that degrades the Estrogen Receptor alpha and inhibits estradiol-dependent ER-directed gene transcription and tumor growth including those harboring ESR1 mutation, has shown that the detection of therapy-resistant subclones before clinical progression can be clinically useful, by basing the treatment strategy on the ESR1 mutation detected in circulating DNA.

Our laboratory, in collaboration with the Department of Pathology and Oncotherapy of the Albert Szent-Györgyi Health Centre has developed a framework for the clinical use of liquid biopsy in the routine care. BC-monitor, our breast cancer-specific ctDNA diagnostic tool is focusing on the most relevant genetic alterations that guide targeted treatment, or underlie the most frequent resistance cases, like ESR1 gain. Processing more than 300 clinical follow up samples, we detected ESR1 oncogenic mutations in more than 30 cases before or during Aromatase Inhibitor treatment. Taken together, ctDNA monitoring during treatment and follow-up with our liquid biopsy tool can predict disease progression four–six months earlier than conventional methods, that has high competence in clarifying preclinical metastases, relapses, and therapeutic resistance.

University of Pécs, Medical School, Dept. of Laboratory Medicine, Pécs, Hungary.

Previously we have demonstrated that the relationship between plasma glucose levels and Hemoglobin A1c (HbA1c) is accurately described by the Michaelis-Menten equation. Using this equation, glycemic control and personalized monitoring can be significantly improved. The aim in our present study was to further improve HbA1c prediction by considering red blood cell parameters as well.

Anemia, especially iron deficiency anemia has been a concern when interpreting HbA1c results, however neither the impact was quantified, nor the exact mechanisms identified. Therefore, we have analyzed plasma glucose, HgbA1c and CBC values from 207.050 simultaneous tests from the past 15 years.

We have found that average HbA1c levels were directly proportional to RBC and Hgb levels of the patients, but inversely proportional to MCV and MCH levels. Interestingly, there was no “stable” zone of CBC parameters where HbA1c levels were unaffected, i.e., the linear or inverse linear relationships continued in the normal reference ranges. Overall, approx. 1.5 % difference of HbA1c was detected between the 2 extremes. Moreover, follow-up of patients with multiple, subsequent laboratory visits revealed that when their MCV values increased by at least 5-10 fL between visits, the HbA1c levels were ∼0.5 % lower than expected.

Our results were generated from a significantly larger database than contemporary studies and may help to better understand the regulation of RBC turn-over. Also, fine-tuning HbA1c interpretation by CBC values may improve the daily monitoring and therapy of diabetic patients.

¹Department of Laboratory Medicine, Faculty of Medicine, University of Debrecen, Debrecen, Hungary

²Institute of Internal Medicine, Faculty of Medicine, University of Debrecen, Debrecen, Hungary

Myeloperoxidase (MPO), a myeloid cell-specific heme-containing peroxidase, is involved in the destruction of foreign cells and microbes. MPO deficiency can be primary or secondary and can be detected by measuring the protein level or by determining MPO activity.

In our case study we present an 82-year-old male patient who was diagnosed with essential thrombocythemia and a partial MPO deficiency. The partial MPO deficiency was observed in the patient based on the Siemens Advia Hematology analyser results. Due to the partial MPO deficiency, the analyser classified a subset of myeloid cells in the large unstained cell (LUC) gate. The patient first received Litalir therapy, because of the high count of platelets (PLT: 1005 x 10⁹ L). After Litalir therapy, the platelet counts normalized, but the MPO deficiency persisted. However, after a change of therapy (Thromboreductin) the MPO deficient clone disappeared. For five years, although the patient was hospitalized for infection, MPO deficient myeloids were not detectable. Then the MPO deficient myeloids reappeared, and at the same time the patient’s condition worsened. He required several hospitalisations.

An additional interesting feature of this case is that a rare calreticulin mutation (complex variant: c.1123_1125insdelTGTTT (p.Lys375fs)), previously described only twice, was detected in the patient.

The case suggests that the reporting of MPO deficient myeloid clone on blood test may be a useful prognostic marker in myeloid malignancies. Furthermore, this case may help in the prognostic assessment of this rare calreticulin mutation.

¹Division of Clinical Laboratory Science, Department of Laboratory Medicine,

²Department of Transplantation, Institute of Surgery University of Debrecen, Debrecen, Hungary

The purpose of our research is to corroborate the role of CYP3A enzyme in developing individual medication therapy via measuring medicine levels in patients’ blood samples.

This retrospective analysis studies 15 kidney transplant recipients. We carried out genotyping (CYP3A5, CYP3A4) after isolating DNA and RNA in patient and donor blood samples; we also determined CYP3A4 messenger RNA expression in case of recipients. Tacrolimus blood levels, dosage, and tacrolimus concentration normalized by dose and the body weight (C0/D ratio) were evaluated. The recipients were divided into 2 groups. Those who carry CYP3A5*1 allele (*1/*1 or *1/*3) are CYP3A5 expressors, whereas those who are homozygous for the nonfunctional CYP3A5*3 allele are CYP3A5 nonexpressors. There were 3 patients with functioning CYP3A5 enzyme (patients with CYP3A5*1/*3 genotype) where increased tacrolimus metabolism was expected. Our data show that C₀/D ratio of CYP3A5 nonexpressors was around 3 times higher than of CYP3A5 expressors. Looking at CYP3A4 enzyme, we found 1 patient carried CYP3A4*22/*22 genotype where we expected decreased CYP3A4 expression. This patient had adequate therapy medication levels (9.50 mg/L) despite having received very low dosage of tacrolimus (0.03 mg/weight/d). Our results confirmed the importance of determining CYP status of recipients after a transplant because individual differences were observed in tacrolimus treatment that were partly influenced by CYP status of recipients.

¹Department of Laboratory Medicine, Semmelweis University, Budapest, Hungary,

²Center for Assisted Reproduction, Semmelwei Egyetem, Budapest, Hungary

Immune dysfunction may related to infertility. We provide regular immunological examinations for the Centre for Assisted Reproduction (CAR). We evaluated the prevalence of abnormal results of immunological parameters obtained in 1749 women between October, 2019 and mid-February, 2023.

The number of positive results for different analytes were as follows: anti-C1q antibody (Ab): 139/1464 (9,5 %); antinuclear Ab (>1/80 titre): 210/1481 (14,2 %); Phospholipid Ab (cardiolipin IgG/IgM and Beta2GPI IgG/IgM): 3/72 (4 %); dsDNA Ab: 64/1464 (4,4 %); celiac disease specific Ab (TG IgA/IgG and deamidated gliadin IgA/IgM) 31/1828 (1,7 %); ENA group: SS-A Ab: 32/1480 (2,1 %), SS-B Ab: 22/1480 (1,5 %), Sm Ab: 2/1480 (0,013 %), Scl-70 Ab: 6/1480 (0,04 %), RNP/Sm Ab: 6/1480 (0,04 %) Jo-1 Ab: 0; RF: 49/355 (13,8 %). Among 828 patients, immune phenotype was assessed: natural killer cells (NK) ratio: low: 14 (1,7 %), high: 95 (11,5 %); B lymphocyte ratio: low 32 (3,9 %), high 7 (0,7 %); T lymphocytes: low: 46 (5,6 %), high: 33 (4 %).

We concluded humor and / or cellular immunity is affected in a significant number of CAR patients. These results can be useful for developing diagnostic protocols for other ARC centres.

¹Department of Laboratory Medicine, Semmelweis University, Budapest, Hungary,

²Faculty of Health Sciences, University of Pécs, Pécs, Hungary,

³University of Physical Education, Budapest, Hungary

Professional athletes are regularly exposed to extreme mental stress during training or competitions. Stress activates hypothalamus–hypophysis-–adrenal axis. We investigated the impact of acute extreme mental stress on adrenal, gonadal, and peripheral steroidogenesis in 40 healthy male professional athletes. The subjects were exposed to a simulated armed street assault in a military tactical room. Peripheral blood samples were taken before entering the tactical room (baseline), at the peak load and, after leaving the room, 30 min of restitution. The concentrations of 14 steroid hormones [aldosterone, androstendione, dehydroepiandrosterone, dehydroepiandrosterone-sulfate, 11-deoxycortisol, 21-deoxycortisol, 11-deoxycorticosterone, cortisone, cortisol, corticosterone, 17α-hydroxypregnenolone, 17α-hydroxyprogesterone, testosterone, dihydrotestosterone] were measured in serum using a previously described LC-MS/MS method with minor modifications. The results of the nonparametric univariate statistical analysis showed significant changes regarding the concentrations of 7.2 %, 35.7 %, and 50.0 % of the assayed steroids at baseline vs. restitution, baseline vs. peak, and peak vs. restitutiton, respectively. The concentrations of androstendione, dehydroepiandrosterone-sulfate, 11-deoxycortisol and testosterone increased significantly from baseline to peak and decreased significantly from peak to restitution. Multivariate statistical analysis did not reveal, however, isolated changes in hormone concentrations. The detected changes were remarkably different from those seen in a vita maxima physical exercise described by our research team previously. Conclusions: significant changes in the steroid hormone concentrations were detected between the various phases except for aldosterone, dehydroepiandrosterone and 17α-hydroxyprogesterone. The overall alterations in the steroid levels as a result of mental stress were not as remarkable as those published during extreme physical exercise.

¹Clinical Chemistry and Immunology Laboratories, Synlab Diagnostic Centre, Budapest,

²Faculty of Health Science, University of Pecs,

³Department of General Internal Medicine, Markusovszky University Teaching Hospital Szombathely,

⁴Department of General Internal Medicine of Javorszky Hospital, Vác, Hungary

Interpretation of thyroid hormone levels when do not fit the clinical picture and contradict the feed-back mechanism often causes confusion. These are mainly caused by substances in the sample that interfere with the analysis. Their recognition is only possible with the cooperation of clinical and laboratory specialists. As an example of this, the authors present a case. Methods: measurements of TSH, thyreoid hormones in native and PEG treated sera, TPO-Ab, TRAK measured on Siemens, Roche and Beckman system. Additional tests: ELFO, paraprotein, RF, IgG. Analysis of the dilution test compared to the dilution results of the serum of 5 hyperthyroid patients, which served as a control for dilution analysis. SHBG was measured by Roche system. Patient: 46-year-old man, diagnosed 7 years ago, with normal TSH (1.79 mIU/L) and high free thyroid hormone levels (average of 8 measurements by four methods: fT4=87.4 pmol/L; fT3=8.7 pmol/L). He was clinically euthyroid. After excluding sample exchange, results with four methods using different antibodies and measurement technics were consistent with the previous, both in native and PEG-treated serum. SHBG level (31 nmol/L) was in reference range, which confirms pseudo-hyperthyroidism. Result of the dilution test (mutant albumin’s affinity for T4 is 60 times stronger!) draws attention to the presence of a protein with abnormal binding capacity in the sample. Family screening with biochemistry was performed: his brother and parents have normal hormones. Genetic tests are in progress. Based on the findings so far, a diagnosis of dysalbuminemic hyperthyroxinemia arises, the prevalence of which is 0.01 %-1.8 %.

Hospitaller Order of Saint John of God, Hospital of Buda, Budapest, Hungary

ISO 15189:2022 specifies requirements for quality and competence in medical laboratories. This standard contains requirements for the medical laboratory to plan and implement actions to address risks and opportunities for improvement. In the laboratory, monitoring the pre-analytical, analytical, and post-analytical processes and meeting the documentation requirements is a serious challenge. It is no longer possible to meet this with traditional means.

The manufacturers’ digital solution helps to allow lab to identify, address and solve your lab’s biggest challenges, using a single, easy-to-use solution. The eLab needs digital solution empowers team with software that consolidates the lab data onto one platform. The eWorkflow engine integrates the entire sample flow, from pre- to post-analytics on a single lab software solution. eAnalytics provides an easier way to track, review, and identify operational trends and challenges that can reduce waste, increase efficiency, and drive financial and operational value. Gathering, organizing, and analyzing testing volumes, lab efficiency, and trends can be time-consuming and can also lead to delays and inaccuracies from manual data gathering. eOrder lets lab easily monitor the progress of the orders, without the need to call a customer service team. eLabdoc up-to-date paperless product information available any time. Customers will be informed about new or modified technical documents according to their preferences. eService provides interactive user documentation and guidance to assist the user in his daily routine operating Online support offers end-to-end issue management in a digital logbook, faster support. eLearning delivers high-quality trainings to customers to achieve operational safety for laboratories, operators, and patients.

University of Szeged Albert Szent-Györgyi Health Centre, Institute of Laboratory Medicine, Szeged, Hungary

The activated protein C resistance (APC-R) is the most frequent hereditary defect associated with deep vein thrombosis. Over 95 % of APC-R phenotype can be explained by the Factor V Leiden (FVL) mutation. This defect is caused by point mutation in the FV gene. For identifying the phenotype expression of the defect there are plasma based functional assays. Our aim was to compare two different types of APC-R functional methods. The Pefakit^® APC-R FVL (Pentapharm) is a plasma-based functional clotting assay and differs from other functional APC-R test by acting specially at the prothrombinase comlex level. FV activator from Daboia russelli snake venom added to sample plasma, to convert FV into FVa. The clotting times (CT) are recorded and as a final result ratio (normal cut-off: >2) is calculated. In the STA-Staclot^® APC-R (Diagnostica Stago) system the principle of the assessment is based on an unusual small prolongation of the CT of the tested plasma in the presence of APC and in calcified medium. Coagulation of the diluted test plasma is achieved in the presence of FV deficient plasma and of Crotalus viridis helleri venom. This venom acts as an activator of FX and therefore triggers the coagulation cascade downstream from FX. In this assay the unit is expressed in second (normal cut-off: >120 sec). The comparative measurements of the two methods were done on 96 plasma samples and confirmed all by genetic tests. Technically the measurement by the STA-STACLOT^® kit is faster. We found correspondence in the case of the Pefakit kit in 98 % and in the case of the STA-STACLOT kit in 87 % at the FVL genotype determinated by PCR. The negative functional test results which are near to the cut-off value always should be confirmed with PCR (Pefakit ratio: 2-3; STACLOT CT: 120-180 sec).

SYNLAB Laboratory Székesfehérvár, Székesfehérvár, Hungary

Hepatitis is a common disease worldwide with various possible causes. One of the most common causes of hepatitis is viral infection but several other agents or conditions can play role in the development of that, for example alcohol abuse, drugs, autoimmune diseases. However, acute viral hepatitis is most frequently caused by hepatitis viruses (hepatitis A, B, C, D, and E), hepatitis can also rarely be caused by other viruses, such as Epstein-Barr virus, cytomegalovirus, or others.

In this case study, we present a 73-year-old female patient, who was referred to the hospital by her primary care physician for laboratory testing due to fatigue and loss of appetite. The laboratory test results showed extremely elevated liver enzyme activities (aspartate aminotransferase 2471 U/L, alanine aminotransferase 2575 U/L, gamma-glutamyl-transferase 206 U/L) and lactate dehydrogenase activity was also very high, 5322 U/L. The complete blood count was roughly normal, but a blood smear examination was performed based on morphology flags of the hematological analyzer. Lymphocyte morphology was similar to that often observed in mononucleosis: lots of reactive lymphocytes with abundant mid- or dark blue cytoplasm, more indented nuclear shape lymphocytes with prominent nucleoli.

Due to the marked differences in liver enzyme activity, acute viral hepatitis was suspected, therefore serological tests were also performed that confirmed hepatitis E virus infection (hepatitis E IgM antibodies were positive and additionally reverse transcriptase polymerase chain reaction (RT-PCR) was performed to detect the hepatitis E virus RNA in blood with the result of 5990 IU/mL).

We would like to call attention to that rare viral-induced hepatitis like hepatitis E infection can cause morphological changes in the blood.

¹SYNLAB Laboratory Székesfehérvár,

²Department of Hematology, Szent György Hospital, Székesfehérvár, Hungary

Myelodysplasia (MDS) is a heterogeneous group of clonal hematopoietic stem cell disorders, characterizing by ineffective and dysplastic hematopoiesis, peripheral cytopenia, hyper- or hypocellular bone marrow. MDS may involve one, two, or all three myeloid hematopoiesis cell lineages. Most of the cases myelodysplasia progresses to bone marrow failure, but high risk of transformation to acute myeloid leukemia is also known.

In this case study we present a 55 years-old-female patient, who visited the hematologist because of her persistent macrocytaer anemia. Her hemoglobin concentration and mean corpuscular volume were 79 g/L and 119 fL, respectively. Folic acid, or vitamin B12 deficiency were not shown (folic acid > 45.4 nmol/L, vitamin B12 > 1085 pmol/L). During physical examination normal status was found, but mild hepatosplenomegaly was detected by ultrasound. Blood and bone marrow smear examinations were also performed. Dysplastic differences were seen in all three myeloid cell lineages in the blood: anisocytosis, polychromasia, dacryocytes, pseudo-Pelger cells, hypogranular neutrophils, monocytes with atypical shape of nuclei, some mastocytes, considerable platelet anisocytosis with giant platelets, hypogranular platelets and micromegakaryocytes.

There were elevated number of megakaryocytes in the bone marrow with dysplasia: multiply nuclei, hyperlobulated nuclei. Several mast cells were also found, but systemic mastocytosis by histological examination of the bone marrow was not confirmed. Isolated deletion of chromosome 5q without excess of blasts was identified with cytogenetic analysis. This case study represents that mastocytes can rarely occur both in peripherial blood and in bone marrow in association with hematological neoplasms, such as MDS.

¹Semmelweis University, Department of Laboratory Medicine,

²Semmelweis University, Department of Anesthesiology and Intensive Care, Budapest, Hungary

One of the most challenging tasks of the intensive care units is treating chronically critical ill (CCI) patients. CCI is a condition with prolonged need for mechanical ventilation. The survival and recovery of these patients are jeopardized by altered immunological status.

In our laboratory we perform lymphocyte immune phenotyping on daily basis. Using our existing data classic (CD16-) and non classic and intermediate monocytes (CD16+) immune phenotype populations with flow cytometry were distinguished and related its change to the course of CCI in 3 patients.

We differentiated CD16-positive (CD16(+)) monocyte from CD16-negative populations. CD16(+) populations also show a bit brighter CD45 phenotype. We found that in CCI patients CD16(+) monocyte ratio increased manifold higher than baseline and normalized slowly, with time. Increased ratios were detected in different stages of the of the mechanical ventilation treatment (3. – 14. day). The absolute numbers of CD16+ monocytes were increased with the ratios in one patient, was at baseline in another one and fluctuated in the third one. All patients survived and emitted home.

Our pilot results indicate that monocyte phenotyping could provide an insight into the kinetics of CCI and, possibly, could be used for a better understanding of this condition.

Central Hospital of Southern Pest, National Institute of Hematology and Infectious Diseases, Budapest, Hungary

In case of lymphoproliferative disorders and infectious diseases, cold agglutinins (mostly IgM antibodies) may appear which activate at low temperature and cause red blood cell (RBC) agglutination. To avoid this, peripheral blood samples must be transported to the laboratory for complete blood count at 37 °C and further incubation is needed which takes time. Our aim was to examine whether the optical determination method of RBC parameters of Sysmex XN-9100 – which is carried out at 41 °C in the RET channel – could replace the manual incubation, this way sample procession would be less timeconsuming. We compared the RBC count and mean corpuscular hemoglobin concentration (MCHC) value of fifteen K3-EDTA anticoagulated blood samples with cold agglutinins before incubation using optical method (RBC-O and MCHC-O) and after incubation using the original impedance method (RBC-I and MCHC-I) as reference method. We used linear regression and Bland-Altman analysis to evaluate the results. RBC-I and RBC-O correlated well (r=0.9688, r²=0.9385), from the Bland-Altman, plot, limit of agreement was -0.54-0.22 T/L (95 % confidence interval) with the bias of -0.16. However, MCHC-I and MCHC-O showed no correlation and poor association (r=−0,6414, r²=4114). According to our preliminary study, the incubation of cold agglutinin containing samples can not be omitted and the optical method of determining RBC parameters can only be capable of replacing the determination of RBC counts by the impedance method yet.

North-Pest Center Hospital – Military Hospital, Central Department of Laboratory Diagnostics, Budapest, Hungary

In our laboratory, the most common indications for testing in the hemostasis department are pre-operative examination, monitoring of anticoagulant therapies, processing of samples received from the emergency department. Moreover, the examination and follow-up of patients coming from the National Hemophilia Center, which is operating in our hospital. Screening and special hemostasis tests are performed on Siemens BCS XP devices, using optical method for determination. In 2022, the annual number of tests performed in our department was 120,400 for screening tests (PT; APTT; TT) and 41,952 for other tests (thrombophilia, hemophilia). Due to the collaboration with the National Hemophilia Center in our Hospital, we often come across many rare and interesting cases. Based on the experience gained here, we are more likely to suspect any hemostasis disorder in cases with deviations in routine test results. Prolongation of clotting time cannot predict possible post-operative bleeding in case of a surgery or an invasive intervention. In case of prolongation of PT, APTT, or TT screening, we can recommend further testing to the clinician, including the determination of specific coagulation factor levels, if necessary. In case of a positive result, these tests can contribute to the clinician’s decision to appropriate preparations and treatment, including the prevention of bleeding complications with pre- and post-operative factor substitution administered to the patient. In this work, we demonstrate the importance of hemostasis screening tests through specific cases, including factor-deficient states with prolonged screening tests, fibrinogen deficiency identified during an APC resistance test, and significant aPTT prolongation without bleeding symptoms.

¹SYNLAB Laboratory Kiskunhalas, Kiskunhalas, Hungary,

²SYNLAB Laboratory Szekesfehervar, Székesfehérvár, Hungary^,

During routine laboratory work, extreme hematology results are relatively rare. In such cases, it is important to know the limits of the method (e.g., linearity ranges) and to distinguish false results due to preanalytical errors from truly abnormal ones. In this report, three special cases are presented. 1. Extreme leukopenia: white blood cell count (WBC) of a 48-year-old man was extremely low, 0.05 G/L. The sample was measured also in “low WBC mode” and it was also analyzed a citrate anticoagulated sample of the patient, for WBC count. WBC result was just as low. Finally, a peripheral smear examination was performed to exclude leukoagglutination. 2. Hyperleukocytosis: WBC count of an 18-year-old man was 734.8 G/L for the first time. This result was above the upper limit; therefore, WBC count was also performed in two- and tenfold dilutions of the original sample. WBC results calculated from dilutions were 797.2 G/L and 809.8 G/L. 3. Cold agglutinin disease (CAD) is a relatively rare condition associated with agglutination of erythrocytes in cold environment, resulting in spurious results of red blood cells (RBC), mean cell volume (MCV), mean cell hemoglobin (MCH) and mean cell hemoglobin concentration (MCHC). Initial test results of a 71-year-old man were as follows: RBC 0.03 T/L, MCV 133 fL, but MCH and MCHC were not measurable. After incubation of the sample at 37^oC for one hour the mentioned results were not corrected. As CAD was suspected, the blood sampling was repeated at 37^oC, and blood tests were performed immediately after the sampling. The results showed: RBC 4.45 T/L, MCV 96 fL, MCH 33.3 pg and MCHC 347 g/L. Supplying reliable laboratory results is very important to prevent life-threatening complications and to give adequate therapy at the proper time.

Semmelweis University, Department of Laboratory Medicine, Budapest, Hungary

DIC is a frequent complication of sepsis, affecting up to 20 % of patients. DIC panel including routine blood clotting test such as prothrombin time (PT), activated partial tromboplastin time (APTT), D-dimer levels, fibrinogen levels and platelet count (PLT) is an essential tool for the identification of DIC. In our retrospective database we analyzed whether DIC panel is used for the recognition of DIC

Data of patients with procalcitonin (PCT) values above the reference range and a history of microbiological tests were analyzed in the period between January and February 2023 in a local laboratory serving pediatric patients at the Department of Semmelweis University.

During the analyzed period, routine blood clotting tests were ordered for 173 patients of age between 1 and 18 years. Of these, 15 patients had elevated PCT levels. All 15 patients had levels of dimer D above cutting value. PT and APTT exceeded reference range in 5 and 4 cases, respectively. Fibrinogen, however, was determined just in 4 cases with results in the upper third of the reference range. PLT was below and above the reference range in one and one patient, respectively. Of note, DIC panel was ordered in just for 2 of 15 patients suggestive for sepsis. Of those, one with acute lymphoblastic leukemia exhibited characteristic findings for DIC; the other patient, findings excluded DIC.

This analysis indicates that in patients with severe infection DIC panel is not ordered routinely, despite their elevated risk for DIC. These data encourage a stronger cooperation between laboratory and clinicians treating septic patients.

Department of Laboratory Medicine, University of Szeged, Szeged, Hungary

Fragmented Red Blood Cells (FRC, fragmentocytes, schistocytes) are most seen in cases of mechanical injury of red blood cells, and their detection in peripheral blood is a needful aid for the diagnosis and follow up of primary and secondary thrombotic microangiopathies (TMA), and other types of haemolytic anaemia. Their measurement has been made possible using new generation of haematology analyzers. However, according to literature data, this automated fragmentocyte count is not suitable for reporting since the sensitivity of the measurements is sufficiently high, but the specificity is low generating increased manual blood smear evaluation in our laboratory. The main objective of our research was to increase specificity by setting up an algorithm for the quantitative evaluation of the fragmentocyte count in the peripheral blood, and to compare the FRC values obtained by the haematology analyzer and the values extracted from the digital cell morphology analyzer with those obtained by manual microscopy, and to evaluate possible correlations between them. We used Sysmex XN-20 FRC % value, and we evaluated the percentage of fragmentocytes from peripheral blood smear according to the International Council for Standardization in Haematology (ICSH) Guidelines, using CellaVision Advanced RBC software and by counting of 1000 red blood cells in optical microscopy as a gold standard method.

Based on our obtained data, we set up and present an algorithm for the objective management of fragmentocyte flag generated by the hematology analyzer, and for the reporting of the fragmentocyte count in the peripheral blood smear.

B-A-Z County Hospital, Miskolc, Hungary

The effects of the widely used DOAC therapy on the assessment of standard coagulation tests and thrombophilia tests must be taken into consideration. They can complicate the evaluation of the findings and can hide pathognomonic results causing delay in the diagnostic process. The direct thrombin inhibitor dabigatran can have a prolonging effect on the aPTT and TT even in therapeutic dose¹. The patient in our case presentation took dabigatran at a dose of 150 mg twice a day because of a previous deep venous thrombosis. Despite the anticoagulant therapy, he was hospitalized for pulmonary embolism. Initial coagulation tests showed significantly prolonged aPTT and TT. The patient treatment was changed from dabigatran to therapeutic dose LMWH after admission to hospital. Although thrombophilia testing is not recommended in an ongoing thromboembolic event in this case antithrombin, protein C, protein S, activated protein C resistance and lupus anticoagulant assays were requested. At the time of blood collection, the patient took dabigatran. The result of the lupus anticoagulant assay could be interfered with the effect of dabigatran, so the test was not evaluable. Later the significantly elevated titer of anti-beta2-glycoprotein I antibodies and anticardiolipin antibodies raised the suspicion of antiphospholipid syndrome. Eventually the patient was diagnosed with SLE by immuno-serological assays and clinical findings. Despite of the testing for thrombophilia is not recommended during current thrombotic event and continuing vitamin K antagonist or DOAC therapy we often receive such requests ignoring that the test results may cause uncertainities in the decison - making algorithm.

¹ prescribing information for PRADAXA Capsules.

https://content.boehringer-ingelheim.com/DAM/c669f898-0c4e-45a2-ba55-af1e011fdf63/pradaxa %20capsules-us-pi.pdf

Institute of Laboratory Medicine, Semmelweis University, Budapest, Hungary

The purpose of the poster is to demonstrate the importance of laboratory hemostasis tests performed before, during, and after liver transplantation through a case study.

Hemostasis tests were performed on a 63-year-old man who has undergone a liver transplantation due to alcoholic liver cirrhosis. The measurement results of the samples were followed before and during the transplantation and for 8 post-transplant days.

The examination of prothrombin (PT), APTT, ATIII, fibrinogen, and some factors (FV, FVII, FX, FXIII) was performed on the Siemens 2000 CSi automate with the reagents provided by the manufacturer by clot detection method and measurement of the NADH-NAD conversion.

The following results were obtained: Results measured as for recipient: PT: 19 sec.; APTT: 54.8 sec.; ATIII: 53 %; Fibr.: 1.9g/L; FV: 39 % FVII: 48 %; FX: 36 %; FXIII: 120 %; during the transplantation: PT: 23 sec.; APTT: 72.1 sec.; ATIII: 80 %; Fibr: 2.0 g/L; FV: 27 %; FVII: 31 %; FX: 37 %; FXIII: 119 %. Factor replacement was carried out. During the 8 days after surgery the following changes were detected: FV, FVII, FX changed to 87 %, 22 %, 71 %, FXIII: decreased to 44 %. The PT was initially prolonged, then shortened, and finally stagnated (PT: 20.1 sec.). The value of APTT was prolonged due to regular heparin administration (APTT: 72.1 sec.) and halved on day 8 (APTT: 40.6 sec.) There was no significant change in the fibrinogen results (1-2g/L).

The emergency laboratory hemostasis tests contributed to the fact that thanks to the adequate factor replacement used, there was no need for blood transfusion either in connection with the liver transplantation or in the postoperative period.

¹Hungarian National Blood Transfusion Service, Budapest, Hungary

²Bio-Rad Magyarország Kft., Budapest, Hungary

Blood group antibody titers (ABTs) reported in titer values vary by test method. The introduction of new testing methods, such as automated methods, requires appropriate method comparison. In this study, the IH-500 automated blood bank system using column agglutination (CAT) and conventional manual tube method for ABT of indirect antiglobulin test (IAT) were compared.

Antibody titration was performed using heterozygous red blood cell suspension as the target antigen and a serial twofold dilution of serum by both column and tube methods. ABT was performed with samples from pregnant individuals (n=50, 54 % with anti-D, 24 % with anti-K, 18 % with anti-c and 4 % with anti-E antibodies). Measured titer values were log₂-transformed on the interval scale and plotted for analysis and comparison.

Forty-eight cases had higher titer values with the CAT. The column method had a median 3-fold higher titer than the tube method. The highest differences (more than 3 dilutions higher) were seen in antibodies directed against antigens of RH system. For anti-K antibodies, the CAT titers were equal or slightly higher depending on the sample tested.

Correlation with the CAT and tube method performed on 50 samples was adequate. The procedure of the CAT method was simpler and could be automated but increased the cost of the test.

Log₂-transformed interval scale values for comparison were useful for interpreting method comparison data sets. Titer results were almost always higher with the CAT. Further testing with the CAT for antibody titration on more samples is needed.

Central Hospital of Southern Pest, Budapest, Hungary

The prevalence of hereditary predisposition to cancer is estimated as high as 5-15 % of all malignant disorders, but the knowledge about the occurrence of HHM is lacking. In case of solid tumours, the comparison of the tumour tissue with peripheral blood provides a simple option to exclude somatic variants, on the other hand the contamination with blood cells may hamper evaluation in hematologic malignancies.

In our laboratory, 346 patients with hematologic malignancy (AML, MDS, MPN) were investigated by targeted next generation sequencing (NGS) with genes involved in somatic and germline hematologic malignancies. In a gene with known germline pathogenic variants, the suspicion of heritability was raised by a variant allele frequency of approximately 50 %. NGS results were confirmed by Sanger sequencing. Germline origin could be excluded from remission samples or somatic cells other than the haematopoietic system (e.g., hair follicles of the patient), while the detection of a pathogenic variant in hair follicles or in a family member confirms hereditability. Congenital pathogenic HHM mutations were detected in 7 cases (2 %) in DDX41 (n=3), RUNX1 (n=3), TP53 (n=1). Additional 5 cases are under evaluation (CEBPA, PTPN11, RUNX1, TERT).

Due to unknown penetrance, HHM is an underdiagnosed condition. The detection of HHM is of therapeutic importance because carrier family members must be excluded from the potential hematopoietic stem cell donor pool and require regular medical attention due to increased risk for malignancy.

Central Hospital of Southern Pest, National Institute of Haematology and Infectious Diseases, Budapest, Hungary

We compared two activated partial thromboplastin time (APTT) assays with presumably varying performance characteristics used in different sites of our laboratory.

Altogether 202 samples requested for Lupus Anticoagulant (LA) testing were evaluated. In addition to dRVVT and SCT screening and confirmatory tests in the same standardized preanalytical circumstances we also measured APTT with Dia-PTT reagent in CoagXL analyser and with Hemosil Synthasil reagent in ACLTop analyser. APTT was expressed as ratio of patient to lyophilized normal plasma pool (Normal Pool- Stago) mixing studies were performed above 1,2 ratio. Additional assays were ordered for the samples that tested negative for LA to measure intrinsic factor activities and possibly interfering LMWH level.

From the total of 202 samples 29 had prolonged APTT (14 %). From all LA positive samples (46, 23 % of total) 11 also had APTT ratio >1,2. Moderate correlation was with SCT screen ratios in patients who had prolonged APTT with Synthasil (R2= 0,613), and no correlation with dRVVT screen ratios. Prolonged APTT results with Dia-PTT showed no correlation with any of screening assays for LA.

From all samples with APTT prolongation 18 were negative for LA. Out of 6 cases that showed prolongation with both reagents 2 had borderline factor activity (IX, XII). From 11 patients who had prolongation only with Dia-PTT 5 had mild factor-activity decrease. Levels were close to lower limit of reference range in factors VIII, IX and XI, 4 cases were detected to have moderate FXII deficiency (activity levels 28-35 %). One patient had slightly prolonged APTT with Synthasil only without any factor deficiency identified.

In conclusion our data reinforced the importance of selection and rational utilization of reagents according to clinical relevance.

Synlab Hungary Ltd., Székesfehérvár, Hungary

In cases of suspected deep vein thrombosis (DVT) and pulmonary embolism (PE), the critical point in the clinical evaluation algorithm is the laboratory value of D-Dimer. The negative predictive value (NPV) of D-Dimer is well used below the test-specific decision threshold (cut-off) to exclude acute thromboembolic events. Beside the positive clinical pre-test probability, elevated D-Dimer results may indicate further confirmatory diagnostic imaging tests (e.g., ultrasound, computed tomography (CT)). Based on the principles of cost-effectiveness and patient safety the frequency of the expensive imaging procedures was examined even if the D-Dimer value was below the cut-off level. In this retrospective study we collected D-Dimer results of 178 patients appeared at the emergency department. These samples were measured on BCSXP system using Innovance D-Dimer immuno-turbidimetric assay (Siemens). The cut-off value for the analytical procedure is 0.50 mg/L, and the negative predictive value is 98.6 %. Anamnestic data, diagnostic procedures and final clinical diagnoses of the patients were compared with their D-Dimer results. D-Dimer values above the cut-off level (0.51- >4.20 mg/L) were found in case of 91 patients. At one third of these patients further imaging tests were performed, but only 6 patients were diagnosed with deep venous thrombosis or pulmonary embolism. Despite that D-Dimer values were negative (<0.50 mg/L) in case of 83 patients, 28 additional imaging examinations (ultrasound, CT, or X-ray tests) were performed to rule out thromboembolic events. Based on our findings, D-Dimer values below the cut-off level can be reliably used in the clinical diagnostic algorithm for low-risk thromboembolic events, significantly reducing the frequency of costly imaging procedures, thereby increasing cost-effectiveness and patient care efficiency.

Department of Laboratory Medicine, Faculty of Medicine, University of Debrecen, Debrecen, Hungary

Hematology analyzers with a body fluid (BF) module could potentially simplify daily routine work in BF analysis and could reduce turn-around-time. In this study we evaluated the agreement of cell counting between the manual chamber technique and the Sysmex XN-1000 and XN-2000 analyzers for white blood cell (WBC) and red blood cell (RBC) counts, as well as for leukocyte differentiation: polymorphonuclear (PMN) % and mononuclear (MN) % in different types of fluids. Within-run precision performed with patient cerebrospinal fluid (CSF) samples was excellent (WBC CV: 3-11 %; range: 6-154 cell/µL and RBC CV: 0-10 %; range: 0-41000 cell/µL). Between-run precision with BF-Check control material tested in 20 consecutive days was: WBC mean 297/µL CV 2.6 %; WBC mean 77/µL CV 5.6 %; RBC mean 76 000/µL CV 2.6 %; RBC mean 26 000/µL CV 4.5 %; PMN % mean 72 % CV 1.0 %; MN % mean 28 % CV 2.6 %. The regression equations of WBC and PMN % in the manual and instrumental methods were WBC in CSF y = 1.0026x + 24.548 (R² 0.86); PMN % in CSF y = 1.1121x – 8.8966 (R² 0.86) and in ascitic fluid (AF) 1.079x – 4.4694 (R² 0.95). We could observe weaker correlation in CSF samples with WBC in the lower range (<100/µL) y = 0.609x + 23.559 (R² 0.56), this phenomenon was more pronounced if WBC was <20/µL (y = 0,3834x + 9,0772; R² 0.16) which is in line with the literature. We can conclude that Sysmex XN1000 and XN2000 BF module provides rapid and accurate counts in clinically relevant ranges of CSF and AF values and PMN and MN percentages, thus providing a valuable alternative to conventional light microscopic analysis.

North-Pest Center Hospital – Military Hospital, Central Department of Laboratory Diagnostics, Budapest, Hungary

Chromogranin A (CgA) is a glycoprotein produced by different neuroendocrine cells. Circulating CgA levels were associated with neuroendocrine differentiation and linked to the tumour mass. The Cisbio Bioassays (France) immunoradiometric assay (CGA-RIACT) is a widely used method for the measurement of CgA. Cisbio Bioassays recently developed an enzyme-linked immunosorbent assay (CGA-ELISA-NG), using the same monoclonal antibodies. Our study aimed to evaluate the analytical and clinical performances of the ELISA assay. We compared the CgA values measured with both RIA and ELISA kits of 150 patients and 6 external quality control samples (UK NEQAS, United Kingdom). For result calculations, we used spline fitting model in RIA assay and cubic spline fitting model in ELISA assay, because the recommended four-parameter logistic 4PL with 1/y² weight fitting model was not available on the ELISA reader (Multiskan EX). With the RIA kit, serum levels of CgA ranged from 11.94 to 23,958.50 ng/mL. 97 patients had normal levels, and 53 had elevated levels (cut-off: 98.1 ng/mL, measuring range 1.5-1,050 ng/mL). With the ELISA kit, serum levels of CgA ranged from 7.81 to 30,287.00 ng/mL, where 80 patients had normal levels, and 70 had elevated levels (cut-off: 101 ng/mL, measuring range 30.6-1,000 ng/mL). Using a linear regression analysis, there was a significant correlation between the CGA-RIACT and CGA-ELISA-NG kits. The limit of detection (LoD) was higher on CGA-ELISA-NG than on CGA-RIACT, which resulted in a higher standard deviation in the lower concentration range of CgA. Switching to a new method requires setting up new baseline values for follow-up patients and information about change in method should be marked on the patient report.

Central Laboratory, Institute of Laboratory Medicine, Semmelweis University, Budapest, Hungary

The determination of total bile acide (TBA) concentration in pregnancy is of importance in the early evaluation of intrahepatic cholestasis of pregnancy (ICP). ICP is a severe pregnancy-associated hepatic disorder with risks of spontaneous and iatrogenic preterm delivery as well as stillbirth.

We introduced the measurement of serum total bile acid (TBA) levels in March of 2022. Since then, 534 samples were analyzed until March 2023. Obstetrics, pregnancy outpatient clinic, other outpatient clinics referred 128, 72 and 118 samples, respectively. External referring institutes sent 216 samples.

For the determination of TBA a colorimetric method was used (DiaSys) on an Atellica automated chemical analyser (Siemens). The recommended reference range of TBA is < 10 µmol/L. However, this reference range is likely unestablished in asymptomatic pregnant women as TBA values in non-fasting state ranged between 9.34 and 21.25 umol/L; TBA levels of 83,3 % of patients ranged between 1,4-17,28 umol/L. These indicate a need for the establishment of an own reference range for each laboratory.

Central Hospital of Southern Pest, National Institute of Hematology and Infectious Diseases, Central Laboratory, Budapest, Hungary

Introduction: During inflammation, in addition to C-reactive protein (CRP), the production of serum amyloid-A (SSA) also increases. In severe infections, within a few hours, the increase in SSA can be 1000 times the initial concentration. Procalcitonin indicates a serious bacterial or fungal infection. SuPAR is an early prognostic factor for severe infections. Ferritin acts as an acute phase marker. IL-6 reacts quickly and sensitively in COVID-19 due to its short half-life. Aim: Through case reports, we aimed to demonstrate the signaling value of inflammatory markers in severe, septic diseases. Patients and methods: For several weeks, we followed the alterations of these markers in 28 patients with severe COVID-19 infection. Among them, we present 4 cases where we compared the SAA/CRP, SAA/Ferritin, SAA/Pct and SAA/suPAR ratios in each individual.

Results: The changes in inflammatory markers in septic patients over time are influenced by several factors. Initial marker levels may have a prognostic value. Using Pearson correlation, SAA/CRP and SAA/Ferritin showed a significant correlation in our patients (correlation coefficient r in the 1st patient was: 0.91 vs. 0.92; in the 2nd patient: 0.99 vs. 0.93; in the 3rd: 0.99 vs. 0.94 and in the 4th patient: 0.97 vs. 0.99 respectively). The SAA/Pct and SAA/suPAR ratios had low correlations among our patients. This may be related to other infections occurring during care, different underlying conditions, and different treatment protocols among our patients.

Conclusion: Calculating the SAA/CRP and SAA/Ferritin ratios could help in defining the true level of inflammation. Supported by other publications, SAA levels can indicate the outcome of a disease earlier than CRP.

¹Central Hospital of Southern Pest National Institute of Hematology and Infectious Diseases, Central Laboratory, Budapest, Hungary

²Heathware Consulting Ltd, Budapest, Hungary

Background: soluble urokinase plasminogen activator receptor (suPAR) is a prognostic inflammatory marker in numerous immune mediated diseases. In COVID-19, high suPAR levels are associated with prolonged hospitalization and more severe patient outcome. Our aim was to study the prognostic value of suPAR during intensive care in comparison with other laboratory markers. Patients and methods: Blood samples were collected from 42 COVID-19 patients at 8-15 different time points throughout the nursing time (448 measurements altogether). SuPAR and other, commonly used laboratory markers (i.e., IL-6, CRP, Pct, ferritin, Ddimer, blood count, troponin, NT-proBNP) were evaluated.

Results: suPAR correlated moderately with only creatinine and NT-proBNP (Pearson correlation coefficient r=0.68 and 0.64 respectively). Random forest regression indicates that high suPAR levels are independent and early predictors of severe COVID-19 outcome. During the observed 4-week period in intensive care, suPAR levels were constantly higher in later deceased patients compared to those who survived. For most patients, suPAR levels rose continuously for 3 weeks, while we observed a decline in the 4^th week. In discharged patients, lower suPAR levels were associated with higher risk of complications and death.

Conclusion: Our data suggest, that suPAR is a useful marker for the monitoring of COVID-19 patients. SuPAR can be used as a prognostic marker, as lower levels in the early stages of COVID-19 indicate lower mortality, while on discharge a low suPAR level could be an indication of a depleted immune response and therefore a potentially more severe outcome.

¹Department of Laboratory Medicine, Semmelweis University, Budapest, Hungary,

²Department of Pediatrics, Semmelweis University, Budapest, Hungary

Aspartic acid (ASA) is a one of the 20 protein-forming amino-acids. ASA is essential for the survival of proliferating cells. In acute lymphoblastic leukemia (ALL) there is an excess of ASA, because of lack of ASA-metabolizing asparagine synthetase enzyme. Hydrolysis of ASA by exogenously administered asparaginase (ASPase) enzyme results in a dramatic decrease of intracellular protein synthesis. As healthy cells are able to synthetise ASA, ASA deficiency affects selectively lymphoblasts that undergo apoptosis.

Therefore, ASPase therapy is fundamental in ALL. In Hungary 2 types of ASPase are available. However, these drugs can cause severe allergic reactions in some cases. Furthermore, they can also lose their effect without overt clinical symptoms. This phenomenon called silent inactivation can only be detected by therapeutic drug level monitoring (TDM) of ASPase.

In our institute, the determination of the ASPase enzyme activity with a RUO ELISA method is available since 2021. Between 2019 and 2021 we used a CE-IVD ELISA method that was discontinued by the manufacturer. During this period, we have analyzed about 700 samples. Each patient had several measurement points. Based on our results less than 5 percent of patients developed a loss of effectiveness due to an allergic reaction or silent inactivation.

The purpose of this poster is to present the background of the investigations as well as to present our experience with measurements.

¹Békés County Central Hospital, Pándy Kálmán Branch, Central Laboratory,

³Department of Cardiology, Gyula;

²Town Hospital, Orosháza, Hungary

According to the 2019 dyslipidaemia guidelines, the target LDL-C level of acute coronary syndrome (ACS) patients was decreased from 1.8 mmol/l to 1.4 mmol/l.

The most ideal method for determination of the LDL-C is the direct measurement by the enzymatic assay, however in our country it is not widely available. In everyday practice to calculate LDL-C levels Friedewald equation was used for decades (when triglyceride levels were below 4.5 mmol/l). Recent evidence suggests there is a significant difference in LDL-C levels calculated with this formula versus the direct method, and Martin-Hopkins estimation of the LDL-C proved to be more accurate and became popular.

We conducted a single-center retrospective observational study of patients who had ACS between 1^st April 2020 and 31^st March 2021. Their LDL-C was calculated on the onset of ACS, and 6 months and 1 year later according to Friedewald (LDL-F) and Martin-Hopkins (LDL-MH). LDL-F was then compared to LDL-MH.

In the period of one year there were 531 patients underwent percutaneous coronary intervention because of ACS, 312 males (58.8 %) 219 females (41.2 %). The mean age (±SD) was 66.9±12.2 and 72.4±11.2, respectively. The median (±IQR) levels at the event, at 6 and 12 months were 3.34 (2.35-4.29), 1.65 (1.01-2.26), 1.59 (1.16-2.19) mmol/l using the Friedewald formula, and 3.32 (2.35-4.27), 1.92 (1.33-2.37), 1.73 (1.36-2.43) mmol/l with Martin-Hopkins estimation, respectively. Compared with the LDL-F, the LDL-MH difference was -0.5 % at the time of ACS event, +16 % at 6, +9 % at 12 months later (statistically significant differences at 6 (p=0.044) and at 12 months (p=0,014).

The results suggest that the considerable difference between the widely used LDL-F and more accurate LDL-MH might negatively influence the actual LDL-C target level attainment. It would be best if the direct LDL method became common, but in the meantime, it would be advisable to use LDL-MH because of its accuracy and reliability.

Department of Laboratory Medicine, Faculty of Medicine, University of Debrecen, Debrecen, Hungary

Aims: We retrospectively analyzed serum level of human epididymis protein 4 (HE4) as a lung-derived inflammatory biomarker in severe COVID-19 patients in association with disease severity and outcome.

Methods: Eighty severe COVID-19 patients (40 critically ill and 40 severely ill) and as controls 25 age- and sex-matched non-COVID-19 sepsis subjects were included. Serum HE4 was measured by an immunoassay (Architect i1000SR, Abbott) in the baseline samples of all study participants obtained at ICU admission and 30 follow-up sera within the COVID-19 cohort. Associations were studied between HE4, routinely available laboratory parameters and clinical characteristics.

Results: Initial HE4 level was significantly higher (p<0.0001) in critically ill COVID-19 patients (524.7 [300.1-1153.0] pmol/L) than in severe COVID-19 subjects (157.4 [85.2-336.9] pmol/L), while similarly high HE4 concentrations were found in non-COVID-19 sepsis conditions (1118.0 [418.3-1953.0] pmol/L, p=0.056). Serum HE4 levels significantly correlated with age, SOFA-score, inflammation-dependent biomarkers, and the degree of lung manifestation evaluated by chest CT. Based on RUC-AUC curve analysis and logistic regression analysis, baseline HE4 independently indicated the severity of COVID-19 with an AUC value of 0.816 (95 % CI [0.723-0.908]; p<0.0001). Furthermore, non-survivors showed even higher baseline HE4 levels without a substantial change under treatment vs survivors (p<0.0001). Finally, high HE4 level (≥331.7 pmol/L) predicted a larger risk for mortality (Log-Rank p<0.0001).

Conclusions: Elevated serum HE4 level correlates with COVID-19 severity and predicts disease outcome.

¹Dr. László Elek Hospital and Clinic, Central Laboratory, Orosháza, Hungary,

²University of Szeged, Albert Szent-Györgyi Health Centre, Institute of Laboratory Medicine, Szeged, Hungary

For economic reasons, it is still problematic for smaller laboratories to carry out certain tests, so such samples are sent on to centers with a larger capacity. The hospital laboratory in Orosháza currently performs the hemoglobin A1c (HbA1c) determination itself, but due to the uncertain economic situation, the possibility of forwarding the specimen to the regional laboratory (University of Szeged, Institute of Laboratory Medicine) may arise in the future. Therefore, in this study, we aimed to compare the HbA1c results obtained from the two laboratories using different measurement methods. We investigated the usefulness of the results for clinical decision making, in particular regarding the possible existence of hemoglobin derivatives or variants that may influence HbA1c levels. In total 101 whole blood specimens without hemoglobin variants (hemoglobin F<4 %) and 101 specimens containing hemoglobin derivatives or variants were analyzed. All specimens were collected in EDTA-coagulated tubes. HbA1c levels were determined in Szeged by Variant II Turbo automated cation exchange HPLC method (Bio-Rad), while in Orosháza they were measured by an immunoturbidimetric test (DxC 700 AU automated chemical analyzer; Beckmann Coulter). For samples without hemoglobin variance (HbF<4 %), no clinically significant difference was observed between the results obtained by the two methods (y=1.0053x-0.162, r=0.9966; ẟ<0.5 %). Similarly, no significant difference was observed for labile A1c derivatives (ẟ<0.5 %). However, in the presence of hemoglobin F variant (>4 %), a clinically relevant difference may be observed (ẟ>0.5 %), whereas a clinically significant difference was confirmed for samples containing a P3 peak (>10 %) (ẟ>0.5 %). Our results confirm that in samples without hemoglobin variance (hemoglobin F<4 %) and in the presence of labile A1c derivatives, the results obtained by the two methods are comparable and can be used for clinical decision making. If the presence of a specific hemoglobin derivative or hemoglobin variant is suspected (its frequency in relation to an Orosháza-sized hospital patient population is estimated to be about 1-2 patient/month), it is necessary to determine the HbA1c level by the gold-standard HPLC assay.

Department of Laboratory Medicine, Faculty of Medicine, University of Medicine, Debrecen, Hungary

Introduction: Glucose-6-phosphate dehydrogenase (G-6-PDH) catalyzes the first reaction step in the pentose phosphate pathway to generate NADPH in red blood cells (RBC). In G-6-PDH deficiency, which is a common X-linked disorder worldwide, hemolytic anemia can be more severe in response to certain drugs (e.g., primaquine, antibiotics, NSAIDs) or oxidative stress (e.g., favism).

Aim: Our aim was to introduce the first quantitative method for the determination of G-6-PDH enzyme activity in Hungary.

Methods: For this purpose, we used the Trinity G-6-PDH reagent kit. In this procedure NADP is reduced by G-6-PDH in the presence of glucose-6-phosphate. The rate of formation of NADPH is proportional to the G-6-PDH activity. G-6-PDH stability was tested in whole blood refrigerated up to 1 week. We analyzed the effect of common interfering factors and that of elevated cell counts as well as age and gender.

Results: Red cell G-6-PDH was stable for 1 week stored at 2-8 °C but freezing of blood is not recommended. Grossly elevated WBC (>50 G/L), platelet (>550 G/L) and reticulocyte count (>300 G/L) as well as lipemia (T g>70 mmol/L) caused falsely elevated G-6-PDH activity (P<0.05), however, hemolysis (up to 10 %) and icterus (TBil up to 150 μmol/L) did not interfere with the reaction. We detected slightly increased enzyme activity in children compared to adults (132 ± 39 % vs. 103 ± 38 %, P=0.025, respectively) due to reticulocytosis. Out of 24 clinical samples 2 showed decreased G-6-PDH level (<60 %), and further genetic verification is required.

Conclusion: G-6-PDH enzyme activity test completes the diagnostics of hemolytic disorders and aids in avoiding inappropriate drugs.

¹Clinical Chemistry and Immunology Laboratories, Synlab Diagnostic Centre, Budapest,

²Faculty of Health Science, University of Pecs,

³Department of General Internal Medicine, Markusovszky University Teaching Hospital Szombathely

⁴Department of General Internal Medicine of Medical Centre, Hungarian Defence Forces Budapest, Hungary

The measured thyroid hormone levels may confuse the interpretation when they do not fit the clinical picture. Some substances in the patient’s sample may interfere with the analysis. Their recognition is only possible with the cooperation of clinical and laboratory specialists. As an example, the authors demonstrate a case. Methods: Measurements of TSH, freeT3, and freeT4 hormones in native and polyethylene-glycol (PEG) treated sera, and thyroid antibodies (TPO-Ab, TRAK) were performed on Siemens and Roche platforms. Serum electrophoresis, paraprotein analysis, rheumatoid factor, immunoglobulin-G, dilution test, PEG precipitation, and sexual hormone-binding globulin were additionally made with Roche tests. Patient: A 55-year-old man’s blood samples were analyzed more times during the past ten years. TPO-Ab levels were extremely high in each case besides the normal TRAK levels. As his hormone levels periodically confirmed hypothyroidism, he was previously treated with L-thyroxine (L-T4). Later, he seemed to be clinically euthyroid since he stopped taking L-T4 for over a year. However, the hormone results, which were measured by two methods, confirmed high TSH (54 mIU/L) and high freeT4 (77 pmol/L), and freeT3 (23 pmol/L) levels. After PEG treatment, 67 % of TSH, 88 % of fT4, and 85 % of fT3 could be precipitated, so biologically active hormone levels confirmed hypothyroidism (TSH=20 mIU/L, freeT4=9.1 pmol/L; freeT3=3.3 pmol/L) therefore the patient received 50 ug/die substitution. After four months, measured from his native serum, the values of all three hormones were also high, but based on the hormone levels after PEG treatment (TSH=7.9 mIU/L; freeT4=12 pmol/L; freeT3=3.3 pmol/L), the patient was adequately substituted. Conclusions: During multiple measurements, relevant anti-TSH and significant anti-fT4 and fT3 and or heterophilic antibodies interfered with immuno-analysis. Thus, we can only rely on the results measured from the sample treated with PEG to confirm the correct substitution of the patient.

National Laboratory on Human Reproduction, University of Pécs, Pécs, Hungary

Cytokines have a crucial role during pregnancy and in various autoimmune diseases. Autoantibodies against cytokines regulate the availability and effectiveness of cytokines, and they have been described in healthy individuals and patients with autoimmune diseases. We were the first to screen multiple anti-cytokine autoantibodies in serum samples of healthy pregnant women (HP) and pregnant women with Hashimoto’s thyroiditis (HTP) compared to age-matched healthy women (HC) using a multiplex assay with the Luminex MAGPIX instrument. The advantage of the method is the detection of multiple autoantibodies from 25μl 1:100 diluted serum samples in a single run. According to our results, the levels of autoantibodies to TNFα, IL-8 and IFNγ are significantly decreased in the first trimester of HP compared to HC, but these differences were missing in HTP. Therefore, these anti-cytokine autoantibodies may have a crucial role in early physiological pregnancy, which appears to be altered in HTP. Inflammatory and Th1 cytokines are known to have essential roles in early pregnancy, which is supported by our results showing differences in the levels of autoantibodies to such cytokines in the first trimester of pregnancies. This multiplex method can be useful for following the regulatory role of anti-cytokine autoantibodies during physiological and pathological pregnancies.

This project was supported by RRF-2.3.1-21-2022-00012 "National Laboratory on Human Reproduction”.

¹Division of Clinical Laboratory Science, Department of Laboratory Medicine, Faculty of Medicine, University of Debrecen, Debrecen, Hungary,

²Medical Center of Hungarian Defense Forces, Budapest, Hungary

Polyethylene glycol (PEG) is often conjugated to proteins and drugs to improve solubility, and half-life and to reduce the immunogenicity of therapeutic molecules. However, due to the presence of PEG in everyday products, persons can develop antibodies against PEG, which can be problematic when a PEGylated drug is administered.

We aimed to develop quantitative methods for the measurement of human anti-PEG antibodies (anti-PEG abs).

We developed indirect ELSA methods in which multi-PEGylated BSA captured the anti-PEG antibodies from the samples and standards. IgG- and IgM-type humanized monoclonal antibodies were used to calibrate the assays. HRPO-labeled anti-human IgG and IgM were used to detect the bound anti-PEG abs. To overcome the inhibitory effect of PEG-based surfactants, CHAPS was used in the assay buffer to reduce non-specific binding. We analyzed serum samples of 20 controls and 2 hemophilia A patients with accelerated clearance after treatment with PEGylated FVIII.

The median(min-max) anti-PEG IgG and IgM levels in the control group were 83(<1.5-9569) ng/mL and <3(<3-12301) ng/mL, respectively. Using the 50 ng/mL Ig level as a cut-off, 67 % and 33 % of controls were positive for anti-PEG IgG or IgM antibody. Both patients had high levels of anti-PEG abs: 266 ng/mL and 241 ng/mL anti-PEG IgM and 1072 ng/mL and 887 ng/mL anti-PEG IgG. Since no anti-FVIII antibody was detected in the patient’s samples, anti-PEG abs are presumably responsible for the accelerated clearance of the PEGylated FVIII. Our results draw attention to the fact that anti-PEG antibody positivity is high in the general population, so it may be worthwhile to test for positivity before using PEGylated drugs.

St Imre Teaching Hospital, Central Laboratory, Budapest, Hungary

Based on literature data, a glucose value above 34.8 mmol/L causes pseudohyponatremia on the ABBOTT Architect c16000 system’s indirect ion selective electrode (ISE). Our goal was to verify the manufacturer data on serum samples on our system for ions and additional analytes that arise based on the literature - which we also routinely examine.

We prepared a series of dilutions (0-25-50-75-100 %) from a 2000 mmol/L glucose infusion solution with distilled water, and then each dilution was mixed at 5 % with the pre-selected normal patient samples. The deviations of the averages obtained from 3 parallel runs were compared to the Reference Change Value (RCV %). Measurements were performed on the Abbott Architect c8000 system and, except for the ion measurements, Diasys reagents were used.

We did not experience an effect causing pseudohyponatremia even at the highest glucose concentration (94.6 mmol/L). Clinically significant differences were found only with the creatinine Jaffé method, so we repeated the investigation at 4 different creatinine concentrations. Based on our measurements, the interference resulting (exceeding RCV95 %) creatinine (µmol/L); glucose (mmol/L) pairs are as follows: (57.8; 20.87), (94.58; 33.24), (185.27; 77.16), (622.77; 270.8). We must consider the disturbing effect at a lower creatinine range, however, glucose concentrations that also affect high creatinine values are not realistic among human serum samples.

The interference described in the literature, which is the base of our tests, was confirmed in our system only with the creatinine Jaffé method where we also found a concentration-dependent effect. Our results draw attention to the fact that glucose interference is a phenomenon that should be confirmed within your measurement system.

Department of Laboratory Medicine, Faculty of Medicine, University of Medicine, Debrecen, Hungary

Analytical errors may occur during routine diagnostic testing of endocrinological parameter using the immunoassay methodology. Erroneous test results caused by antibody interference, particular to the named methodology, may cause reason for concern and probably lead to misdiagnosis, unwarranted further diagnostic investigations and prescription of inappropriate treatment modalities. In vivo macrocomplex formation has been implicated for a number of hormones but have been best characterized for determination of prolactin and thyroid stimulating hormone (TSH). Most commonly, analyte specific IgG-type autoantibodies form complexes with hormone molecules, nonetheless, the underlying mechanism may be heterogeneous. With complex formation the half-life of the hormone is increased, while its antigenicity is preserved, and its physiological role blunted. Although gel filtration would be best recommended to detect macrocomplexes, it is time consuming and not routinely performed. The most commonly used approach is to measure the hormone level is contention before and after precipitation of the large molecular weight complexes using polyethylene glycol. When the endocrinological laboratory results are inconsistent with the symptomology of the patient, a critical interpretation and probable macrohormone interference should be considered. In such scenarios, communication between the laboratory and the physician is paramount. Till date, there is no universal consensus as regards to the diagnostic work-up of spurious hormone results. We present a summary of the various approaches enumerated in the scientific literature

¹Department of Laboratory Medicine, Faculty of Medicine, University of Debrecen, Hungary

² ESZFK Nonprofit Kft, Budapest, Hungary

Introduction: Macro creatine kinase (macro-CK) is a complex formed by polymerization of CK isoenzymes with 2 subtypes. Type I is the association of CK-BB or CK-MM with immunoglobulins while Type II enzymes are oligomers of mitochondrial CK. Their presence in plasma results in false elevations of total CK and CK-MB isoenzyme activities, which often leads to unnecessary investigation for myocardial ischemia.

Aims and Methods: The prevalence and characteristics of macro-CK cases in Hungary is still unexplored. To address this issue, we reviewed the medical records of 867 patients, for whom CK agarose gel electrophoresis and quantitative scanning was carried out at the Department of Laboratory Medicine at the University of Debrecen between 2012 and 2022.

Results: Of these 867 patients 124 had macro-CK1 (14 %) and 23 had macro-CK2 (2,6 %). The 124 cases with macro-CK type I (77 females and 47 males) had an average age of 64 years (0-93). The mean of CK levels were 861 U/L (151-7491 U/L) with a mean macro-CK1 of 41 %. The 23 cases with macro-CK type II (10 females and 13 males) had an average age of 49 years (0-90). The mean of CK levels were 575 U/L with a mean macro-CK2 of 18 %.

Conclusions: Consistent with the results of other studies, macro-CK is not a rare phenomenon in laboratory practice in Hungary. Physicians should be aware of the presence of macro-CK so that invasive or costly procedures are not undertaken and the cause of increased serum CK levels should be explored.

National Institute of Oncology, Central Laboratory, Budapest, Hungary

Prealbumin (transthyretin) appears before albumin during serum protein electrophoresis. Produced by the liver, prealbumin binds triiodothyronine, thyroxine and the holo-retinol binding protein with retinol or vitamin A in the circulation and is excreted through the kidneys and the gastrointestinal tract. Prealbumin levels can be increased by exogenous corticosteroids, acute renal insufficiency, renal tubule damage and dehydration and decreased by acute phase response due to infection, trauma, malignant disease, starvation, and other acute or chronic diseases. We examined prealbumin levels measured in outpatients and inpatients, who either suffered from various forms of cancer or underwent lung transplantation, to assess the correspondence with literature data in terms of correlation with negative or positive acute phase proteins, and to evaluate if prealbumin levels provide added information when measured along with other acute phase proteins. Prealbumin levels measured from January 1 to December 31, 2020, at the National Institute of Oncology were collected retrospectively and analyzed. The correlation of prealbumin levels with C-reactive protein, ferritin, albumin, and transferrin was analyzed with Spearman’s rank correlation. 126 prealbumin measurements were completed for 109 patients (21 % outpatients and 79 % inpatients). Prealbumin levels were not followed up in 86 % of the patients. Prealbumin levels were decreased in 48.5 % of inpatients. Prealbumin levels showed strong positive correlation with albumin and transferrin levels. Prealbumin levels showed significant negative correlation with positive acute phase proteins C-reactive protein and ferritin (r_s = – 0.66 and – 0,24 respectively, p <0.001); and significant positive correlation with negative acute phase reactants albumin and transferrin (r_{s =} 0.72 and 0.58 respectively, p <0.001). Given the strong correlation of prealbumin with other acute phase reactants, prealbumin measurement could be omitted from the list of laboratory tests, especially in the hospitalized patient group, however, the main advantage of prealbumin is its short half-life (1.9 – 2.5 days).

Funding: GINOP-2.3.2-15-2016-00043, NKFI-K120633

University of Szeged, Department of Pediatrics, Szeged, Hungary

Cystic fibrosis (CF) is the most common autosomal recessive inherited disease among Caucasians. The incidence of this disorder is approximately 1:4000 in Hungary and 1:2500 worldwide. By now newborn screening (NBS) programs in most European countries have included CF because it is a well-established public health strategy with international standards. Therefore, the prevalence is constantly increasing. In Hungary CF was included in the NBS program in 2022. Our country screening approach is immunoreactive trypsinogen (IRT)/IRT* pancreatitis-assiciated protein (PAP)/IRT + Safety Net (SN) strategy. The first-tier test used the IRT measurement where the cut-off value was 65 ng/ml based on our own pilot study (2017;2020) and international data. If the first IRT value was above 120 ng/ml, the newborn was reported as positive (safety net). If IRT was 65-120 ng/ml, PAP measurement was performed as second-tier analysis and the obtained value was multiplied by the average of the IRT values. If the multiplication value was over the cut-off (IRT*PAP > 160) we asked for a new blood sample for another IRT measurement. The obtained results from the second DBS were evaluated by three cut-off values, based on weeks of life. Cases with a positive screening result were referred to the CF Centres for sweat testing. If the sweat chloride ion cencentration (mmol/L) was above the reference range, in the intermediate range or undeterminable, the CFTR mutation analysis was performed as the next diagnostic step. Since the beginning of the CF screening, we examined 41140 newborns, of these 119 were CF-screen positive and 7 of them were confirmed to have CF. Based on data so far the applied screening protocol (IRT/IRT*PAP/IRT+SN) had a sensitivity of 100 % and a specificity of 99,73 %. However, since this approach was associated with a relatively large number of false positive cases in the first year of screening, a modification of the cut-off values or a third-tier test with extended genetic analysis is suggested to improve the positive predictive value (PPV).

Synlab Hungary Ltd., Laboratory of Molecular Diagnostics, Budapest, Hungary

Leiden mutation analysis of the coagulation factor V gene is one of the most frequently performed tests in molecular genetic diagnostic laboratories. According to the Hungarian Ministry of Health’s professional protocol, "random screening of the general (normal) population for FV Leiden mutation is not recommended", and testing before starting oral contraception is not listed among the indications for genetic or functional testing.

The aim of this retrospective statistical study was to determine the positivity rate of Leiden tests ordered by gynecologists as opposed to those requested by doctors in other specialties.

Of the Leiden test requests performed by our laboratory in the past 4 years, we indiscriminately included in our study those for which we were able to identify the specialty of the referring practice or physician. Data were processed in Excel and statistical significance was calculated using chi-square tests.

Our results show that 9.5 % of the 8486 Leiden cases sent by 403 gynecologists from 329 providers were heterozygous, which is not significantly different from the positivity rate of 9,8 % of the general Hungarian population (p=0.85). However, it is highly significantly different from the heterozygote rate of 13 % (p<0.00001) among the 3895 requests by non-gynaecologists, and, in particular, from the 21.7 % positivity rate requested by public internal medicine clinics (p<0.00001).

It is concluded that, overall, in the Hungarian ob-gyn practice Leiden mutation testing is requested as a screening test, presumably mainly before initiating hormonal contraception. The results of the tests requested by them also provide an upper limit for the population prevalence in Hungary, which complements the data available so far based on a small sample size.

Central Hospital of Southern Pest, Budapest, Hungary

Multiple myeloma (MM) is the second most common hematologic malignancy, characterized by the proliferation of clonal plasma cells (PC) in the bone marrow (BM). In ∼15 % of cases, t(4;14) translocation (IGH::NSD2 gene fusion) can be detected in the background of MM, which has an unfavourable prognosis, but is sensitive to proteosome-inhibiting therapies. The presence of measurable residual disease (MRD) in MM is also a parameter, that influences therapy and indicates a favourable prognosis. Our goal was to develop a quantitative PCR (qPCR) technique for IGH::NSD2 mRNA quantification, which can detect MRD with high sensitivity. By testing diagnostic and follow-up samples (n=160), 100 % concordance was found between qPCR and traditional qualitative PCR methods. IGH::NSD2 fusion mRNA could be identified in 96.5 % of t(4;14) patients with qPCR both on LightCycler480II and on droplet-digital PCR (Biorad QX200) platforms. Altogether 333 BM, PB (peripheral blood) or PC samples from 107 MM patients with t(4;14) translocation were evaluated in our laboratory between 2006 and 2021. We investigated 36 PB samples from 25 patients. With this sensitive system we could prove the presence of myeloma cells in the circulation in several cases, especially in case of extramedullar involvement. In general, we concluded, that IGH::NSD2 fusion transcript of PB samples showed 1000 times lower sensitivity comparing to BM samples.

Central Hospital of Southern Pest, Budapest, Hungary

Somatic hypermutation (SHM) status of the immunoglobulin heavy chain variable region (IGHV) is a significant prognostic indicator for CLL. In case of ≥ 98 % identity to the reference sequence, SHM is classified as ’unmutated’, which is associated with adverse prognosis. However, the amplification efficiency of primers from ERIC consortium’s recommendations that match leader gene sequences are not ideal for Sanger sequencing. Next generation sequencing (NGS) can increase this efficiency. Since 2019, 673 IGHV tests were performed in our laboratory, 597 with Sanger method, and (from 2023), 76 samples using NGS (LymphoTrack Leader Assay). This number of SHM determinations allowed us to compare the two systems. SHM status could not be determined in 14 % of cases tested with the Sanger method, using leader primers only. 4.4 % of the tests did not lead to any result, even with framework primers (due to nonamplification or inefficient sequence quality). NGS method could significantly reduce the number of aforementioned cases, for example by identifying biallelic/biclonal rearrangements involving the same type of genes. We identified SHM status in every patient (8/8) carrying such gene rearrangements. In contrast, NGS LymphoTrack Leader assay, only 5.3 % of samples required a confirmatory primer system and only 2.6 % failed to determine SHM status. In addition, both sequencing and data evaluation were significantly faster and more efficient. In summary, NGS provides solutions for the difficulties of the Sanger method and greatly improves the efficiency of the recommended leader primer system. According to its sensitivity, this new method also leads to several problems due to new cases with minor alleles, so further comprehensive studies and development of guidelines are needed in the near future.

¹Bács-Kiskun County Teaching Hospital, University of Szeged, Kecskemét, Hungary,

²Department of Pharmacology and Pharmacotherapy, University of Pécs, Medical School, Pécs, Hungary,

³Eötvös Loránd Research Network, Chronic Pain Research Group, University of Pécs, Hungary

Anxiety and depression are among the most common mental illnesses in the United States, approximately 40 million adults suffer from an anxiety disorder. Clinical studies show that women show a much higher prevalence than men. Even though mood disorders are more common in women, most animal studies were conducted in male animals. Differences between male and female brains play an important role in psychiatric disorders. Our aim was to study the behavior and memory of female and male mice, compare them at group and individual levels and set up a scoring system to investigate the relationship of memory functions and behavior.

3–5-month-old mice were divided into 4 groups: male and female C57BL/6 wild-type and Tac4 gene deficient mice. Behaviour and memory functions of these groups were compared using different behavioural tests (Tail suspension, Forced Swim, Elevated Plus Maze, Sucrose Preference and Open Field Tests) and memory tests (Novel Object Recognition, Y maze tests).

Female Tac4 gene-deficient animals showed significantly worse memory function in Y maze test and significant depression-like behavior compared to their wild-type counterparts. In the open-field test, both male and female gene-deficient animals spent significantly less time in the central zone of the box, suggesting higher anxiety level compared to the wild-types.

Our results suggest that animals (especially females) with Tac4 gene deficiency show worse memory and depression-like behaviour.

Division of Clinical Laboratory Science, Department of Laboratory Medicine, Faculty of Medicine, University of Debrecen, Debrecen, Hungary

Background: Endothelial protein C receptor (EPCR) plays an important role in the activation of protein C, one of the most important physiological anticoagulants. The rs867186 single nucleotide polymorphism G>A in the PROCR gene (EPCR p.Ser219Gly) with an estimated allele frequency of 10 % in the general population may influence the risk of venous thromboembolism (VTE). We performed a case-control study and a meta-analysis of the association between rs867186 and the risk of VTE.

Objective and methods: For case-control study we enrolled n=263 VTE patients and n=320 unrelated healthy controls. Clinical and demographic data were collected, and SNaPshot assay was used for genotyping. For the meta-analysis the total number of cases and controls were 5768 and 30017, respectively and it was performed by Stata 17 and by a new, online MetaGenyo Statistical Analysis System software. Furthermore, a reproducibility study was conducted to validate our results.

Results: EPCR p.Ser219Gly independently and significantly increased the risk of VTE in individuals wild type for rs8119351, another PROCR SNP. In the meta-analysis a significant association was found between EPCR p.Ser219Gly polymorphism and VTE under dominant model (OR=1.27, 95 % CI:1.11-1.46, p=0,0006) recessive model (OR=1.60, 95 % CI:1.26-2.04, p=0,0001) GG versus AA contrast model (OR=1.64, 95 % CI:1.28-2.09, p=0,0001) GA versus AA contrast model (OR=1.24, 95 % CI:1.08-1.43, p=0,002).

Conclusion: These studies suggest a significant role of EPCR p.Ser219Gly polymorphism in the development of VTE.

¹Central Hospital of Southern Pest – National Institute of Hematology and Infectious Diseases, Laboratory of Molecular Genetics,

²Department of Hematology and Stem Cell Transplantation, Budapest, Hungary

Multiple myeloma (MM) is an incurable hematologic malignancy caused by proliferation of neoplastic plasma cells in the bone marrow. Risk-stratification and initial therapy selection largely depend on chromosome abnormalities (CA) detected in MM cells. Chromosome trisomies and translocations involving immunoglobulin heavy locus (IGH) gene are considered as primary pathogenic cytogenetic abnormalities, while multiple secondary CA-s may also arise along the course of MM. We examined 227 bone marrow samples from MM patients newly diagnosed between 2018-2023, for the presence of CA-s using interphase fluorescence in situ hybridization (iFISH). Immunomagnetic positive selection (EasySep™, STEMCELL Technologies) of CD138⁺ plasma cells was performed on all samples to increase FISH sensitivity. Trisomies were found in 42.7 % (97/227), while IGH-translocations in 41 % (93/227) of the samples, both in accordance with the literature. Co-occurrence of trisomy and an IGH-translocation was identified in an additional 6.6 % (15/227) of the samples. In the IGH-translocated subgroup, t(11;14) (19.8 %, 48/227) and t(4;14) (10.6 %, 24/227) were the most frequent. Secondary cytogenetic abnormalities gain(1q21) and del(17p) were identified in 36.6 % (83/227) and 15.9 % (36/227) of the samples. The association of high-risk CA-s with shorter overall survival could not be demonstrated, which can be explained by relatively short follow-up time (median 1.8 years) and effective, novel therapeutic modalities.

University Debrecen,

¹Department Laboratory Medicine,

²Department Medical Microbiology,

³Emergency Clinic,

⁴Department Infectious Diseases of the Pediatric Clinic,

⁵Emergency Department Gyula Kenézy Campus, Debrecen, Hungary

Introduction: The SARS-CoV-2 epidemic is constantly presenting new challenges to specialists involved in the screening and diagnostics of infected patients. Three Vivalytic (Bosch) bedside CoV-2 PCR analyzers were installed in three patient care institutions of the University of Debrecen (Emergency Clinic; Department of Infectious Diseases, Pediatric Clinic; Emergency Department, Kenézy Gyula Campus).

Objective: Comparison of SARS-CoV-2 PCR test results provided by the Vivalytic analyser and the Department of Medical Microbiology (DMM).

Materials and methods: Vivalytic CoV-2 test - using nasopharynx/ oropharynx sample after 30 minutes inactivation - could be measured directly from the inactivating medium with a PCR cartridge. The sample volume was 300 µL, the measurement time was approx. 45 minutes. Some of the samples were also tested using the routine diagnostic method (multiplex RT-PCR) in the DMM.

Results: 2,835 measurements were performed with the Vivalytic device, of which 218 (7,7 %) were positive, 2,332 (82,3 %) were negative and 285 (10,0 %) could not be evaluated. The DMM received 420 samples (Vivalytic results - 333 negative, 4 positive, 83 inconclusive), of which 24 were positive, 379 were negative and 17 were borderline. The samples that provided different test results by the 2 methods (Vivalytic vs DMM): 1 positive vs 1 borderline, 27 negative vs 9 borderline + 18 positive, 83 inconclusive vs 3 positive + 2 borderline + 78 db negative. Based on the Vivalytic positive and negative results the 2 methods showed 92 % agreement.

Conclusion: The 4 Vivalytic positive tests were also positive/borderline with the PCR of the DMM, showing the reliability of a positive Vivalytic result. At the same time positive results were also found in the case of negative Vivalytic samples. In the case of a negative sample, it is a subject of individual consideration, but in all cases with inconclusive measurements confirmation by routine RT-PCR at the DMM is recommended.

Department of Laboratory Medicine, Faculty of Medicine, University of Debrecen, Hungary

The pneumatic tube system (PTS) is a quick and reliable way to transport laboratory samples. A questionnaire survey was carried out to assess the operation and use of PT systems in Hungarian laboratories.

Fourteen laboratories completed the questionnaire, with 7 Swisslog, 5 Sumetzberger and 2 Aerocom systems. The first PTS started to operate in January 2007, while the latest one, already in use, was installed in November 2019. PTS lengths are highly variable and range from 250 to 9372 metres, with 6 to 56 clinical endpoints. On average, 100-1700 capsules are sent every day and 7 systems have automatic capsules. In case of two PT systems, the operator is exclusively an external company, in other cases the hospital or university, operates it in partnership with an external company. Except for 4 systems, the operator is available in 24/7. In the last 3 months, the average downtime of the PTS was 180 minutes per day in the worst case. No information is available for 10 laboratories on the rate of operational failures exceeding 1 hour, while in case of 4 laboratories it was 0-2.2 %. Eight institutions also use the PTS to send other types of shipments, mainly pathological samples, and medicines. In 3 laboratories, new lines or stations have been added since the installation of the PTS in addition to the troubleshooting.

In conclusion, the PT systems used by Hungarian laboratories are rather heterogeneous, and many laboratories have insufficient information on the extent of malfunctions and in most cases the operator is only engaged in troubleshootings while improvements are only rarely made.

Respondents to the Questionnaire: Hungarian Military Hospital, Semmelweis University, University of Szeged, University of Pécs, University of Debrecen, Hospital Laboratories at Kecskemét, Berettyóújfalu, Eger, Nyíregyháza, Miskolc, Szolnok, Veszprém, Szombathely, Sopron.

Szabolcs-Szatmár-Bereg Megyei Kórházak és Egyetemi Oktatókórház, Department of Laboratory, Fehérgyarmat-Mátészalka-Vásárosnamény-Nyírbátor¹ , Nyíregyháza² , Hungary

Laboratory results that indicate potentially life-threatening conditions -critical risk results (CRR)- must be reported by laboratories to the requesting clinician faster than in routine. Arrangements of CRR reporting including laboratory parameters and thresholds for reporting (CRR list) vary from laboratory to laboratory. We reviewed CRR lists of four satellite laboratories of our laboratory network considering current professional guidelines and CRR list that is used in the core laboratory of our network with a goal to harmonise CRR lists within our laboratory network. The revised CRR list with some questions about the clinical need and administration of CRR reporting were sent to 26 hospital physicians and 97 GPs using the four satellite laboratories of our network. The questionnaire was answered by 17 hospital physicians (65 %) and 21 GPs (22 %). All respondents agreed that CRR reporting was important and almost all agreed with the practice that the fact of reporting should be included in the report. In agreement with guideline recommendations, we found that clinicians needed alerting with less parameters. On average 7 (min:3; max:15) and 6 (min:0; max:14) parameters out of 28 parameters of the reviewed CRR list were found useless by hospital physicians and GPs, respectively. Parameters that were found important to be reported by most respondents were K⁺, glucose, troponin, INR, and hematological parameters. However, clinicians of this survey were satisfied with our revised alert thresholds. On average 15 (min:12; max:17) and 19 (min: 16; max: 20) out of thresholds of 20 parameters were accepted by hospital physicians and GPs respectively. None of those who disagreed with any thresholds on the list provided different thresholds to be applied.

Findings of these survey will be used in harmonisation of CRR list in our laboratory network.

Szabolcs-Szatmár-Bereg megyei Kórházak és Egyetemi Oktatókórház, Medical Laboratory, Fehérgyarmat-Mátészalka-Vásárosnamény-Nyírbátor¹ , Nagykálló-Nyíregyháza² , Hungary

Szabolcs-Szatmár-Bereg megyei Kórházak és Egyetemi Oktatókórház (SZSZBMK) is the integrated teaching hospital of Szabolcs-Szatmár-Bereg County consisting of 5 hospitals and 1 outpatient service center in 6 main geographical locations of the county. Historically all sites used to have their own medical laboratories operating separately with phlebotomy sites and connections to GP’ offices. Functional integration of laboratories including patient-focused medical and financial processes have been started more than a decade ago and by now medical laboratories of SZSZBMK are operating in a structured, integrated manner under the direction of a chief medical officer.

A short historical overview will be presented to demonstrate major steps of integration process of laboratories. Details on how the entire laboratory process from pre-analytics to post-analytics are organized in the integrated laboratories will be shown. Special focus on preanalytical processes will be given, when guideline recommendations, types of sampling devices and pre-analytical transport equipment will be discussed. How laboratory algorithms have been introduced in some clinical areas to assist clinicians in better test requesting to achieve diagnosis faster and to avoid unnecessary testing will be presented. Methods of postanalytical harmonization without full methodological harmonization to achieve comparable results and to avoid unnecessary retesting when patients move in between hospitals will be shown. Finally, integration in the fields of financial processes, HR, and quality management will also be presented.

In summary, in the era of high-tech laboratory analyzers and advanced IT, integrative thinking when organizing tasks of laboratories is unavoidable. SZSZBMK laboratory network is a high quality, well-functioning example on the potential solutions for integration. Laboratory professionals’ responsibility to assist decision makers in finding the best alternative in integration which fits with the local clinical needs and serves best the interests and safety of our patients.

Synlab Hungary Ltd., Székesfehérvár, Hungary

In the interest of the effective patient care and well-established clinical decision-making it is necessary to ensure the highest quality requirements even in the preanalytical laboratory phase. Ensuring optimal temperature during transportation of the samples greatly affect the reliability of test values. In this study the effects of various transport temperatures on the accuracy of results of several analytes (Alanine aminotransferase-AST, Direct bilirubin-Dbi, Glucose-Glu, Calcium-Ca, Potassium-K, Creatine kinase-CK, Lactate dehydrogenase-LDH, Magnesium-Mg, Total bilirubin-Tbi, Triglyceride-Tg) were modeled. Blood samples (with gel separator) were collected from 28 volunteers. Chemical parameters were measured on Beckman Coulter AU5800. After complete coagulation, one blood collection tube from each patient was centrifuged and the chemical parameters were analyzed afterwards. The other primary tubes were also centrifuged and then were stored 3 hours at different temperatures (-5 °C, 4 °C, 25 °C and 45 °C). After that the chemical parameters were analyzed. For examination of the analytical accuracy, it was determined the level of bias as the percentage of deviation to the target value measured at 25 °C. Based on the expected deviation of reproducibility of parameters (2-3 %), it was found that Ca, Mg, and Tg values remained stable at all temperatures. Most of the analytes remained stable at 25 °C, except Glu and Dbi. At -5 °C, K and CK levels increased by 27.87 % and 3.2 %, respectively. At 4 °C, Glu decreased by 6.04 %, while K and CK increased by 11.9 % and 5.04 %, respectively. The level of most analytes increased at 45 °C compared to 25 °C (7 % for AST, 15.48 % for LDH, 22.05 % for Dbi, 3.12 % for Tbi, 80.44 % for K), but Glu and CK showed some decrease, 40.52 % and 4.77 %, respectively. Our results reflect that proper preanalytical quality assurance is crucial for reliable results, so patient safety, cost-effectiveness and patient care efficiency may increase.

St. Imre Teaching Hospital, Central Laboratory, Budapest, Hungary

As an independent control user, we find that changes in the reagent lot sometimes cause a shift in the controls results. The Clinical and Laboratory Standard Institute (CLSI) EP26 2022 guideline summarizes the procedures that a laboratory should perform before using a new reagent lot. If the difference in the results measured with the new and old reagents is within the rejection limit (RL), the new lot can be used. The procedure was tested when there was a change in the lot of the glucose reagent. As a first step, the number of samples required for comparison must be determined, for which the required data must be entered in the Excel workbook (WB) provided as an appendix to the directive: repeatability, reproducibility, the critical difference (per concentration level), type 1 and 2 error targets, rejection limit proportion (k). Instead of the resulting 1-1-1 sample, we chose 3-3-3 patient samples with concentrations similar to the control values and measured these samples in 3 duplicates with the old and the new lot of reagents on the same device. The obtained results were entered in the WB table, where we got the average deviation in % between the results measured with the old and new reagents. Data show results of target concentration (mmol/L)/RL ( %)/mean deviation ( %) by levels: Level 1: 3.21/10.79/1.63; Level 2: 6.23/10.52/0.98; Level 3: 18.85/10.45/2.01. Control data measured with the old and new reagents were also entered in the table, where we obtained different results for the average deviations ( %): Level 1: -2.34, Level 2: -1.62, Level 3: -1.54 %. Based on the investigation, the new lot of glucose reagent was considered acceptable. Within all three concentration ranges, we experienced significant differences between results, so it was useful to compare 3 samples instead of the minimum 1. We were also able to demonstrate that the behaviors of control samples were different compared to patient samples.

St Imre Teaching Hospital, Central Laboratory, Budapest, Hungary

Based on the recommendations and objectives of the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) and the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM), quality indicators (QI) have been collected mainly manually per department (chemistry, hematology, hemostasis, emergency) since 2012, which means recording 67 indicators daily. The preliminary analysis was aggregated and evaluated based on the indicators for which interpretable data were available in the last ten years (n=20). We plotted the results on a Pareto chart and selected the indicators that accounted for 80 % of the errors. These were as follows: 1) hemolytic samples were 10 % of the samples received for chemical analysis; 2) coagulated samples account for 8.9 % of anticoagulated samples; 3) few samples, 4.2 % of the samples received for chemical analysis; 4) incorrect blood/citrate ratio is 4.3 % of samples containing citrate, 5) samples not received are 2.1 % of all samples. Examining the individual years separately, despite the regular phlebotomy education and other measures, we did not find a clear trend among the indicators. The data of the automatic registration of the hemolysis index were compared with the manual data collection of the last five years. The difference was minimal in 2018-2019 (0.6 %, 0.42 %), but a more significant difference was experienced between 2020 and 2022 (1.76 %, 1.44 %, 3.2 %). Based on these, it is conceivable that manual data collection resulted in similar inaccuracies in other areas. Based on the experience of the last ten years, we have reduced the number and range of QIs to be collected so that we can focus on the most common and problematic errors. By reducing the number of QIs and automating data collection as widely as possible, our goal is to achieve more accurate quality indicators, which makes the response to nonconformities more traceable.

¹Vaszary Kolos Hospital, Central Laboratory, Esztergom, Hungary,

²National Institute of Oncology, Clinical Central Laboratory, Budapest, Hungary

Daratumumab, a human anti-CD38 monoclonal antibody, is used as a treatment in multiple myeloma therapy with a growing number of indications. In our institute serum protein gel electrophoresis and immunofixation is used as a routine part of the diagnostic process and the monitoring of disease progression and/or therapeutic efficacy in multiple myeloma by the detection of M-protein. Given that daratumumab is a human monoclonal IgG kappa antibody, daratumumab treatment can interfere with disease monitoring via standard serum electrophoresis and immunofixation. The Hydrashift daratumumab assay provides a way to distinguish daratumumab from the patient’s endogenous M-protein in serum immunofixation. The procedure is similar to standard immunofixation, with an additional step, when antidaratumumab antiserum is added to the samples. During immunofixation, if daratumumab is present in the sample, antidaratumumab /daratumumab immune complex is formed and migrates to the alpha-1 region, while the endogenous M-protein remains in the gamma region. 7 samples from patients with multiple myeloma treated with daratumumab were selected at our laboratory, whose electrophoresis and/or immunofixation pattern raised the suspicion of daratumumab interference, based on the presence of an M peak where daratumumab characteristically appears. Immunofixation was performed using the Sebia Hydrasys 2 analyzer and the Hydrashift daratumumab assay according to the manufacturer’s instructions. In 6 of 7 samples, the presence of daratumumab could be identified and distinguished from the endogenous M protein when present. Ongoing daratumumab treatment was confirmed via consultation with the treating physician. The Hydrashift assay is relatively new, and there are only a few reports are available with its use. We expect the number of patients receiving daratumumab treatment at our institution to increase, therefore, the Hydrashift daratumumab assay could be used as a simple procedure to avoid interference. Laboratory professionals as well as clinicians should be aware of this possible interference and consult with each other to avoid diagnostic confusion.

Semmelweis University, Szent Rókus Laboratory, Budapest, Hungary

Infrared (IR) spectroscopy is a novel approach to analyse qualitative and quantitative analysis of renal stones. We obtained a Jasco FT/IR-4X Fourier transform infrared spectrometer at the Department of Laboratory Medicine, Semmelweis University In order to introduce this technique into laboratory we started to construct an own patient kidney stone library. This library along with that offered by the company can be used later for the assignment of spectra to renal stone composition of our own patients.

Therefore, we analyzed the spectra of synthetic uric acid, calcium oxalate monohydrate, and kidney stones from patients. We prepared mixtures containing 1, 2 or 3 components of identified stones. The recorded spectra were compared with the original Jasco library (this library is based on Japanese experience and literature data). The sFT-IR ATR results were confirmed by microscopic and chemical tests ( 161 sample)

Our own library showed a good correlation with the Jasco library and chemical methods (r=0.863, r=0.812, Spearman). The results of the identification of calcium oxalate and uric acid showed complete similarity. Within calcium oxalate stones, however, the assessment of the relative rate of calcium oxalate monohydrate and calcium oxalate dihydrate components indicated a greater variability. The greatest difference between sFT-IR ATR and chemical tests was detected in case of mixed stones containing calcium phosphate. We still have limited data on rare L-cystine and xanthine.

We concluded that FT-IR/ATR spectroscopy is simple and fast kidney stone analysis. Its benefits are moderate reagent cost, and small sample volume requirements. The use of FT-IR/ATR, however, requires a time-consuming creation of an own library representative for the patient population.

SYNLAB Hungary Ltd., Clinical Chemistry and Immunology Laboratory, Budapest, Hungary

Interest in the relationship between nutrition and the intestinal system is on the rise due to increased gastrointestinal symptoms, metabolic disorders, as well as immunological and autoimmune diseases. Therefore, many laboratories also pay attention examining the flora of the intestinal system. The fecal flora status tested by the PCR method provides more information than microbiological cultures, it provides data about many intestinal diseases. More recently, the composition and metabolic functions of gut microbiota have been proposed as being able to affect obesity development. The aim of the study was to map the microbiome deviations, which can support an expert gastroenterologist in development of a personalized therapy.

During the fecal flora status examination, the numerical presence of 9 aerobic and 4 anaerobic bacteria, as well as germinating and filamentous fungi were investigated using the PCR method.

Between 01.06.2022 and 01.01.2023, 93 patients (average age 46.4 ± 17.24; 48 women, 45 men) requested an examination of the stool flora status. The examination was more commonly requested by male patients in the 31-45 age group. A decreased number of bacteria was observed mostly in Enterococcus spp. 46.2 %, Bifidobacter spp. 29 %, Lactobacilli spp. 57 %., while an increased number of fungi was observed in the case of Candida albicans 17.2 %, Candida glabrata 6.5 %.

The distorted aerobic/anaerobic flora is characteristic of intestinal dysbiosis, with the disturbance of the colonization-based resistance of the digestive system, which manifested in the clinical picture of irritable bowel syndrome/food intolerance. Microbial changes in the human gut can be considered as a factor involved probably in obesity development in humans.

Central Hospital of Southern Pest, National Institute of Hematology and Infectious Diseases, Central Laboratory, Budapest, Hungary

Introduction: Chronic hepatitis caused by viral infection, alcoholic liver damage, metabolic disorders could lead to the development of chronic liver disease. As a result, liver fibrosis and, in more severe cases, cirrhosis can develop. The ELF test, which can be measured in the clinical laboratory, combines three serum biomarkers: hyaluronic acid (HA), procollagen type III amino terminal propeptide (PIIINP) and tissue inhibitor of metalloproteinases 1 (TIMP-1). Once quantified from serum, with a help of an algorithm we can generate a single ELF score that shows a good correlation with biopsy, which is the gold standard of liver fibrosis diagnostics.

Aim: Our aim was to compare the distribution of patients according to age, the submitted diagnosis and the results obtained before and after COVID-19.

Patients and methods: In 2018, we received samples mainly from liver fibrosis patients caused by viral hepatitis (n=720). From 2022, the indication of the measurements expanded to include patient’s samples of other non-alcoholic fatty liver disease (NAFLD) and alcohol-induced steatohepatitis (n=822). In 2018, the ELFS avg. was 9.76 %, of which <35 years (n=42, 5.8 %) ELFS avg: 9.52 % In 2022 ELFS avg. was 9.83 %, of which <35 years (n= 118, 14.3 %) ELFS avg.:8.69 %. We also analysed additional data from the patient population under the age of 35 measured in 2018 and 2022. Age, gender, HA, TIMP, PIIINP, ELFS, ALT, AST, GGT, total bilirubin, platelet count, FIB-4, BNO code/.

Results and conclusion: ELF test measurement is an important diagnostic tool for assessing liver fibrosis. By measuring the samples of a younger patient population, liver fibrosis in various liver diseases can be diagnosed earlier. The advantage of the ELF test is that it’s a non-invasive, easily reproducible, and quick method to use, which can be beneficial in hard-to-reach populations like drug users or prisoners.

¹Faculty of Medicine, Department of Laboratory Medicine, Division of Clinical Laboratory Science, University of Debrecen, Debrecen;

²Faculty of Medicine, Department of Laboratory Medicine, University of Debrecen, Debrecen;

³Faculty of Medicine, Division of Clinical Immunology, University of Debrecen, Debrecen

The quality of the immune response is determined not only by the number of antibodies, but also by the strength of their binding. Our goal is to investigate the effectiveness of the immune response against SARS-CoV-2 in myositis patients receiving immunomodulatory therapy, by the binding kinetics of anti-SARS-CoV-2 IgG to the spike structural protein.

Anti-Spike and anti-NC determinations were performed using the ECLIA method from serum samples taken 8 months apart from three myositis patients. On sensor chips immobilized with Spike protein, the most important kinetic parameters (ka, kd, KA, KD) were characterized by SPR.

The previous sample of patient I. can be characterized by a KD value of 9.56x10-9 M, while in the case of the new sample, this value was 6.53x10-10 M. The previous KD value of patient II. was 1.94x10-10 M, while that of the new sample was 1.98x10-10 M. For the III. patient, we obtained KD values of 5.49x10-9 M and 3.31x10-10 M for the previous and the new sample. The KD values of healthy subjects fell in the range of 10-8-10-10 M, depending on the vaccine.

The examined patients developed an effective immune response against SARS-CoV-2 even with immunosuppressive therapy. We can conclude that the immunosuppressant therapy did not deteriorate the quality of the immune response. According to the parameters tested at the previous and recent times, the new samples developed a bond almost an order of magnitude stronger. So, as time progresses, IgG affinity improves, indicating a more effective immune response.

Central Hospital of Northern Pest - Military Hospital, Budapest, Hungary

We present a case of a 63-year-old male who was transported to a local hospital by ambulance service with a 4-day history of fever, headache, and confusion. His CT scan showed a 2.5 cm round shaped low-density region with ring enhancement in the left frontal lobe, causing midline shift, edema, and hydrocephalus. The patient’s white blood cell count was 13,8 G/l (88,4 % granulocytes, 3 % lymphocytes, 8,5 % monocytes), glucose 8,9 mmol/l, hsCRP 224 mg/l, PCT 4,65 ng/ml. Intravenous dexamethasone, meropenem and vancomycin were started for a suspected brain abscess, the patient was intubated and was reffered to our hospital for neurosurgical care. On arrival operative drainage was done and a ventricular drain was left in situ. Postoperative MRI scan showed left frontal abscess rupture into the cerebral ventricles, extensive ventriculitis, plexitis and perifocal edema. Mannitol dehydration was started due to elevated ICPs. EEG was non-reactive without any epileptic signs. Chest, whole abdomen and otorhinolaryngologic examination were all normal. Transoesophageal echocardiography showed patent foramen ovale, but no infective endocarditis. Serological tests for HIV, Aspergillus, Echinococcus, Toxoplasma were negative. Multiple organisms were isolated from the blood and CSF cultures: Streptococcus intermedius, Eikenella corrodens, Fusobacterium nucleatum, Actinomyces meyeri, Campylobacter gracilis, Bacillus circulans. Histopathology findings: intraventricular rupture of brain abscess with source of abscess unknown, probable haematogenous spread. Despite all effort, the patient remained in coma due to irreversible neurological damage and died after withdrawal of care. We considered this case worthy of presentation due to the unusual aerobic, anaerobic, and microaerophilic bacteria association with brain abscess without obvious primary source of infection in an immunocompetent patient.

¹BAZ Count Hospital and University Hospital, Department of Laboratory Medicine,

²University of Miskolc, Faculty of Health Sciences, Miskolc, Hungary

Background: We describe 2 cases from our daily practice and point out the significance of this rule in 2 patients suffering to hemolytic transfusion reactions, after short time of receiving cross-matched red blood cell units (RBCs).

Case report: A 73-years-old man with a traumatic injury received four units of apparently compatible red blood cells. In short time he became uremic, hyperkalemic, anuric, and required dialysis. A 53-years-old polytransfused woman was referred to our rehabilitation center after orthopedic surgery. In phase of the prae-surgical blood testing procedure, 3 irregular antibodies were detected. In the recovering period a repeated transfusion was became necessary, which resulted an intravascular hemolytic transfusion reaction. Laboratory investigations - free plasma hemoglobin (hemoglobinemia), urine hemoglobin (hemoglobinuria), unconjugated hyperbilirubinemia, decreased serum haptoglobin and elevated serum LDH level – confirmed the intravasal haemolyis. Serological tests showed that the blood of the donor and the recipient were fully compatible before the transfusion and no unexpected antibodies were detected.

Conclusions: Sample validity refers to only that period, when a pre-transfusion sample can be preserved for testing and used to produce cross-matched blood. The validity period of the sample depends on the patient’s transfusion-, and obstetric history. The development of hemolytic complication must also be considered when forming a selected blood product according to the rules.

SYNLAB Hungary Ltd., Diagnostic Center Clinical Chemistry and Immunological Department, Budapest, Hungary

Zonulin is a protein that increases the permeability of tight junctions between cells of the wall of the digestive tract. There are diseases that are known to be associated with intestinal permeability, and there is a lot of speculation about other possible diseases that might be connected to it. We aimed to observe immune responses to food antigens (IgG) and examine fecal zonulin levels through which investigated the correlation between these factors. Zonulin and food intolerance test was performed using ELISA method.

In this study we processed zonulin and food intolerance results from 01.2021. to 03.2023. The total number of patients who were screened for zonulin was 957. 53 % of 957 participants was found positive for zonulin. The grand mean of zonulin value considering all the results was found to be 129.12 µg/mL (SD: 81.32) that is higher than the reference range (14.0-108.0 µg/mL). Based on the results an increased zonulin level was found among 52 % of the women and 56 % of the men that took part in this study. From the total number of patients 55 requested both zonulin and food intolerance tests. Egg, milk, yeast, and oat were the most common components which triggered immune response in patients required intolerance test. From the 55 patients 36 had positive result for zonulin from which 31 showed immune response to at least one food component. Compared the most common components which triggered immune response in patients with positive zonulin result only oat had significant effect (p=0.041).

IgG hypersensitivity and increased zonulin level may play important role in the pathogenesis of leaky gut syndrome. We recommend further investigation to study the connection between the leaky gut syndrome and defensine levels to establish a more efficient and personalized therapy in the field of diagnostic.

¹Department of Laboratory Medicine, University of Pécs (UP) Medical School (MS), Hungary

²Department of Anaesthesiology and Intensive Therapy, UP MS

³1^st Department of Medicine, Division of Infectious Diseases, UP MS

⁴János Szentágothai Research Centre, UP

SARS-CoV-2 infection may cause an acute and potentially severe disease with 2 % mortality. Since oxidative stress is involved in the pathogenesis of COVID-19, assessing its degree might indicate disease severity. We quantified the non-enzymatic total antioxidant capacity (TAC) of the sera of COVID-19 patients and that of controls by a previously validated enhanced chemiluminescence (ECL)-based TAC method and elucidated its predictive value in COVID-19. We investigated sera of 67 COVID-19 (n=40 ward; n=27 intensive care unit, ICU) patients, 34 controls measured at admission. ICU patients exhibited the highest TAC levels (median: 386.76 µmol/L), whereas ward patients (315.44 µmol/L; p<0.05) and controls (295.39 µmol/L; p<0.01) had lower TAC values. A newly introduced marker, TAC/lymphocyte ratio differentiated better COVID-19 patients (ICU vs. ward patients: 609.77 vs. 292.47 µmol/G; p<0.05) from controls (34.87 µmol/G; p<0.001) than TAC alone. Receiver operating characteristic (ROC) analysis revealed that, besides the classical markers, both TAC [area under the curve (AUC)-ROC: 0.71; p<0.05] and TAC/lymphocyte ratio (AUC-ROC: 0.77; p<0.01) offered predictive values regarding COVID-19 severity. TAC/lymphocyte ratio also predicted the mortality of patients (AUC: 0.79; p=0.001). We suggest that serum TAC and TAC/lymphocyte ratio have predictive capacities in COVID-19. TAC might also serve as a promising severity marker in other systemic inflammatory diseases.

University of Pécs, Medical School, Department of Laboratory Medicine, Pécs, Hungary

Over the past decade, it has become regular to find more and more synthetic addictive drugs in emergency clinical or forensic samples. Recently, as a novelty, in several cases the peaks of mitragynine and its metabolites appeared on the chromatogram of the serum or urine sample of patients. Mitragynine is an alkaloid of the kratom (Mitragyna speciosa) tree. This evergreen plant, similar to the coffee shrub (Rubiaceae), is native to the Far East: Indonesia, Malaysia, Thailand. In these regions, kratom leaves have been used for centuries chewing as stimulating agent (in smaller doses) and tea brewed from dried leaves for its analgesic, euphoric and narcotic effects (in higher doses). It has appeared in USA and Europe only a couple of decades ago. Dried, powdered leaves is sold in alternative shops. Mitragynine binds to μ-opioid receptors, as selective agonist. It is addictive, but the effect is not as dramatic as heroin. It can be used instead of opiates or to help in withdrawal. Kratom in some countries is banned, in Hungary not illegal.

In our laboratory Mitragynine and its derivatives were detected in late 2020 in a patient sample from the emergency department. We could identify them both in serum and urine with our Shimadzu TOXIS.II HPLC-DAD device. Since then, there have been several cases.

Mitragynine concentrations were between 0,05 μg/ml and 1 μg/ml in serum samples. An even wider concentration range was observed in urine samples. In some cases, we could detect only the alkaloids of kratom, in others they occurred together with other agents.

In clinical laboratory toxicology practice, we often find substances in biological samples we have no experience with, so it is necessary to acquire new information and standards. We do not know how often an unexpected result will be repeated.

¹Department of Pharmacology and Pharmacotherapy, Medical School, University of Pécs, H-7624, Hungary,

²Pain Research Institute, University of Liverpool, United Kingdom,

³PharmInVivo Ltd., H-7629, Pécs, Hungary,

⁴Bács-Kiskun County Teaching Hospital, University of Szeged, Kecskemét; Hungary

Complex Regional Pain Syndrome (CRPS) is a severe chronic pain condition that develops after a small injury. The most common symptoms are hyperalgesia, edema, and autonomic disorders. Since its therapy is unsatisfactory, there is a great need to explore the key mediators and identify novel drug targets. However, CRPS is a chronic disease and we aimed to investigate whether the main symptom of CRPS, hyperalgesia, can be sustained in a longer term, experiment of up to 45 days. We planned experiments of 13, 20 and then 45 days. Purified IgG from CRPS patients and healthy volunteers was injected into female C57Bl/6 mice after microinjury (plantar skin-muscle incision) was induced, and the mechanical pain threshold was measured by dynamic plantar aesthesiometer. In the 13-day experiment, we investigated astrocyte and microglial activation also.

After the incision, there was a 45-55 % decrease in the mechanical pain threshold in both treatment groups (CRPS and healthy IgG). In a 13-day experiment, daily IgG treatment caused persistent mechanical hyperalgesia. CRPS IgG increased astrocyte and microglia activation in the spinal cord dorsal horn. In the 20-day trial, hyperalgesia was maintained by day 9, while the 45-day trial resulted in by day 21.

Autoantibody-mediated processes may play a significant role in the early phase of CRPS development, and it is hypothesised that in the chronic stage, minor exposure may be sufficient to maintain symptoms.

University of Pécs, Medical School, Department of Laboratory Medicine, Pécs, Hungary

Suicide with pharmaceuticals is common in our toxicological laboratory practice, but it is usually survived. The most frequent group of poisoning agents are the psychopharmacons. However, they cause death very rarely. The second most usual agent group are the drugs for hypertension and other circulating or heart problems. They are much more life-threatening poisons. One of them is amlodipine which is a Ca channel blocking drug.

We found amlodipine nearly 70 times yearly in the past few years. This drug intoxication sometimes occurs with several other drugs, e.g., betoxolol, benzodiazepines or psychopharmacons. The highest concentration of serum amlodipine was 414 ng/ml in our practice. Therapeutic range is 3-15 ng/ml. The patient was an 84-year-old man, he died in drug poisoning. The amlodipine poisoning was validated after his death in the forensic toxicology laboratory of our university.

The measurement and monitoring of drug concentrations helps doctors to decide which treatment will be the best and to predict the changes of the state of patient.

Department of Immunology and Biotechnology, Clinical Center, University of Pécs Medical School, Pécs, Hungary

Background: There is increasing evidence that in addition to disease-specific effects, vaccines against infectious diseases also exert nonspecific effects (NSEs) on the ability of the immune system to handle other pathogens and may also induce dynamic changes in the autoantibody repertoire. Although epidemiological information regarding the NSEs of vaccines is accumulating, the immune-serological evidence is still scarce.

Aims: Therefore, we decided to investigate potential connections between vaccine efficacy and natural autoantibody (nAAb) levels.

Methods: Clinical residual samples of our cross-border co-operation with Croatia were used for the assessment of MMR and COVID-19 vaccine-elicited immune response (IR) (n=1500), as well as for the investigation of the immunization-associated expansion of the nAAb pool. In-house ELISAs (anti-citrate synthase, anti-MMR) and commercial ELISAs (anti-SARS-CoV-2 ELISAs IgG, IgA, NeutraLISA and IFN-γ release assay ‘IGRA’) were applied.

Results: We found significant differences in the IR given to different COVID vaccines. Moreover, significant positive statistical connections were found between nAAb levels and vaccine induced antibody positivity, suggesting a certain level of plasticity of the nAAb pool in response to immunization.

Conclusion: Our results may support the theorem about the beneficial NSEs of vaccination and may raise further questions regarding individual variability in vaccine responsiveness.

¹Department of Laboratory Medicine, University of Pécs, Pécs, Hungary

²2^nd Department of Internal Medicine and Nephrology-Diabetes Center, University of Pécs, Pécs, Hungary

The coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 infection is characterized by an exceptionally diverse manifestation. Identification of novel laboratory markers could improve diagnostics and therapeutic decision-making. We aimed to investigate the diagnostic and prognostic value of urinary orosomucoid (u-ORM), cystatin-C (u-CYSC) and neutrophil gelatinase-associated lipocalin (u-NGAL) in association with COVID-19. This retrospective study involved 244 adults with confirmed SARS-CoV-2 infection treated at the Emergency Care Unit of University of Pécs. Patients were classified based on different documented clinical outcomes. Blood and urine samples of each participant were taken for routine laboratory testing at the time of admission. U-ORM, u-CYSC and u-NGAL were determined by automated immune turbidimetric assays from samples stored at -80 °C. U-ORM was effective in the prediction of the need for intensive care and artificial ventilation (AUC ROC: 0.864 and 0.834 respectively, p<0.001). Higher levels of u-ORM were observed in non-survivals (medians: 275.9 vs. 68.8 mg/L, p<0.001) and in those who displayed disease progression (191.9 vs. 60.4 mg/L, p<0.001) as well. U-CYSC and u-NGAL were predictive of renal replacement therapy (RRT, p=0.002, p=0.023) and u-NGAL also predicted the development of oligo/anuria (p=0.003). Our findings show that besides conventional inflammatory parameters, u-ORM seems to be a beneficial additional approach in predicting outcomes of COVID-19. U-CYSC and u-NGAL as tubular markers may reflect kidney involvement in COVID-19.

Central Hospital of Southern Pest, National Institute of Hematology and Infectious Diseases, Budapest, Hungary

We developed and validated a novel and reliable High-Performance Liquid Chromatography-Ultraviolet (HPLC-UV) method for determining the antiepileptic drug, levetiracetam (LEV) levels in plasma samples (therapeutic range: 12-46 µg/mL) based on FDA recommendations which is compatible with our Rigol L-3000 system.

LEV was measured by using a reversed-phase (C18) isocratic method at 195 nm. Least squares analysis was used to assess the AUC of the seven calibration curves in each of the eight standard solutions (2.5-100 µg/mL) for determining the correlation coefficient (Y=6.7491 X) of the average curve of LEV (r²=0.9999). During the study of accuracy, precision and sensitivity, we calculated the average concentration [µg/mL], relative standard deviation [%] and average concentration accuracy [ %] by performing five analyses of LLOQ 2.5 µg/mL (2.61 ± 0.1, 3.97, 104.53), LQC 12.5 µg/mL (12.98 ± 0.19, 1.47, 103.84), MQC 25 µg/mL (25.04 ± 0.45, 1.79, 100.15), and HQC 50 µg/mL (50.37 ± 0.1, 0.82, 100.74) as within-run study; and performing five analyses on five days of LLOQ (2.69 ± 0.19, 6.92, 107.57), LQC (12.85 ± 0.29, 2.23, 102.81), MQC (25.34 ± 0.48, 1.88, 101.34) and HQC (50.09 ± 1.45, 2.89, 100.18) as between-run study. Recovery rate was over 97 % after protein precipitation. In the specificity study, LEV was determined in samples containing 50 different drugs, 4 infusion solutions and one dietary supplement without any interference. Whole blood samples were stable for 24 hours at 25 °C and plasma samples for 3 weeks at -20 °C. The stock solution of LEV and caffeine (internal standard) was stable for one year at -80 °C.

Our validation process proved that our novel HPLC-UV method is suitable for the determination of levetiracetam in plasma samples as a routine laboratory test.

Markhot Ferenc Teaching Hospital, Central Laboratory, Eger, Hungary

Objective: We examined what kind of serostatus can be measured in a population that is mostly vaccinated and highly exposed to infection at the end of the third year of the COVID-19 pandemic, in the descending phase in Hungary.

Method: Antibody determinations were made from the venous blood serum of volunteer health workers and their relatives. Reagent: DiaSorin SARS-CoV-2 TrimericS IgG antibody indirect immunoassay (intact homotrimer S protein). Platform: LIAISON XL chemiluminescent immunoanalyzer. We evaluated the results by comparing them with data from the questionnaire (vaccination, infection, transfusion anamnesis, immunosuppressive treatment).

Results: In the examined period (September 2022 - December 2022), a total of 918 people (men: 205 - 22 %, women: 713 - 78 %), age mean 49.7 years (9-97) IgG antibodiy against protein S determinations were carried out. In 16 cases, sampling was done at several times to monitor intercurrent infection or revaccination. In 911/918 (99.2 %) cases, we measured seropositivity above the cutoff. 7/918 (0.8 %) samples proved to be seronegative.

Conclusion: A high proportion of healthcare workers and the population living in family relationships with them have been vaccinated and/or have experienced an infection in the past, which explains the exceptionally high (99.2 %) serological positivity. Among the seronegatives (0.8 %), the humoral response induced by the vaccine could not be verified in 5 samples: one trivaccinated patient is receiving immunosuppressive therapy due to multisystemic inflammation, in 4 cases the vaccination series was incomplete, or 10 months had passed since the last dose. Vaccination was not performed in 2 seronegative cases.

Central Hospital of Southern Pest, National Institute of Hematology and Infectious Diseases, Central Laboratory, Budapest, Hungary

Background: The indirect immunofluorescence assay (IFA) is the gold standard for ANA screening, and it can detect more than 100 different antibodies, such as anti-PCNA as well as anti-cytoplasmic antibodies. AESKU HELIOS® is the first fully automated system that prepares the immunofluorescence slides, takes pictures with a built-in LED microscope and camera, then evaluates them automatically. For ANA and ANCA slides, the HELIOS® automatic also suggests a pattern for the positive samples. The tissue slides are evaluated by the laboratory staff manually based on the pictures taken.

Aims: In our work we would like to present our experience with the automated method.

Methods: On HELIOS^® system we use Hep2/ ANA, ANCA, EMA, LKS IIF slides. In all cases, the results of the automated IIF readings were compared to the manual microscopic results. 172 patient samples were compared for Hep2 slides and 115 for ANCA slides.

Results: For Hep2/ANA slides, 94 % of the samples recommended as negative by HELIOS (112/105) and 97 % of the samples recommended as negative by ANCA (99/96) were also reported as negative by the microscopic evaluation. However, the recommended ANA patterns were only considered valid in 68 % of the samples. In most cases, the preparation of the tissues is adequate and can be well visualised manually with a microscope.

Conclusion: The HELIOS® system can correctly discriminate between ANA and ANCA positive/negative samples. The use of automation reduces the time needed to complete each test. It helps laboratory staff, although manual microscopic evaluation cannot be eliminated, but the time spent on microscopy can be significantly reduced.

¹Semmelweis University, Department of Laboratory Medicine, ²Semmelweis University, Department of Anesthesiology and Intensive Care, ³Semmelweis University, Department of Pharmacy and Pharmacy Administration, Budapest, Hungary

Colistimethate is a controversial last-resort polypeptide antibioticadministered as a prodrug in life-threatening infections caused by carbapenem-resistant Enterobacteriaceae. Due to its severe neuro- and nephrotoxicity and narrow therapeutic window, the therapeutic moni- toring of the active moiety colistin (polymyxin A and B) is crucially important. A method was developed and fully validated for the simul- taneous quantitation of polymyxin A and B concentrations in human serum, ascitic fluid and hemodialysate. All validation criteria were met. Samples could be stored at 2-8 °C, -18 °C and -70 °C for 7 days, 4 weeks and 2 months, respectively, without any sign of polymyxin degradation. In chronic critically ill patients, large inter- and intraindi- vidual variability of systemic colistin exposure was observed. Colistin concentrations in ascitic fluid and hemodialysates attained 63.4±26.1 % and 31.9±6.8 % of those observed in serum, respectively. The systemic exposure to colistin is subject to dynamic changes depending on renal function, the approach to hemodialysis, and the formation of third spaces. The concomittant monitoring of colistin concentrations in these fluid spaces can be an efficient supporting tool for the clinical evaluation of patient status and for making therapeutic decisions.