59th National Congress of the Hungarian Society of Laboratory Medicine

The Hungarian JENDRASSIK-award lecture

Antibody interferences causing false results in the laboratory diagnosis

E. Toldy

Institute of Diagnostics, Faculty of Health Science, University of Pécs, Central Laboratory of Markusovszky University Teaching Hospital, Szombathely

Conventional immunometric assays are usually highly sensitive, but their weakness is the susceptibility to antibody interferences. The presence of interfering substances in patient’s sample may lead to erroneous test results, which can have disastrous consequences for the patient (unnecessary further investigations and therapies, false awareness of illness etc.). This communication presents data focusing on interferences caused by endogenous antibodies against biomolecules (troponin, thyroglobulin, prolactin, TSH), forming macromolecules and changing the reactivity of protein binding methods. In addition, the measurement of some enzyme activities also can lead to erroneous test result (amylase, CK, GOT). The presence of these endogen autoantibodies do not cause disease, but may cause disparate laboratory results. Macro-hormones and macro-enzymes are high molecular weight conjugates of hormones or enzymes. These molecular forms are heterogeneous, but they mostly consist of hormones and anti-hormone autoantibodies. Generally, their half-life is longer and their biological activity is limited. The presence of macromolecules could be confirmed in practice by gel filtration chromatography, however this is not routinely available in most laboratories. PEG-precipitation is more commonly available technique to investigate the macromolecules. Laboratories should do routine screening in cases with previous history of autoantibodies. In any other cases, the clinician should communicate to the laboratory any clinical suspicion of discordance between the clinical and the laboratory data. The author summarizes these interactions of autoantibodies against analyte based on her own practical experiences and the present literature.

The Hungarian JENDRASSIK-award lecture

Why is medical research important also in routine laboratories?

É. Ajzner

Jósa University Hospital, Central Laboratory, Nyíregyháza, Hungary

Research activities are traditionally linked to academic environment in Hungarian laboratories. However, the shift from a knowledge-based to a problem-solving medicine with the need for adaptation to evolving technology are increasing the need for more research that is put to practical use (applied research) also in hospital/routine laboratories. In the presentation some examples of applied research that were initiated and run partly or completely in hospital environment will be discussed with a goal to demonstrate that applied research is feasible also in routine laboratories.

The clinical need of causative treatment of bleeding patients resulted in molecular genetic and biochemical characterisations of rare haemostasis disorders (Factor V and XIII deficiencies), which will be presented in the first group of studies. The next study will be about developing a method for intraoperative monitoring anticoagulation with hirudin of a patient suffering from heparin-induced thrombocytopenia. The last group of studies in the presentation will be based on case histories with relevant laboratory test results and will demonstrate investigations on how laboratory tests playing crucial role in the investigated clinical decision-making are applied in everyday practice.

Scientific research and scientific co-operation with physicians are indispensable requirements in the training program for the speciality of laboratory medicine in Europe. Thus, applied research activity could be integrated also into routine responsibilities of specialists of hospital laboratories. In the author’s view, the more successful the laboratories in answering clinical questions with assistance of applied research the better their pivotal role on healthcare delivery recognised in the future which can possibly also improve both visibility and appreciation of laboratory medicine among medical professions.

Clinical significance of low cholesterol and high

7-dehydrocholesterol in inherited metabolic disorders

A. V. Oláh¹, G. P. Szabó², I. Balogh¹

¹ University of Debrecen, Department of Laboratory Medicine,

²Sport Diagnostic, Living and Therapy Center, Debrecen, Hungary

Smith-Lemli-Opitz syndrome (SLO) is a multiple congenital anomaly with severe mental retardation due to 7-dehydrocholesterol reductase (7DHCR) deficiency. Low cholesterol (Cho) and high 7DHC lead to developmental disorders or miscarriage. Therefore, we introduced a photometric 7DHC assay in 2002, and prenatal genetic testing in 2009. We studied the relation between the phenotype and biochemical markers. According to Kelley’s clinical scores, 15 patients (age: 0-18 years) were divided into three phenotypes. Lipid parameters were monitored four times yearly. Deficiency of cholesterol correlates well with severity scores (r=0.793). In typical SLO Cho was decreased (1.47±0.7 mmol/L) and 7DHC was high (202±77 mg/L, reference range of 7DHC <0.15 mg/L). In severe SLO Cho was extremely low (0.64±0.2 mmol/L) with elevated 7DHC (180±52 mg/L). Initial serum Cho value below 1 mmol/L or Cho/7DHC<2 suggests bad prognosis, life expectancy is less than one year. Cholesterol supplementation elevates Cho/7DHC ratio which improves behaviour, great motor skill, walking distance, sleep patterns. Statins might inhibit the synthesis of DHC, but it was suspended, when the liver function impaired. Average activities of ALT and AST were approx. 2 times higher in severe and typical SLO than in mild type. Recently we evaluated a special GC-MS method for detection of 7DHC in 15 carriers. We detected slightly elevated serum 7DHC (0.3-5.5 mg/L) in 12 carriers, but 7DHC was normal in 3 carriers.

Lipid markers have prognostic value in the classification of SLO. Although therapeutic results are limited, monitoring of liver function is necessary. Extremely low Cho level in a pediatric sample compared to the age-dependent reference range can be considered as pathological.

Urinary orosomucoid as a novel laboratory marker of the inflammatory activity in Crohn’s disease

B. Szirmay¹, P. Kustán¹, Z. Horváth-Szalai¹, A. Tárnok², P. Sarlós³,

N. Szigeti⁴, A. Ludány¹, T. Kőszegi¹

¹Department of Laboratory Medicine, ²Department of Pediatrics,

³1st Department of Internal Medicine, ⁴2nd Department of Internal

Medicine and Nephrology Centre, University of Pécs, Medical

School, Hungary

Crohn’s disease (CD) as a chronic inflammatory bowel disorder requires a lifelong patient care. Laboratory markers have an essential role in the management of CD. We aimed to measure urinary levels of orosomucoid in relation to the inflammatory activity of CD and to compare it with clinical scores and conventional laboratory parameters. Blood and urine samples of 55 adult and 31 pediatric patients with CD and 68 healthy individuals as controls were analyzed. Patients were categorized according to their clinical indices (Harvey-Bradshaw Index (HBI) or Pediatric Crohn’s Disease Activity Index (PCDAI)). Urinary orosomucoid (u-ORM) was measured by automated immune turbidimetric assay and values were referred to urinary creatinine (u-ORM/u-CREAT, mg/mmol). U-ORM/u-CREAT values showed a 7-fold elevation in children with active CD (0.50 vs. 0.07 mg/mmol, p<0.001) and a 2-fold elevation in adults (0.32 vs. 0.14 mg/mmol, p=0.01) compared to patients with inactive disease. U-ORM/u-CREAT correlated well with conventional inflammatory markers (hs-CRP, serum ORM, WBC; p<0.001) and activity scores (HBI, p=0.018; PCDAI, p<0.001). U-ORM/u-CREAT had similar discriminative performance to hs-CRP and serum ORM in the differentiation of active from inactive pediatric CD patients. Our findings suggest that urinary quantification of ORM can provide valuable information for the follow-up of CD patients. The automated measurement can give results rapidly and the non-invasive property of sampling is especially advantageous for children.

Development and application of a liquid chromatography-mass spectrometry method to create population pharmacokinetic models of atorvastatin and rosuvastatin – demonstration of the pivotal role of the clinical laboratory in precision pharmacotherapy

G. Karvaly¹, K. Kovács¹, T. Holczer¹, I. Karádi², A. Zsáry², B. Vásárhelyi¹

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

² 3^rd Department of Internal Medicine, Semmelweis University, Budapest, Hungary

The antihyperlipidaemic substances atorvastatin and rosuvastatin are among the most widely prescribed entities worldwide. To assist their administration in the framework of precision pharmacotherapy, an ultra-high performance liquid chromatography-tandem mass spectrometry method was developed and validated both according to the guideline of the European Medicines Agency and the requirements set forth for modeling using the Pmetrics™ package of R and the BestDose™ drug therapy individualization software (Laboratory of Applied Pharmacokinetics and Bioinformatics, University of Southern California, Los Angeles, USA) [1]. Assay error polynomials were constructed separately for hemolytic, hyperbilirubinaemic and lipaemic specimens and showed that assay performance deteriorated when it was applied to lipaemic samples but was not influenced by hemolysis and high bilirubin content. The method was employed for the construction of the nonparametric Bayesian population pharmacokinetic models of atorvastatin and rosuvastatin in hyperlipidemic patients. This work demonstrates the fundamental role of clinical laboratories in precision pharmacotherapy.

[1] Jelliffe R, Neely MN (eds). Individualized drug therapy for patients. 1^st edition. Elsevier, 2017.

Macrophage activation syndrome. Complicated disease diagnosed with “routine” laboratory tests

B. Fodor^1,2, I. Gilányi², R. Simon² G. Marton²

¹University of Miskolc, Faculty of Healthcare Studies, ²BAZ-County’s Central Hospital and University Teaching Hospital, Miskolc, Hungary, University of Miskolc, Miskolc, Hungary

Introduction: Monocyte Activation Syndrome (MAS) is a rare, but potentially life-threatening, predominantly childhood disease. Its primary characteristic is the uncontrolled regulation of T cell and monocyte activation, leading to cytokine storm. It is commonly associated with sJIA, SLE, dermatomyositis and Kawasaki disease. Secondary hemophagocytic histiocytosis (HLH) also may develop on the basis of MAS.

Patients and methods: In this study we present 3 cases, two of them suffering from secondary HLH (due to infection and immunodeficiency) and a child from primary HLH. In all three children, the MAS persisted for several months and according to the activity of disease following laboratory values were measured respectively: ferritin> 7000, CRP:> 300 mg/l, D-dimer:> 6000 ng/ml, fibrinogen: 1.95 g, ASAT:> 100 U/l, ALT:> 80 U/l, se. cholesterol:> 7 mmol/l, se. triglyceride:> 4 mmol/l, se. LDH:> 3000 U/l, S100:> 20 mg /l (ref. value: 0 to 0.105!!).

Discussion: persistent, therapy-resistant infection, hemorrhagic conditions, high fever, etc., should raise the possibility of HLH and/or MAS. The “routine” laboratory have a fundamental role in the recognition of these (potentially fatal) disorders. According to the current diagnostic criteria, high ferritin levels, decreased platelet counts, triglyceride levels above 3 mmol/l, ASAT in excess of 48 U/l, and decreased fibrinogen levels are certainly directed to MAS. Furthermore, according to our experiences elevated S100 protein and LHD level is an additional sensitive marker of MAS.

Investigation of estrogen metabolism in pregnancy

K. Kovács, B. Vásárhelyi, I. Kocsis, G. B. Karvaly

Department of Laboratory Medicine, Semmelweis University, Budapest, Hungary

Estrogen metabolism takes place along three pathways in humans, yielding several substances with notable physiological effects. Due to the technological complexities, very few authors have published approaches for the determination of estrogen metabolome so far. These authors focused on postmenopausal women with tumors, inflammatory diseases or autoimmune processes.

The levels of the systemic estrogen metabolome during pregnancy have not been addressed so far. In our research, the estrogen profile was evaluated in plasma samples collected from pregnant women (n=189). To this end, a sensitive and high-throughput method was developed and validated for the simultaneous analyses of the free and conjugated levels of estrogen hormones and 13 metabolites in plasma samples using two-dimensional ultrahigh performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS).

The estrogen hormones and their metabolites were found predominantly in conjugated form, except for estradiol, 2-methoxyestrone and 2-methoxyestradiol, which proved to be present mainly as free substances, unlike in earlier studies. All estrogens except 17-epiestriol, 4-methoxyestrone and 4-methoxyestradiol, showed typically, but not in all cases, a significant increase in their levels by the 24^th gestational week. The predominant pathway was hydroxylation on carbon atom 16, which is in line with earlier observations.

Our conclusion is that monitoring the estrogen metabolome can be useful for the early diagnosis of adverse events in pregnancy.

Our work was supported by the National Office for Research, Development and Innovation through the National Bionics Program. (Government Regulation No. 1336/2017)

Analysis of plasma human epididymis protein 4 (HE4) for monitoring CFTR potentiator ivacaftor therapy in cystic fibrosis

B. Nagy Jr¹, Z. Bene², Z. Fejes¹, S. L. Heltshe³, N. J. Ronan⁴, E. Joseloff⁵, S. C. Bell⁶, G. Balla², M. Macek Jr⁷, B. J. Plant⁴, M. D. Amaral⁸, J. Kappelmayer¹, I. Balogh¹

¹Dept. of Laboratory Medicine, University of Debrecen, Debrecen, Hungary; ²Dept. of Pediatrics, University of Debrecen, Debrecen, Hungary; ³Dept. of Pediatrics, University of Washington, School of Medicine, Seattle, Washington, USA; ⁴Cork Adult Cystic Fibrosis Centre, Cork University Hospital, Cork, Ireland; ⁵Cystic Fibrosis Foundation, Bethesda, Maryland, USA; ⁶QIMR Berghofer Medical Research Institute and The Prince Charles Hospital, Brisbane, Australia; ⁷Dept. of Biology and Medical Genetics, Charles University, Prague, Czech Republic; ⁸University of Lisbon, BioISI-Biosystems & Integrative Sciences Institute, Lisbon, Portugal

There are no reliable blood biomarkers for monitoring CFTR modulating therapy efficacy in cystic fibrosis (CF) as yet. In this clinical study, 60 CF patients in 3 independent cohorts were enrolled who carried at least one Class III CFTR mutation (p.Gly551Asp) and were treated with CFTR potentiator ivacaftor. Plasma HE4 levels were retrospectively measured before treatment and after 1-6 months of ivacaftor therapy. FEV₁ values, sweat chloride and C-reactive protein (CRP) were correlated with HE4. HE4 levels were significantly lower after 1 month than at baseline and sustainably decreased up to 6 months. Significant negative correlation between HE4 and FEV₁ (r=-0.5376; P<0.001), and positive correlation with sweat chloride (r=0.2411; P=0.001), and CRP (r=0.4613; P<0.001) were observed. HE4 had a significant ROC-AUC value (0.722 95% CI: 0.581-0.863; P=0.029) to predict improved lung function by ivacaftor. Overall, the severity of lung disease can be assessed via plasma HE4 measurement in CF patients under ivacaftor therapy.

Protein O-GlcNAc modification; implications for laboratory medicine

T. Nagy

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

O-linked N-acetylglucosamine or O-GlcNAc is the result of a post-translational modification that involves the reversible transfer of an N-acetyl-glucosamine molecule to a Ser/Thr OH-group of intracellular proteins. The number of proteins known to undergo O-GlcNAc modification is rapidly increasing; transcriptional factors, nuclear pore proteins, cytoskeletal proteins, chaperons, etc. O-GlcNAc modification is essential for several cellular functions thus its dysregulation may lead or contribute to pathological events. It is also linked to cellular metabolism since the production of its substrate, UDP-GlcNAc is influenced by the availability of various metabolites such as glucose and glutamate.

Since the formation of O-GlcNAc is a highly dynamic process and it seems to be connected to crucial intracellular regulatory events, analysis of actual O-GlcNAc levels in biological samples could be of diagnostic value. Analysis of O-GlcNAc has been proposed for the detection, assessment and monitoring of various diseases including diabetes, Alzheimer’s disease, malignancies and inflammation. O-GlcNAc may also inform about the stress-tolerance of otherwise healthy individuals (e.g. in exercise physiology).

Several techniques have been developed to measure O-GlcNAc, such as protein electrophoresis, flow cytometry or mass spectrometry. However, these methods are predominantly used in research only and relatively little work has been done to implement them in the clinical laboratories. In this presentation, advantages and disadvantages of various detection approaches are demonstrated and the potential diagnostic value of O-GlcNAc analysis is discussed. We also present current data of our own efforts to develop and validate a quantitative laboratory test for O-GlcNAc.

Novel immune turbidimetric detection and predictive values of serum actin-binding proteins in sepsis

Z. Horváth-Szalai¹, P. Kustán¹, B. Szirmay¹, T. Huber³, B. Bugyi^3,4, D. Mühl², A. Ludány¹, A. Miseta¹, T. Kőszegi^1,4

¹Department of Laboratory Medicine, ²Department of Anaesthesiology and Intensive Therapy, ³Department of Biophysics, ⁴Szentágothai Research Centre, University of Pécs, Hungary

Sepsis is a multifaceted clinical syndrome where both clinical and laboratory parameters are mandatory for rapid intervention. Serum gelsolin (GSN) and Gc globulin are essential actin scavenger proteins, the depletion of which could contribute to the development of organ failures in sepsis. We sought to validate a new, automated immune turbidimetric assay for se-GSN and to determine the predictive values of se-GSN and Gc globulin in sepsis when comparing them to classic clinical and laboratory parameters. Validation of se-GSN assay was performed according to the second edition of Eurachem guidelines. Serum GSN and Gc globulin levels were determined by automated immune turbidimetric assay on a Cobas 8000/c502 analyzer. Septic (n=46) and non-septic (n=28) patients were followed for 5 days, while 35 outpatients served as controls. Patients were retrospectively categorized according to the sepsis-3 criteria, and 14-day mortality was also assessed.

In our new se-GSN assay, low sample volume (7 μL), 0.72 mg/L detection limit and 10-minute assay time were achieved. Similar to first-day procalcitonin (ROC area under the curve: 0.98), GSN also differentiated sepsis from non-sepsis (0.88). In addition to first-day Sequential Organ Failure Assessment (SOFA) clinical score (0.88), GSN (0.71) also discriminated septic survivors from non-survivors, whereas septic shock was predicted by lactate (0.99) and Gc globulin (0.76). Higher Gc globulin levels were measured in sepsis than in septic shock (p<0.01) all along the follow-up period. Consequently, both GSN and Gc globulin seem to be valuable predictive markers in sepsis which may facilitate clinical decision making.

Clinical and laboratory aspects of novel oral anticoagulants; novelties and experience of a university laboratory

Z. Bereczky, A. Kerényi, A. Veszprémi, E. Nagy, R. Szabó, M. Zoaka, É. Ajzner, J. Kappelmayer

University of Debrecen, Faculty of Medicine, Department of Laboratory Medicine and Division of Clinical Laboratory Science, Debrecen and A Jósa Teaching Hospital, Nyíregyháza, Hungary

The novel oral anticoagulants (NOACs) are now widely used. Among them dabigatran, rivaroxaban, apixaban and edoxaban are now approved for various clinical indications using different dosing schemes. Elimination rates of them are differently dependent on kidney function. Laboratory monitoring of NOACs is not required routinely, however in certain clinical situations the knowledge of their plasma concentration is necessary. The coagulation screening tests (PT and APTT) are not recommended for monitoring, however – depending on their sensitivity – some PT or APTT reagents could be useful for demonstrating the presence of the drug in the plasma in emergency situations.

A survey was performed about the practice of NOACs measurements by a questionnaire sent to Hungarian laboratories. Additionally, we analyzed the NOACs levels of 187 patients and compared the sensitivity of 4 different PT and 3 APTT reagents to each other and to our routine reagents in normal pooled plasma spiked with dabigatran, rivaroxaban and apixaban and in “real life” patients.

According to the results of the questionnaire NOACs are measured only in 7 laboratories with different methods and with heterogeneous interpretations. The NOACs concentrations in „real-life” patients are in a wide range both for peak and trough levels that may have an impact on clinical outcome. The different PT and APTT reagents showed highly different prolongations in the presence of the same drug’s same concentration, moreover, some of them did not show significant prolongations even at high doses. None of the tested PT and APTT reagents were suitable for detecting apixaban. These findings underline the responsibility of the laboratory to have knowledge about the behavior of their reagents in the presence of NOACs.

Bereczky Z, Oláh Z, Ajzner É, Kappelmayer J. Orv Hetil 2017;158:1930-45.

Clopidogrel therapy: how to monitor?

Z. Bagoly

University of Debrecen, Faculty of Medicine, Department of Laboratory Medicine, Division of Clinical Laboratory Sciences, Debrecen, Hungary.

Dual antiplatelet therapy with aspirin and a P2Y12 ADP receptor antagonist is the cornerstone treatment in the prevention of recurrent ischemic events. Although randomized clinical trials have proved that newer P2Y12 inhibitors, e.g. prasugrel and ticagrelor are more effective in acute coronary syndrome, clopidogrel is still the most widely used P2Y12 inhibitor because it causes fewer bleeding events, it is well tolerated and costs less. Nevertheless, patients treated with clopidogrel have substantial variability in their platelet inhibition, and clinical studies suggest that approximately 30% of patients are resistant to clopidogrel therapy. Poor response to clopidogrel can be detected by various laboratory methods and can be at least partly explained by genetic polymorphisms encoding CYP2C19, the hepatic enzyme involved in the biotransformation of clopidogrel to its active metabolite. Some experts advocate individualizing P2Y12 inhibitor therapy based on the laboratory results of platelet function tests and genetic polymorphisms. Here we focus on our current understanding of the value of laboratory monitoring and genetic testing to identify poor responders to clopidogrel. Advantages and disadvantages of laboratory tests that are specific/non-specific to P2Y12 inhibition will be highlighted with an emphasis on tests suitable for testing patients on dual antiplatelet therapy (not influenced by concomitant aspirin treatment). The role of detecting CYP2C19 loss-of-function alleles (*2 and *3) and gain-of-function alleles (*17) will be explained based on the literature and on our personal experiences when testing Hungarian patients.

Introduction of an automated global coagulation assay, the coagulation inhibitor potential method (CIP)

B. Réger, H. Losonczy, Á Nagy, E. Kátai, Á. Péterfalvi, N. Farkas, A. Miseta, O. Tóth

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

Thrombophilia can be defined as an increased, persistent tendency to hypercoagulability and venous thrombosis. Diagnosis of hereditary thrombophilia using the current routine laboratory methods is time consuming and expensive. A single laboratory test for detecting multifactorial thrombophilia would be useful. The Coagulation Inhibitor Potential (CIP) assay is considered to be a promising global thrombophilia screening test. In the CIP assay, which is a modified version of the Overall Haemostatic Potential (OHP) method developed by Blombäck at al., pentasaccharide and Protac are added to the plasma in order to enhance the inhibition of coagulation by accelerating the activation of antithrombin and protein C. The aggregation of fibrin after the addition of tissue factor is monitored and compared to aliquots in which inhibition is not enhanced.

The aim of the study was to adapt the manual application of the original CIP method to an optical coagulation analyser (ACL family of Coagulation Analyzers from Instrumentation Laboratory). By this automation the test would be easier, faster and more precise allowing the performance of simultaneous analyses of up to eighteen samples. The CIP assay could be used to detect a thrombophilic condition in blood samples by monitoring fibrin formation and measuring the inhibition of coagulation. We measured the CIP values of known thrombophilia patients and healthy controls. We studied the analytical performance, cut-off value and usefulness of the test in the diagnosis of hereditary thrombophilias. The new method seems to be appropriate and reliable for the detection of antithrombin-, protein C- and protein S deficiencies, homozygous FV Leiden mutation and also for combined deficiencies.

Novel approach to the laboratory diagnosis of antibodies against coagulation factors, with special reference to anti-factor XIII antibodies

L. Muszbek,¹ K. Pénzes,¹ É. Katona,¹ M. Kun,¹ A, Bonnefoy,² Z. Vezina,² N. Szuber,² K. Rázsó,³ Z. Bereczky,¹ A. Kerényi,¹ B. Bécsi,⁴ F. Erdődi,⁴ GE. Rivard²

University of Debrecen, Faculty of Medicine, Debrecen, Hungary: ¹Division of Clinical Laboratory Science, Department of Laboratory Medicine, ³Department of Internal Medicine, ⁴Department of Medical Chemistry, ²CHU Sainte-Justine, Montréal, QC, Canada

Factor XIII (FXIII) consists of two catalytic A subunits and two inhibitory/protective B subunits. Alloantibodies against FXIII subunits rarely develop in inherited FXIII deficiency but make its management very difficult. Acquired FXIII deficiency due to anti-FXIII autoantibodies is a rare but severe bleeding diathesis usually with life-threatening soft tissue bleedings. Neutralizing anti-FXIII-A antibodies may interfere with FXIII activation and/or inhibit activated FXIII (FXIIIa), while non-neutralizing antibodies accelerate the clearance of FXIII. The traditional method for detecting neutralizing antibodies is the mixing study and its semi-quantitative variant, the Bethesda-Nijmegen assay. There is no established method for the diagnosis of non-neutralizing antibodies. Our aim was to introduce new methods in the diagnosis and classification of anti-FXIII antibodies. For quantitatively assessing the inhibitory capacity of an antibody we determined the 50% inhibitory concentration (IC50). For the determination of the antibody’s binding affinity to FXIII and its subunits surface plasmon resonance measurement was introduced. For targeting the effect of neutralizing antibody, a three-step assay system was designed. The cleavage of FXIII-A by thrombin was investigated by Western blotting, the effect on Ca²⁺ induced activation and FXIIIa was investigated by modified FXIII activity assays. The clearance of FXIII was assessed by activity measurement following the administration of FXIII concentrate. Anti-FXIII-A antibodies from three patients were characterized using the above methods. The IC50 for IgGs varied between 0.05 and 0.34 mg/mL. The antibodies bound with the same high affinity (Ka: 10⁸-10⁹ M^-1) to FXIII-A₂ and FXIII-A₂B₂, but not to FXIII-B₂. The three-step activity assay and the clearance study revealed different types of anti-FXIII-A antibodies: the alloantibody and one autoantibody exerted a combined effect; they inhibited FXIII activation and FXIIIa activity and accelerated the clearance of FXIII. The main effect of a second autoantibody was the inhibition of FXIIIa. Introduction of new methods in assessing anti-FXIII antibodies improved their functional characterization and allowed us to design a new classification.

When modern technologies encounter: optical illusion?

Two cases of ‘pseudo-eosinophilia’.

A. Peterfalvi, A. Békési, A. Jauk, I. Litter, A. Miseta

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

Absolute and relative eosinophilia may be due to several reasons, including allergies and asthma, eczema, parasitic infections, hematopoietic disorders. Here we present two cases with eosinophilia detected optically with an automated clinical hematology analyzer, but with no eosinophilia when checked for in a blood smear with light microscopy.

Case 1 is a 61-year-old woman undergoing endovascular embolization of a cerebral arteriovenous malformation with Onyx® Liquid Embolic System. This consists of 3 components: ethylene vinyl-alcohol copolymer (the embolic material), dimethyl-sulfoxide (solvent) and micronized tantalum powder (for radiographic visualization). In the laboratory the patient’s blood was presented with 42.7% eosinophilia in an automated analyzer the day following the neurosurgical procedure, which proved to be 1% with light microscopy. The day before the operation no eosinophilia was detected with the analyzer. The ‘pseudo-eosinophilia’ could be detected for more than a week.

Case 2 is an 80-year-old man with bladder cancer undergoing urethrocystoscopy and transurethral resection of the tumor and the prostate gland with laser vaporization. Similarly to the previous case, 40.2% eosinophilia was measured the next day, while no eosinophilia but neutrophil granulocytes with regular morphology could be seen with light microscopy.

Despite the lack of the full understanding and explanation of the underlying mechanisms that led to the false measurement results – ‘pseudo-eosinophilia’ –, our objective is to draw the attention to the possibilities of such cases, and to highlight the importance of double-checking eosinophilia in order to report true lab results.

Hematologic markers of platelet activation in atrial fibrillation

A.H. Shemirani^1,2, Z. Csanádi³, O. Hajas³, N.K. Tóth², A Kiss³, E Nagy-Baló³, F. Sarkady², L Csiba^4,5, Z. Bagoly^2,5

¹Erzsébet Hospital Central Laboratory, Sátoraljaújhely, Hungary; ²Division of Clinical Laboratory Science, Department of Laboratory Medicine, ³Institute of Cardiology, ⁴Department of Neurology University of Debrecen, Faculty of Medicine, Debrecen, Hungary; ⁵ MTA-DE Cerebrovascular and Neurodegenerative Research Group, Debrecen, Hungary

Atrial fibrillation (AF) is the most common arrhythmia associated with a high risk of thromboembolism. We studied the relation between the risk of stroke in AF as defined by the most commonly used clinical scoring system (CHA₂DS₂-VASC) and mean platelet volume (MPV) and platelet distribution width (PDW) as indicators of platelet activation. Patients were recruited between September 2014- January 2017. We enrolled 86 (56 male, 30 female) patients with AF. Mean age was 55.5±10.2 years. 31 patients received anticoagulants, all were antiplatelet-free. Patients were divided into 2 groups based on their risk for stroke according to CHA₂DS₂-VASC (0-1 score “low risk”: group 1 and 2-6 score “high risk”: group 2). Group 2 had higher MPV than group 1 (10.3±0.9 vs. 11.0±0.9 fL, P=0.002). PDW demonstrated the same trend (12.6±1.9 vs. 13.9±1.9 fL, P=0.005). Platelet count (PLT) showed no significant difference between two groups (223±63 vs. 212±53, P=0.430). PLT had no significant correlation with MPV, but showed an inverse correlation with PDW (r=-0.233, P<0.05). A positive correlation between MPV and PDW was also observed (r=0.944, P<0.001). Soluble E-selectin levels measured from platelet poor plasma revealed no correlation with MPV, PDW or PLT. C-reactive protein showed a weak correlation with PLT only (r=0.289, P=0.012). Our results indicate that platelet size and activation markers such as MPV and PDW are associated with an elevated risk of stroke in AF patients.

Acute myeloid leukemia (AML): evaluation of genetic background and stability during relapse

P. Kövy¹, N. Meggyesi¹, A. Bors¹, A. Kozma¹, E. Ádám¹, J. Dolgos², N. Lovas², I. Vályi Nagy², P. Reményi², H. Andrikovics¹

¹Laboratory of Molecular Genetics, Central Hospital of Southern Pest (DPC), Budapest, Hungary; ²Dept. of Hematology, DPC, Budapest, Hungary

AML is a malignant hematopoietic stem cell disorder. The WHO classification (2016) is based on genetics including molecular- and cytogenetic factors. The aim of our study was to assess the frequency and stability of cytogenetics, fms-like tyrosine kinase 3 (FLT3) and nucleophosmin1 (NPM1) mutations. We applied standard karyotyping, FISH, PCR followed by fragment analysis or RFLP at diagnosis and relapse in a cohort of 491 adult patients (106 diagnosis and relapse sample pairs). The frequency of t(15;17) was 8.8% (n=43), t(8;21): 4.9% (n=24), inv(16): 4.9% (n=24), normal karyotype (NK): 40.5% (n=199), t(v;11q23): 2.6% (n=13) and complex karyotype: 14.9% (n=73). FLT3-ITD positivity was 22% (108/491) in the whole cohort and 34.7% (69/199) in normal karyotype (NK) AML. FLT3-TKD occurred in 7.8% (37/473) most commonly with inv(16) (18.2%). NPM1 positivity was 24.4% (118/484), which increased in NK-AML to 49.2% (97/197). NPM1 was mutually exclusive with t(15;17), t(8;21), inv(16), t(v,11q23) in accordance with WHO2016 classification. Stable karyotype was observed in 67%, clonal regression in 9% and clonal evolution in 24% out of 100 sample pairs. FLT3-ITD status proved to be stable in 93.4%, regressed in 1.9% (n=2/106) and progressed in 4.7% (n=5/106). Similar ratios for regression were detected in case of NPM1 [1.9% (2/106)], but no emergence of NPM1 happened at relapse. In summary, our data showed great agreement with expected frequencies known from the literature. The stability of molecular genetic markers is comparable to that of cytogenetics. The project was funded by NVKP_16-1-2016-0005 and NKFIH K104903 grants.

Do we know enough about microbes including viruses?

G. Reuter

University of Pécs, Dept. Med. Microbiol Immunol, Pécs, Hungary.

Our knowledge about microbes including viruses is far from complete and, nowadays, it is changing fundamentally. Beside the classical clinical view that “viruses are (human) pathogens”, there is increasing knowledge that viruses are the most abundant, (bio)diverse life-like entities in the biosphere and are “major players in Earth’s early life and in the present-day ecology and evolution of life”. In addition, the microbiome – community of more than 100 trillion microorganisms – that colonizes the human body, contains 100-400 times more genes than our genome, helps keep us alive. These host-microbe interactions influence the host normal physiology and disease in several, mostly presently unknown ways. The modern up-to-date methods like viral metagenomics, high-throughput sequencing and bioinformatics provide novel opportunities to generate an unbiased characterization of the viral population (virome) in various organisms and environments. This review summarizes the current knowledge related to viruses in the light of the results of the modern detection techniques.

Novel neuroinvasive astrovirus infections in pigs with encephalomyelitis, weakness and paralysis in Hungary

Á. Boros, M. Albert, P. Pankovics, H. Bíró, PA. Pesavento, TG. Phan, E. Delwart, G. Reuter

Government Office of Baranya County, Public Health Department, Laboratory of Virology, Pécs, Hungary.

Astroviruses (AstVs) with a single-stranded RNA genome are generally known to be associated with gastroenteritis in humans and other vertebrates including pigs. Recent studies indicate the extra-intestinal presence and novel neuroinvasive (Ni) potential of certain AstV strains in human as well as in mink, ovine and bovine cases. Although research data about the genomic diversity and host species spectra of Ni-AstVs is continuously increasing little is known about the disease course, potential transmission and neuropathology of these viruses especially not in pigs.

Our research group has recently discovered a novel neuroinvasive porcine AstV (Ni-PoAstV) strain which belongs to the PoAstV type 3 genotype, in multiple newly weaned pigs, showing signs of encephalomyelitis, posterior weakness and paralysis, during prolonged outbreaks in Hungary taking place between 2011 and 2017. Our results indicate that Ni-PoAsV-3 can cause viremia and disseminated infections involving certain parts of the central nervous system and multiple internal organs. Furthermore, Ni-PoAstV-3 was generally undetectable in the faeces/intestinal samples, while it could be detected frequently in the respiratory system of affected pigs raising the possibility of the respiratory route of transmission/infection of this virus. Histopathological changes including neuronal necrosis and microgliosis could be observed in the brainstem, cerebellum and spinal cord, where the neuronal localizations of viral RNA could also be detected by in situ hybridization. Based on these data, Ni-PoAstV-3 could be associated with prolonged outbreaks of encephalomyelitis and could have a veterinary and economical importance by threatening livestock.

The clinical significance of human parechovirus infections in newborns and infants

Zs. Hamarics, Á. Boros, Z. Liptai, Z. Nyul, P. Pankovics, G. Reuter

University of Pécs, Dept. Med. Microbiology and Immunol, Pécs, Hungary.

Human parechoviruses (HPeVs, family Picornaviridae) can cause gastroenteritis but most of the infections are asymptomatic. Recent reports show that HPeVs could also be associated with severe central nervous system symptoms and sepsis-like syndromes in infants. The clinical significance of HPeVs in Hungary had not been investigated before. The aim of this retrospective study was the detection and typization of HPeVs in faecal samples of children using RT-PCR and sequencing methods. The faecal samples collected from children with unknown gastroenteritis were divided into three groups: group A) hospitalized children younger than 10 years of age (N=75); group B) infants (N=208) and group C) children younger than 18 years of age, with sepsis-like/neurological symptoms. HPeV was found in 3 (4%) of the 75 specimens in group A (HPeV1 N=2; non-typeable N=1) from infants between the age of 7 and 11 months. In group B, HPeV was detected in 14 (6.7%) of the 208 samples (HPeV1 N=5; HPeV3 N=4; non-typeable N=5). While most of the infants with mild HPeV1 infection (4/5) required only home care, 85% of the HPeV3 infected infants (3/4) required hospitalization in this group. In group C, 4 HPeV strains were detected (HPeV1 N=2; HPeV3 N=2) in ≤2-months-old infants hospitalized with sepsis-like/neurological symptoms. The leading symptom of HPeV1 was diarrhoea, although heart murmur and neurological symptoms (somnolence, languor) also occurred in infants. HPeV3 infections were more common among newborns. The main symptoms of severe HPeV3 infection were: gastroenteritis (5/6), fever ≥39^oC (5/6), lack of appetite (5/6), somnolence/languor (3/6), rash (2/6), respiratory symptoms (2/6) and sepsis-like syndrome (2/6). HPeV1 infections were more common and less severe in infants than HPeV3 infections.

Salivirus outbreak in newborn babies with acute gastroenteritis in a neonatal hospital unit in Hungary

N. Bolba, Á. Boros, M. Raáb, É. Károly, A. Karai, A. Kátai, P. Pankovics, G. Reuter

University of Pécs, Dept. Med. Microbiol Immunol, Pécs, Hungary.

Each year, 1.5 million people die because of diarrheic infectious diseases in the World, of which the etiological agent is unknown in 40% of the cases. Salivirus is a non-enveloped picornavirus (family Picornaviridae) with positive, single-stranded RNA genome, discovered in 2009. Salivirus could be an etiological agent of gastroenteritis in humans. The aim of this retrospective study was to identify the etiological agent of an outbreak of gastroenteritis with unknown etiology by molecular methods. Between July 7 and 13 2013, 5 (26%) of the exposed 19 newborns between age of 1.5 and 5 days had symptoms of acute gastroenteritis (diarrhea 100%, fever 40%, vomiting 40%, loss of appetite 40%) in a neonatal hospital unit in Bács-Kiskun county. The routine laboratory diagnostic tests were negative for bacteria (Salmonella spp., Shigella spp., Campylobacter spp., E. coli, Y. enterocolitica, S. aureus), viruses (rotavirus, adenovirus, norovirus, sapovirus, astrovirus, Aichi virus, human parechovirus) and parasites. Two years later, salivirus was detected from the archived faecal samples with 99.7% nucleotide identity to each other in the immunodominant viral capsid protein (VP1) region. This is the first report of the determination of the complete (8021nt long) viral genome sequence of salivirus (KT240115). High viral loads (2.1-2.6x10⁹copies/g) were detected in newborn faeces by real-time RT-PCR method. Newly discovered virus could be identified in archived specimens collected from an outbreak of gastroenteritis with unknown origin. The high viral nucleic acid copy numbers and the highly identical viral genome sequence support the etiological role of salivirus in this outbreak. This is the first documented outbreak of gastroenteritis associated with salivirus in the literature.

Is culture an obsolete method?

Gy. Mestyan, K. Kovacs, A. Nyul, S. Melegh

Department of Medical Microbiology and Immunology, University of Pécs, Pécs, Hungary

The backbone of the daily practice of bacteriological diagnostics is still determined by the principles and laboratory procedures worked out in the 19th century. Usage of microscopes, staining procedures, media and culture methods are considered the basis of our work. Studying the cultural characteristics, the micro- and macromorphology of bacteria is a time-consuming task. Evidence-based modern medicine has been sending the message that our laboratory diagnostics is sluggish and inefficient from clinical point of view. The rapid laboratory diagnosis can result in a faster recovery into health and in a smaller degree of antibiotic pollution in our inner and external environment. The new molecular methods (hybridization, amplification, sequencing) as the modern forms of Leeuwehoek’s magnifying lenses help us not only in searching for pathogens but also in the detection of their virulence and resistance genes directly in the clinical samples. The speed of the new methods makes the future prospects of diagnostic bacteriology very attractive, in which the usage of classical methods is gradually decreasing. However, the molecular methods mentioned not only gradually replace the old ones in some area but in other fields of bacterial diagnostics they have fruitful effects on updating the old, culture based examinations keeping them as useful diagnostic tools of the future clinical microbiologists.

Typing of Klebsiella pneumoniae isolates with MALDI-TOF MS

S. Melegh, K. Kovacs, A. Nyul, Gy. Mestyan

Department of Medical Microbiology and Immunology, Clinical Centre, University of Pécs, Pécs, Hungary

Extended spectrum β-lactamase (ESBL) producing K. pneumoniae isolates belong to the most frequently encountered nosocomial Gram-negative pathogens, therefore monitoring their spread is necessary. The currently used molecular typing methods (pulsed field gel electrophoresis, multilocus sequence typing, whole genome sequencing) are reliable technics but they are time-consuming and expensive, therefore their utility in the daily work of infection control is limited. For that reason, additional typing methods are being sought that can serve as a basis for an inexpensive and real-time surveillance system in the clinical microbiology laboratory.

The aim of our study was to develop a mass spectrometry-based typing method, that can help to rapidly detect the most common ESBL-producing K. pneumoniae clones. Altogether 201 matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) measurements were performed for 60 K. pneumoniae clinical isolates belonging to several clonal groups (27 ST15, 10 ST101, 8 ST147 and 15 sporadic isolates). We identified 19 peaks that were characteristic of either of the groups. Based on the intensity of these peaks, the mass spectra were sorted with support vector machine (SVM). The SVM classification showed 89.2% and 91.8% accuracy during the training and the test period, respectively. The results of this study indicate that MALDI-TOF MS based typing shows potential to establish a real-time surveillance system for the monitoring of the most frequent ESBL-producing K. pneumoniae clones. To the best of our knowledge this was the first study to identify K. pneumoniae clones ST15, ST101 and ST147 with MALDI-TOF MS.

Chapters from the history of bacterial endotoxic lipopolysaccharide research in the Department of Medical Microbiology and Immunology, University of Pécs, Pécs, Hungary

B. Kocsis

University of Pécs, Clinical Center, Dept. of Medical Microbiology and Immunology, Pécs, Hungary.

1st period: Serotyping of members of Enterobacteriaceae.

Principal investigator: prof. dr. Károly Rauss [1].

2nd period: Chemical composition of Shigella sonnei endotoxins:

Kontrohr T. identified 2-amino-2-deoxy-L-altruronic acid as a constituent of Shigella sonnei phase I lipopolysaccharide [2].

3rd period: Biosynthesis of heptose region of endotoxins:

Kontrohr T. & Kocsis B. isolated Adenosine 5’-Diphosphate-D-glycero- and L-glycero-D-mannoheptose [3,4]: and they also developed an assay for the detection of ADP-D-glycero-D-mannoheptose-6-epimerase [5]

4th period: Analysis of the Gram-negative bacterial outer membrane

 proteins by electrophoresis & microchip technology.

 Kustos I., Kocsis B., Kilár F. [6]. Kilar A., Kocsis B., Kustos I., Kilar F., Hjerten S.: used capillary electrophoresis to monitor endotoxins by protein complexation [7]

5th period: Analysis of S- and R-type endotoxins extracted from S.

 sonnei. Makszin L., Kilár A., Felső P., Péterfi Z., Kocsis B., Kilár

 F.: developed a quantitative microfluidic method for the

 detection of S- and R-type endotoxin components by chip

 capillary electrophoresis [8]. Bui A., Kilár A., Dörnyei Á., Poór

 V., Kovács K., Kilár F. analysed the carbohydrate composition of

 endotoxin from R-type isogenic mutants of Shigella sonnei by

 capillary electrophoresis and GC-MS [9].

6th period: Mass spectrometric analysis of endotoxins. Sándor V.,

 Kilár A., Kilár F., Kocsis B., Dörnyei Á. determined the fine

 structure of lipid A component of endotoxins [10, 11].

7th period: Nagy L., Fekete Cs., Kocsis B.: analysed the genomic

 structure of S. sonnei 53G and S. sonnei 4303 based on MAUVE Multiple Genome Alignment software.

Reference: [1] Rauss K. Dysenteria 1955. [2] Kontrohr T. Carbohydr. Res. 1977, 58, 498–500. [3] Kontrohr T. J. Biol. Chem.: 1981, 256: 7715-18. [4] Kocsis B. J. Biol. Chem. 1984, 259: 11858-60. [5] Kontrohr T. 1. J. Chromat. 1986, 354: 417-23. [6] Kustos, I. Expert Rev. Proteomics 2007, 4: 91-106. [7] Kilár A. Electrophoresis 2006, 27: 4188-95. [8] Makszin L. Electrophoresis 2012, 33: 3351-60. [9] Bui A. Croatica Chemica Acta: 2011, 84: 393 – 405. [10] Sándor V. J. Mass Spectrometry: 2016, 51: 615-28. [11] Sándor V. J. Mass Spectrometry: 2016, 51 1043-63.

Treatment options of Clostridium difficile infection: our latest experiences with fecal microbiota transplant

A. Varga^1,2, B. Kocsis¹, D. Sipos², Sz. Vigvári², P. Kása³, Sz. Pál³, É. Mikó¹, L. Szereday¹, F. Bechtolsheim¹, Z. Péterfi²

¹University of Pécs, Clinical Center, Dept. of Medical Microbiology and Immunology, Pécs, Hungary.

²University of Pécs, Clinical Center, 1st Dept. of Internal Medicine – Dept. of Infectology, Pécs, Hungary.

³University of Pécs, Faculty of Pharmacy, Institute of Pharmaceutical Technology and Biopharmacy, Pécs, Hungary.

Clostridium difficile infection (CDI) is widely known as a consequence of broad-spectrum antibiotic use. The currently available antibiotics are getting less effective due to resistant clones. The hyper-virulent ribotype 027 produces a binary toxin besides the considerably higher production of toxin A and B, therefore it has a tendency to cause pseudomembranous colitis much more frequently.

All three commonly used drugs have major drawbacks: metronidazol has a decreasing success rate; by now it is evident that the use of vancomycin facilitates the spreading of vancomycin resistant Enterococcus fecalis/faecium (VRE), and although fidaxomicin is known for its decent results in the treatment of CDI, it is too expensive to be widely accessible.

Fecal microbiota transplantation (FMT) on the other hand has similar success rate to that of fidaxomicin for a much lower price. We are seeking a reliable and more tolerable formulation of FMT in order to facilitate the use of the method.

After more than 100 successful FMT administered through nasogastric tube, we are working on soft gelatin capsules filled with freeze dried fecal supernatant. The first few experiments ended with promising results: the new formulation seems to be effective and much more tolerable than our formerly used method.

Bacteria classification problem in the laboratory diagnosis of black pigmented anaerobic bacterial infections

T. Nagy¹, T. Galli², G. Nagy¹, E. Szántó¹, J. Simon¹

¹⁾Medical Centre, Hungarian Defence Forces, Department of Diagnostic Laboratory, Budapest, Hungary ²⁾Centre for Computational Intelligence, School of Computer Science and Informatics, Faculty of Technology, De Montfort University, Leicester, United Kingdom

Some species of Prevotella and Porphyromonas genus produce characteristic brown-black pigment on blood agar and are named collectively as black-pigmented anaerobes (BPA). BPA isolated from relevant samples of patients treated during the last six months were investigated by our laboratory. The sample population (n=76) with BPA underwent three different analysis (1) Vitek 2 ANC Card (bioMérieux), (2) Rapid ID 32A (bioMérieux), and (3) Bruker-Maldi-Tof Mass Spectrometry to detect the exact bacteria species. The three methods were expected to yield the same results in each case. In contrast, the listed methods produced significantly different results in our samples. Even if misclassifications are ignored to compare the classified and not classified cases only, the statistical significance p=0.0002013 (χ²=17.021) is achieved. For describing the consistency quantitatively, we established a simple indicator: inter-classification reliability to highlight to what extent the three methods classified the samples in the same manner: Vitek 2 ANC Card 7.89%, Rapid ID 32A 7.89%, and Maldi-Tof MS 16.67% when the non-classified cases at Maldi-Tof MS were considered, which improved the indicator value at this method. The pairwise comparison demonstrates better inter-classification reliabilities: Vitek 2 ANC Card - Maldi-Tof MS: 7.89%, Maldi-Tof MS - Rapid ID 32A: 21.05%, and Vitek 2 ANC Card - Rapid ID 32A: 40.79%. In conclusion, the three methods investigated including the pairwise comparisons show poor reliabilities on the BPA samples. Further research is necessary to verify the bacteria misclassification on a larger population and to analyse the biological causes of the deviations. In addition, general improvement in the bacteria identification process could be achieved by regularly updating the local identification databases with the descriptions of the novel bacteria species available online.

Early diagnosis of endocrine conditions

Márta Korbonits

Barts and the London School of Medicine, London

Prevention of disease or severe complications is the intended hallmark of modern medicine. Currently available diagnostic methods allow the recognition of an increasing number of diseases allowing treatment and hopefully better long-term outcomes.

Couples where the offspring could be predisposed to an endocrine disease can be supported in various ways.

1. In case of severe genetic diseases, pre-implantation diagnostics can eliminate the risk of a carrier/homozygote baby (e.g. VHL, CAH, XLAG).

2. In CAH, treatment was provided in pregnancy until genetic diagnosis was made, but more recently this approach has been questioned.

3. Neonatologists can be prepared for rapid testing of the newborn (babies of mothers with Graves’ disease, hypocalcaemia, hypercalcaemia, diabetes insipidus, hypoglycaemia, diabetes).

4. Clinical genetic counselling can be offered for the offspring at the appropriate time (MEN2A/B, MEN1, AIP, Carney).

5. Ultrasound findings of the foetus during pregnancy can draw attention to possible diseases (cysts in kidney, enlarged thyroid, small for gestational age).

6. Neonatological routine examination and screening can detect disease where intervention is important (hypothyroidism, hypopituitarism, phenylketonuria, Turner syndrome, Familial glucocorticoid deficiency, virilisation, ambiguous genitalia, small for gestational age).

Modern diagnostics and increasing knowledge of genetics will ensure timely diagnosis and state of the art management resulting (hopefully) in better outcomes.

Laboratory work-up of an infant with ambiguous genitalia

Henriett Butz and Attila Patócs

Semmelweis University, Department of Laboratory Medicine and HAS-SE Lendület Hereditary Endocrine Tumours Research Group (Budapest, Hungary)

A newborn with ambiguous genitalia represents a serious diagnostic challenge. Various disorders of sex development, congenital adrenal hyperplasia, defects in androgen biosynthesis or metabolism are those diseases which need to be evaluated. During laboratory workup different laboratory procedures including chromosomal and molecular biological analyses, clinical chemistry and hormone laboratory tests have to be used for diagnosis and differential diagnosis. In addition, of hormone laboratory tests some very special (steroid hormone profiles measured by high pressure liquid chromatography and mass spectrometry) techniques should also be used. Through this presentation we aim to summarize all those laboratory methods which will be required for the accurate diagnosis of a newborn with ambiguous genitalia. The topic will cover genetic and hormone laboratory methods and through presentation of two cases suffering from congenital adrenal hyperplasia and androgen insensitivity we will present how these tests are used in every day clinical practice.

New definition and cutoffs for childhood metabolic syndrome (results from the IDEFICS Study)

D. Molnár

Department of Pediatrics, Medical School, University of Pécs, Pécs, Hungary

INTRODUCTION: Due to the epidemic of obesity in childhood and the consequent „tsunami of chronic diseases”, the MS was extended to the paediatric population, though it was difficult to apply the adult cutoffs to children. The lack of consensus is due in part to our evolving understanding of normal developmental changes associated with childhood and puberty, and to the lack of age and gender specific reference values for HDL-C, triglyceride, insulin, HOMA, waist circumference, etc.

OBJECTIVE: To develop new definition and reference standards (cutoff points) for metabolic syndrome (MetS) in European children and to establish a quantitative MetS score.

DESIGN AND METHODS: Population-based survey in eight European countries, including 18745 children from 2.0 to 10.9 years of age, recruited during the survey. Anthropometry (weight, height and waist circumference), blood pressure and fasting serum triglycerides, HDL cholesterol, glucose and insulin were measured.

RESULTS: Among the various definitions of MetS, the highest prevalence (5.5%) was obtained with our new definition requiring close observation (monitoring level). Our more conservative definition, requiring pediatric intervention gives a prevalence of 1.8%. In general, prevalences were higher in girls than in boys. The prevalence of metabolic syndrome is highest among obese children. The metabolic syndrome score shows a positive trend with age, particularly regarding the upper percentiles of the score.

CONCLUSIONS: According to different definitions of pediatric MetS, a non-negligible proportion of prepubertal children are classified as affected. We propose a new definition of MetS that should improve clinical guidance. The continuous score developed may also serve as a useful tool in pediatric obesity research.

New diagnostic possibilities of embryo viability

G.L. Kovács^1,2, G. Montskó², Á. Várnagy³ and J. Bódis³

¹Department of Laboratory Medicine, ²Szentágothai Research Centre and ³Department of Obstetrics and Gynecology, University of Pécs, Pécs, Hungary

Due to infertility, there is an increasing need for in vitro fertilization procedures. Since multiple embryo transfer increases the prevalence of multiple gestation, single embryo transfer gains ground. Therefore, finding the embryo with the best implantation potential is crucial. In retrospective experiments a previously identified possible biomarker - the alpha-1 fragment of human haptoglobin (HptA1) - was quantitatively measured in spent culture media of in vitro fertilized human embryos. A novel polypeptide marker was found to differentiate between viable and nonviable embryos during in vitro fertilization. This molecule was identified with MS as the α-1 fragment of human haptoglobin. The final goal is to adapt a complex mass spectrometric assay to a lab-on-a-chip measurement. HptA1 in spent embryo culture medium samples (n=201) was measured using liquid chromatography coupled mass spectrometry (LC-ESI TOF MS) in retrospective, blind experiments. Haptoglobin and also HptA1 are present in the culture medium and the concentration increases during in vitro embryonic development. The difference in the concentrations of HptA1 according to outcomes “live-birth” and “no-birth” was significant (p<0.001). The clinical sensitivity was 100%, while specificity 55%, area under ROC curve was 0.906. The increased amount of HptA1 in culture media samples of in vitro fertilized embryos negatively correlates with implantation potential. By combining the traditional morphological evaluation with the mass spectrometric assay, an increment in live birth rate was found in retrospective experiments. The HptA1 assay might serve as an additional tool to increase the success rate of in vitro fertilization.

Culture of human granulosa cells as a model for ovarian steroid production: evaluation of the aromatase activity

I. Földesi¹, D. Kata¹, Zs. Csáti², J. Zádori²,

¹University of Szeged, Department of Laboratory Medicine, and ²Assisted Reproduction Centre – Kaáli Institute, Szeged, Hungary

Human granulosa cells collected from patients undergoing treatment for in vitro fertilization (IVF) are extensively used for the evaluation of ovarian function. Aromatase inhibitors are widely used to block estradiol biosynthesis in estrogen-dependent breast carcinoma and nowadays as a premedication (alternative to clomiphene citrate) in the course of IVF. In the present study the aromatase activity of cells was investigated in the presence or absence of aromatizable androgens (testosterone, androstenedione). In addition, the effects of two aromatase inhibitors (anastrozole, examestane) were also evaluated. Our goal is to establish a cell culture model for the investigation of the aromatase expression. Cells were plated at a density of 50,000-100,000 cell/well and preincubated for 1-3 days prior to treatment. Incubation was continued for 4-6 days with medium changes performed every day or every other day depending on the experimental settings. Spent media were collected and stored at -20 C° for the steroid analysis. Close correlation was found between serum estradiol levels measured prior to ovulation induction and the corresponding estradiol produced by granulosa cells of the same patient during the first day of culture. This indicates that these cells maintain their steroidogenic capacity in vitro at a degree which corresponds to the patients’ in vivo sensitivity to gonadotropin stimulation. In the presence of androgens granulosa cells were able to produce large amounts of estradiol for at least 11 days in culture indicating that the aromatase enzyme activity is maintained. Anastrozole inhibited aromatase activity in a time and dose dependent manner. However, the effects of examestane on estradiol production could be evaluated only indirectly since the compound cross-reacted with estradiol kits.

Evaluation of a new test for stimulating TSH receptor autoantibodies: a multicenter study

E. Toldy¹, E. Pintér², J. Konderák², G.L. Kovács³, K. Koller⁴, P. Lakatos⁵, Z. Lőcsei⁴

¹Diagnostic Institute, Faculty of Health Science, University of Pécs, ²Synlab Hungary Ltd, Budapest Diagnostic Center, Clinical Chemistry Laboratory, ³General Department of Internal Medicine, Ferenc Flór Teaching Hospital of Pest County Kistarcsa, ⁴General Department of Internal Medicine, Markusovszky University Teaching Hospital, Szombathely, ⁵First Department of Internal Medicine, Faculty of Medicine, Semmelweis University, Budapest, Hungary

Autoantibodies against the TSH receptor (TRAb) are the major pathological factors in Basedow Graves’ disease (BGD). The routinely used total TRAb (T-TRAb) assay provides information on the three known TRAb variants - the stimulating (S-TRAb), blocking and apoptotic ones. Recently, the first S-TRAb assay method (TSI, Immulite, Siemens) has been marketed as measuring S-TRAb only. The aim of the present study was to evaluate the clinical utility of this new test in comparison with conventional T-TRAb method, using samples of three endocrine outpatient clinics. Methods: Sera of 240 patients (184 women, 56 males, age 53.3±17.3 year) with thyroid diseases were analyzed for T-TRAb and S-TRAb and for thyroid function parameters. 110 subjects with BGD (84 treated), 37 patients with Hashimoto thyroiditis (HT) were included and 93 persons with non-autoimmune thyroid diseases (47 euthyroid struma, 46 toxic adenoma) served as control. The prevalence of endocrine ophthalmopathy (EOP, N=29) and the type and duration of treatments have also been documented. Results: The correlation between the T-TRAb and S-TRAb methods was r=0.72. S-TRAb showed nearly 30 % lower concentration than T-TRAb assay. The specificities of the assays were similar (98%). In BGD and EOP, the clinical sensitivity of the S-TRAb method was better (BGD: 85% vs. 75%, p=0.06; EOP: 66 vs. 83%, p=0.12). When the duration of the treatment was taken into account, significantly (p0.05) higher sensitivity was found in BGD patients without treatment compared to those on long-term therapy (S-TRAb: 96% vs. 78%, p=0.02; T-TRAb: 88% vs 65%, p=0.05). Sixteen percent (6/37) of HT patients were TRAb positive by both methods. Conclusions: The S-TRAb method offers higher sensitivity in BGD and EOP. Since the sensitivity of S-TRAb method was better in BGD patients on long-term treatment, this assay might be more useful in therapy monitoring.

Recent developments in complement analysis: comprehensive genetic workup and functional analysis for patients with complement mediated kidney diseases

Z. Prohászka, N. Garam, N. Veszeli, L. Varga, D. Csuka

Research Laboratory, IIIrd Department of Internal Medicine, and MTA-SE Research Group of Immunology and Hematology, Hungarian Academy of Sciences and Semmelweis University, Budapest, Hungary

Atypical hemolytic uremic syndrome (aHUS) and complement mediated C3 glomerulopathy may develop due to acquired or inherited factors leading to uncontrolled complement activation via the alternative pathway. Recent developments in genetic and serum complement analysis led to increased number of available assays to identify such factors in the affected patients. Besides the measurement of classical and alternative pathway factors and regulators, specific complement autoantibody (anti-FH, anti-C1q and C4-, or C3-nephritic factors) and activation product level determinations may help to distinguish between causes of complement consumption or dysregulation. Two thirds of aHUS and one third of C3G patients carry rare and/or common risk variations in genes of complement factors and regulators. Most rare variations in aHUS and C3G fall to the CFH-CFHR gene cluster on chromosome 1, where copy number variations and appearance of hybrid genes with the expression of proteins with extra copies of functionally relevant complement regulatory domains may result in disturbed alternative pathway regulation and development of aHUS or C3G.

The broad availability of genetic workup for aHUS and C3G patients, together with the introduction of next generation sequencing technology, led to the recognition of high number of novel variations in the target genes, making careful family analysis and functional studies even more important to establish correct interpretation of such variants. The presentation will review the current state-of-the-art of complement analysis for aHUS and C3G with illustrative case presentations.

Hepcidin: regulation, role in diagnosis and therapy

K. Sipos, E. Pandur, R. Pap, E. Varga, G. Jánosa

University of Pécs, Faculty of Pharmacy, Pécs, Hungary

Hepcidin is the hormone which regulates iron metabolism in mammals. It is synthetized mainly in the liver in a prepropeptide form. The regulation of the expression of hepcidin is complex: both physiological (iron level, oxygen availability, demand for hematopoiesis) and pathological (inflammation) circumstances have effects on it. The receptor of hepcidin is ferroportin, an iron exporter of different cells (macrophages, hepatocytes, enterocytes). By binding ferroportin hepcidin inhibits iron release into the bloodstream, so the levels of iron and hepcidin are regulated opposingly. The determination of hepcidin level in the blood as well as in the urine is not routine yet. Nowadays efforts are being made to develop an internationally accepted measurement of blood hepcidin level together with the definition of normal hormone levels. The expression or maturation of hepcidin have been described in different pathological conditions, so the measurement of hepcidin level could be used for differential diagnosis in case of anemias of different origin or types of hemochromatosis. Also, there are clinical trials to use hepcidin or its analogues for therapeutic purposes. In this review presentation we will demonstrate some background information (discovery of hepcidin, functions and regulation of the expression of the peptide) as well as our experimental results, including the potential role of hepcidin in neurodegenerative diseases.

Hepatitis C virus type and subtype distribution in Hungary

J. Gervain

Szent György University Teaching Hospital, Székesfehérvár, Hungary

Hepatitis C virus (HCV) shows great structural variability. Based on genome sequencing and phylogenetical analysis, 7 types and 67 subtypes can be differentiated with varying geographical distribution. It is very important to determine the HCV type/subtype prior to starting direct antiviral therapy (DAA), which has been available since 2014, because the type, dose and optimal length of medication depend on these. In Hungary, the treatment of chronic HCV patients started in 1992 with the relevant special diagnostic tests being carried out in our Molecular Diagnostic Laboratory. Current summary discusses the results of 6092 patients (175 serotypes, 5917 genotypes) based on age, gender, regions and genotype distribution changes over time between 1996 and 2017. Methods: Serotyping: Murex, Abbott (1996–1999). Genotyping: INNO-LiPA HCV II. (1999-2006), Versant HCV Genotype 2.0 (2009–2015), real-time PCR (2016-; Cobas HCV GT, Cobas 4800). Results: Genotype distribution: GT1a: 5.6%, GT1b: 84.6%, GT1a+1b: 5.1%, GT2: 0.1%, GT3: 1.8%, GT4: 0.1%, mixed: 1.6%, GT1 (nondifferentiated subtype): 1.1%. Female/male ratio is 52%/48%. The most common age category is 50–60 years (37% of all cases). There was no genotype asymmetry among the four Hungarian regions and Budapest. Conclusion: There have been no substantial changes in the HCV type/subtype distribution in Hungary over the past 20 years, 1b remaining the most common. The introduction of real-time PCR method for genotyping has resulted in a major quality improvement including only few mixed subtype results, leading to more efficient drug selection.

Significance of sustained deep molecular response in chronic myeloid leukemia – single laboratory experience

N. Meggyesi¹, P. Kövy¹, A. Bors¹, Á. Bátai¹, Gy. Ujj², G. Halm, P. Reményi¹, J. Dolgos, S. Fekete, I. Vályi-Nagy¹, H. Andrikovics¹

¹Central Hospital of Southern Pest, National Institute of Hematology and Infectious Diseases, Budapest, Hungary; ²Hetényi Géza Country Hospital, Szolnok, Hungary

The success of tyrosine kinase inhibitors (TKIs) has led to the possibility of therapy-cessation without relapsing [treatment free remission (TFR)] for CML patients with stable deep molecular response (DMR). To determine patients’ suitability for TFR, several factors need to be assessed. TKI withdrawal is recommended for patients with non-high Sokal score, typical BCR-ABL1 transcript, chronic phase disease, optimal response, more than 8 years on TKI therapy, and minimum 2 years of DMR monitored in a standardized laboratory. According to the recommendation, in case of atypical transcript, prior accelerated/blast phase, TKI failure or no DMR, TKI cessation is not suggested. In this study we studied the suitability of TFR for 177 CML patients. Sokal score was non-high in 60% (107/177) of patients. Most of the patients (94%) had typical BCR-ABL1 transcript. In 105 cases (59%) only chronic phase was detected in previous history, without acceleration. The response to TKI therapy was optimal in 66 (37%) patients. Half of the patients (48%) were treated for >8 years, and 108 (61%) patients achieved DMR. After all, only 10 (6%) patients fulfilled all these criteria, where TKI withdrawal is strongly recommended. The presence of exclusion criteria forbids TFR in 69 patients (39%). In the remaining 98 patients (55%) TKI withdrawal could be considered if significant toxicity or planned pregnancy occurred. Sensitive and standardized BCR-ABL1 molecular monitoring of DMR plays an important role in therapy cessation. Project is partly supported by NVKP_2016.

ADAMTS13 gene sequencing in patients with ADAMTS13 deficiency

B. Takács, E. Szabó, D. Csuka, Gy. Sinkovits, Z. Prohászka

Semmelweis University, 3rd Department of Internal Medicine, Research Laboratory and Füst György Complement Diagnostic Laboratory

Congenital thrombotic thrombocytopenic purpura (TTP), also known as Upshaw-Schulman syndrome (USS), is a rare, life-threatening disease characterized by thrombocytopenia, microangiopathic hemolytic anemia and widespread microvascular thrombosis, which leads to the ischemic damage of multiple organs. USS is caused by mutations in ADAMTS13 gene which encodes a plasma zinc metalloprotease of the ADAMTS family responsible for the cleavage of von Willebrand factor (VWF). Mutations in the ADAMTS13 gene, resulting in severely reduced VWF-cleaving protease activity in plasma are responsible for the autosomal recessive inheritance of the disease.

In the past year we carried out the genetic screening of several patients and the relatives, whose ADAMTS13 activity was deficient without the presence of inhibitors. To search variations, we screened 7 patients’ ADAMTS13 gene with Sanger sequencing. We identified multiple mutations and common variations in every patient. Most of the identified mutations were reported previously, some of them occurred in multiple patients. Three novel mutations have also been identified in our study. Here, we present the differential diagnostic process for the various types of TTP and highlight the importance of the genetic screening in suspicious cases.

MRSA screening by a real time PCR system

V. Németh, A. Petrevszky, M, Kovács, G. Bekő

Uzsoki Hospital, Budapest, Hungary

Infection control of MRSA is a top priority at many hospitals. Standard procedures in clinical microbiology takes 2 or 3 days before definitive MRSA identification can be achieved but a Real-Time PCR System offers a fast and accurate means of MRSA identification.

In October 2017, our hospital was given an opportunity to try a Roche Cobas Liat analyser. The Cobas Liat System was used in the microbiology department of the Central Laboratory of Uzsoki Hospital but it is also suitable for Point of Care Testing (POCT). This real time PCR system detected S.aureus bacteria in the sample in less than an hour and was able to detect MRSA. Samples were collected from the patients of traumatology who had upper-respiratory tract symptoms or did not have previous microbiology test results about MRSA screening. Samples at the intensive care unit (ICU) were also collected from patients who arrived from another department (e.g. emergency department) in a serious condition. We tested 18 samples from which 9 were MRSA screening samples (swabs from the nose or wounds). 3 screenings had positive result (presence of Staphylococcus aureus) and 2 of them were MRSA positive. The other samples were various types of specimens (sputum, cannula, skin lesions and wounds). In this batch 2 MRSA cases were detected from a total of 4 S.aureus positive samples. Positive MRSA screenings were confirmed by culture. Our results accelerated the decision-making process for the further treatment of these patients, including patient allocation (e.g. isolation if needed) and special requirement for their operations.

Based on our data, the device is reliable and user-friendly and we can get accurate results with correct pre-analytical specimen handling.

Detection of antibodies directed against domain 1 of β2 glycoprotein I in patients with antiphospholipid syndrome

A. Kerényi, G. Nagy, P. Antal-Szalmás, J. Kappelmayer

Dept. of Laboratory Medicine, University of Debrecen, Debrecen, Hungary

Antiphospholipid syndrome (APS) is an acquired thrombophilia caused by antiphospholipid autoantibodies (aPL). Diagnosis of APS is based on laboratory and clinical criteria (thrombosis or pregnancy-related complications). The detection of lupus anticoagulant, IgG and/or IgM isotype anti-cardiolipin (ACA) and anti-β2 glycoprotein I antibodies (aβ2GPI) constitute the laboratory tests of APS. The β2GPI protein consists of 5 domains, and the association of thrombosis with the presence of antibodies directed against domain 1 is known from the literature. The aim of this study was to investigate the presence of IgG isotype antibodies targeting domain 1 of β2GPI (aD1β2GPI) in patients having IgG isotype aβ2GPI (APS: n=12; autoimmune disease: n=3; aPL positive asymptomatic patients, n=3) using a chemiluminescence immunoassay. We have shown that the level of IgG isotype aD1β2GPI was higher in thrombotic than in non-thrombotic APS patients. In patient samples the concordance between the IgG isotype aD1β2GPI and aβ2GPI was high (77.7% in the whole group; 75% in APS patients), and the correlation between antibody titers was also strong (r=0.71 in the whole group; r=0.73 in APS patients). We have also calculated the strength of association between the positivity of IgG isotype aD1β2GPI and thrombotic outcome, the OR=16 (95% CI=0.67-383.1), however, due to the low number of samples the association was not significant (p=0.087).

This study provides further evidence that the large majority of IgG isotype aβ2GPI are directed against domain 1. Since the concordance between the two antibody positivities was not 100% and aD1β2GPI negative patients can have thrombosis, we conclude that monitoring aD1β2GPI cannot replace detection of aβ2GPI in the laboratory diagnosis of APS.

EASI: harmonization of autoimmune diagnostics by questionnaires and recommendations

J.G.M.C. Damoiseaux

Medical Immunology, Central Diagnostic Laboratory, Maastricht UMC+, The Netherlands

The European Autoimmunity Standardization Initiative was founded in 2002 in order to optimize the communication between clinicians and laboratory specialists and to harmonize autoimmune diagnostics for the systemic rheumatic diseases. The organization of EASI is based on national teams that are represented in the international EASI Forum. Both on national as well as international level initiatives are undertaken to meet the goals of EASI. The first EASI goal is addressed by publication of booklets with background information and by the organization of EASI symposia at the international autoimmunity meetings. The second goal, harmonization, is achieved by questionnaires for laboratories. They have proven very successful because they have unraveled a large diversity in procedures for autoantibody testing within countries and also between countries. Publication of results, in combination with recommendations, enables harmonization of autoimmune diagnostics. In the last 15 years EASI is well recognized within the autoimmunity network since there are many interactions with other societies involved in harmonization and standardization. Over the years there is an ever-expanding number of countries participating in EASI and, as such, participation of Hungary is more than welcome.

Diagnostic algorithm for antinuclear autoantibody testing - clinical and financial considerations

G. Nagy, I. Csípő, E. Gyimesi, J. Tóth, S. Demeter, P. Antal-Szalmás

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

The diagnostics of autoimmune rheumatic diseases strongly depends on laboratory tests, dominantly on autoantibody determinations. Several techniques and assay types are available for evaluation of the autoantibody profile of these patients. The most widely used indirect immunofluorescence (IIF) assay on HEp-2 cells can identify dozens of antinuclear (ANA) and anti-cytoplasmic autoantibodies, while ELISA-s or immunoblots utilizing mixed or single antigens can identify the exact specificity of them. Since the application (replacement and sequential order) of these tests is rather ambiguous we developed an algorithm for the most frequently used antinuclear and anti-cytoplasmic autoantibodies and evaluated clinical and financial efficacy of this novel system.

The number and results of autoantibody determinations (ANA HEp-2 IIF, anti-dsDNA and anti-ENA tests) between January and June, 2017 were collected from our laboratory information system (GLIMS). The theoretical number of autoantibody tests was recalculated along the following rule: anti-dsDNA and anti-ENA tests were performed only if ANA HEp-2 IIF was positive. Separate analyses were performed taking into account the ANA HEp-2 IIF patterns and titers, too.

Our results showed that the ANA HEp-2 IIF guided selection of anti-dsDNA and anti-ENA ELISAs could reduce the number of anti-dsDNA and anti-ENA tests by 48% and 56%, sparing about 7.1M HUF per year. The rate of ANA HEp-2 IIF negative but anti-dsDNA positive cases was 1.8%, meaning 64 samples, from which 16 patients (0.45%) had significantly higher (>2x URL) anti-dsDNA value. The rate of ANA HEp-2 IIF negative but anti-ENA positive samples was 1.1% (56 cases). 14 and 19 of these patients showed only anti-SS-A or anti-Jo-1 positivity, known to be weakly reactive on HEp-2 cells.

The described sequential application of ANA HEp-2 IIF assay, anti-dsDNA and anti-ENA ELISAs provided high clinical efficacy and proved to be cost-effective.

Evaluation of autoantibody measurements in systemic rheumatic diseases. How do we proceed in Hungary?

E. Nagy, P. Gergely

National Institute of Rheumatology and Physiotherapy, Budapest, Hungary

The measurement of autoantibodies is important not only in the diagnosis and monitoring of systemic rheumatic diseases, but also in differentiation of patient subgroups and in the risk assessment of complications.

In the last decade, the number of available methods for the detection of autoantibodies increased, leading to the replacement of old tests with new ones and appearance of new platforms. This generated a huge variability between laboratories and reproducibility became a serious problem. The biggest challenge for laboratory specialists is to choose the appropriate method for autoantibody testing, in order to obtain the most reliable result.

A great amount of effort has been put internationally in setting up guidelines how to use and interpret the results of autoantibody tests. Owing to this initiative, few years ago an international recommendation for the assessment of autoantibodies to cellular antigens was formulated. As for ANA testing with immunofluorescence method, a consensus on ANA Staining Patterns was achieved internationally (ICAP). Description of the established patterns is already available on-line in several languages.

With respect to the new recommendations, the National Institute of Rheumatology and Physiotherapy, together with other Immunology Centers from Hungary takes the initiative to evaluate the current knowledge and usage of autoantibody tests, primarily ANA, anti-ENA, anti-dsDNA and ANCA in Hungarian laboratories, with the final goal of formulating national guidelines for the usage of these assays. For this purpose, we have already finalized the Hungarian version of the ICAP nomenclature, as well as the international questionnaire on the usage of the above mentioned autoantibodies.

Immunosenescence – aging of the immunity

Zs. Szabó, N. Hartvig, K. Miklós, J. Simon

Medical Center – Hungarian Defense Forces, Department of Laboratory Medicine-Clinical Immunology, Budapest, Hungary

In the western world, longer life time with health and well-being is not just a possibility, but also a challenge for the economy, healthcare system, and for science. Understanding the connected pathway mechanisms of ageing help us to improve the health condition of the elderly population. All stages of health are strictly connected to the work of the immune system. The ageing of the immune system, the so called immunosenescence is researched by great effort in many different research areas.

In old age the decline of immune functions is a natural process that affects both the adaptive and the innate immune response. It significantly contributes to the greater frequency and the severity of infections and the rate of cancer in elderly. Cardiovascular and diabetic diseases, according to recent reviews, are presumably connected to inflammation and immunosenescence as well.

To characterise immunosenescence just few biomarkers are available today. The aim of routine laboratory diagnostic is to find and introduce more of them. It could be like subpopulation of lymphocytes or levels of immunoglobulines etc. Biomarkers are a requirement of therapeutic intervention. Great task of laboratory diagnostics in the near future is to step forward in this field.

Novel strategies to detect neurological ion-channel specific autoantibodies

T. Berki, K. Böröcz, Zs. Csizmadia, Zs. Hayden, P. Balogh

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

Ion channels are complex transmembrane proteins that orchestrate the electrical signals necessary for normal function of excitable tissues, including the central nervous system, peripheral nerve, and skeletal muscle. Progress in molecular biology has allowed cloning and expression of genes that encode channel proteins, while advances in electrophysiology have made the functional assessment of single channel molecules possible.

Neurological channelopathies are frequently acquired through autoimmune mechanisms. All of these autoimmune conditions can arise as paraneoplastic syndromes or independently from malignancies. The pathogenicity of autoantibodies to ion channels has been demonstrated in most of these conditions, and patients may respond well to immunotherapies that reduce the levels of the pathogenic autoantibodies. Paraneoplastic limbic encephalitis has a poor prognosis and is most commonly associated with lung, ovarium, and testicular neoplasms, leading to immune reactions against intracellular antigens (anti-Hu/ANNA1, anti-Ri/ANNA2, anti-CV2/CRMP5 and anti-Ma2/Ta). In contrast, the recently described autoimmune encephalitis subtypes present with a broad spectrum of symptoms, respond to autoimmune therapies well and usually associate with autoantibodies against neuronal cell surface receptors (NMDAR, GABABR, AMPAR) or synaptic proteins (LGI1, CASPR2).

In our laboratory, standard diagnostic methods are introduced to detect these autoantibodies. Transfected cell-based BIOCHIP, and line-blots are used as laboratory tests together with standard indirect ELISA methods to distinguish these conditions and to help diagnose the disease.

Immunological laboratory examinations in the investigation of female and male infertilities

J. Németh

Medicover PLC, Budapest, Hungary

There are many underlying causes for female infertility. The increasing occurrence of infertility problems can be caused by anatomic and genetic disorders, hormonal disruption, infections, blood coagulation problems and many other immunological mutations, which can all prevent pregnancy, fetal development and the outcome of the pregnancy as well. The occurrence of autoantibodies can hinder the reproductive process even without having any autoimmune disorders. It is suggested to examine the antibodies against the thyroid gland, the phospholipid antibodies, the celiac disease-specific antibodies, anti-nuclear antibodies and anti-dsDNA antibodies. In case of implantation failures, it may be important to examine the cellular immune status.

In case of males, the semen diversion makes the immunological examination necessary, which can be the measurement of the inflammation specific markers from the ejaculate or the detection of anti-sperm antibodies.

Searching for immunological incompatibility of a couple can be a factor in the investigation of the immunological background of infertility. The laboratory tests for this include the measurement of mixed lymphocyte culture (MLC) and the detection of blocking antibodies.

The presentation provides detailed information about these immunological tests, their place and their interpretation in the investigation of infertilities.

Component resolved molecular diagnostics in allergology

P. Antal-Szalmás, E. Gyimesi, I. Csipő, J. Tóth, S. Demeter, G. Nagy, J. Kappelmayer

University of Debrecen, Dept. of Laboratory Medicine, Debrecen, Hungary.

The diagnostics of allergic diseases strongly depend on laboratory tests, dominantly on allergen specific IgE determinations. The presence of these antibodies can be identified by the classical skin tests and laboratory assays utilizing allergen extracts. The major disadvantage of these methods is that their reagents contain several allergenic molecules in one “tube”, few of them might be recognized by cross-reacting IgE antibodies, providing false positive results. The exact identification and production/purification of these individual allergenic molecules made possible the development of component resolved molecular diagnostic tests, that can prove the real sensitization against a certain allergen, furthermore, can show the presence of cross-reacting antibodies. Determination of the exact physico-chemical properties of these allergenic molecules can provide further important practical information (e.g. the heat-stability of food allergens can guide the cooking procedures after which these foods can be consumed without a severe allergic reaction). The concentration of these allergen molecule specific IgE antibodies can have prognostic information, too.

In December, 2017, molecular allergic tests for cow milk (casein, alpha-lactalbumin, beta-lactoglobulin), hen egg (ovalbumin, ovomucoid), peanut (Ara h1, 2, 3, 6, 7, 9, 5, Bet v1) and insect venoms (Api m1, 2 and Ves v5) were introduced in our Laboratory. The first results of these determinations, their clinical utility and the extra information compared to classical allergen-specific IgE tests provided by these molecular assays will be demonstrated in 5 different allergic cases.

Drug interactions in the laboratory practice

I. Földesi

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

The ageing of the population, the continuous rise in expected lifespan at birth, the consumption of an increasing amount and types of drugs or other drug-like preparations raise the possibility of interactions among them. Drugs taken together with the patient may cause interactions at the absorption, distribution, metabolism and elimination levels. In addition, depending on the administered/taken dose they may interact with the laboratory tests based on chemical, biochemical or even immunochemical reactions. The competition for the protein binding sites altering the concentrations of certain metabolites could also be expected. With the advanced age the frequency of doctor visits becomes higher in parallel to the diagnostic laboratory test requests. Not only the medication or “drug abuse” increases, but the probability of interactions with laboratory tests. Without recognizing such phenomenon, the laboratory findings may lead to misinterpretation of laboratory data, ordering of further, unnecessary tests, false diagnosis, wrong treatment decision or inappropriate follow up of patients. In summary, it may cause longer recovery, more costs and poor patient satisfaction. In 1978, the prevalence of drug-laboratory effects was estimated as 12%, when patients had such laboratory test results that potentially were affected by drugs (1). Nowadays, more than 40-50 thousand drug interactions are known (2) but this number is increasing almost every day. The lecture presents the most frequent drug-laboratory test interactions.

1. Friedman RB, Young DS, Beatty ES. Clin Pharmacol Ther 1978;24:16–21.

2. Young DS. Effects of drugs on clinical laboratory tests, 5th ed. Washington: AACC Press, 2000

Selecting quality control strategy for clinical chemistry tests based on patient risk

Sz. Szakony

St. Imre Teaching Hospital, Budapest, Hungary

Background: Test performance, testing volume, the risk level of a test and patient risk are all important factors to take into account when selecting QC strategy. We investigated whether the Bio-Rad Mission: Control software system could improve QC strategy with special regard to patient risk. Methods: 23 clinical chemistry tests were selected for analysis. Current QC data (mean, SD), biological variation minimum as allowable total error limit and 1:2s QC rule were used to assess test performance. The expected number of QC events [E(QCE)], the expected number of unreliable patient final results [E(N_uf)] and false rejection rate (P_fr) were calculated for each assay. Results: Based on the test performance three chemistry assays [albumin (ALB), calcium (CA), ALT] were selected for modeling. The calculations were performed on low and high volume testing. In the case of ALB (Sigma: 2.7) the P_fr and E(QCE) were the same (13%; 2.4), but E(N_uf) differed (<1 vs 22.9) for low and high volume testing. In the case of CA (Sigma: 4.2) and ALT (Sigma: 19.9) the P_fr, E(QCE) and E(N_uf) showed the same results (CA:13%;1.7;<1, ALT:13%;1;0) regardless of testing volume. Changing QC rule 1:2s to repeat 1:2s, the P_fr decreased (2.2%) in all cases, but E(QCE) increased for ALB (10.6) and CA (7.8) regardless of testing volume. E(N_uf) increased more in high volume testing (ALB:3.1 vs 77.9; CA: <1 vs 2.6). Increasing QC frequency could decrease E(N_uf) for high volume testing (ALB: 15.6; CA: <1). In the case of ALT the P_fr could be further reduced (0.8%) by changing QC rule (1:3s) while E(QCE) and E(N_uf) did not change for low or high volume testing. Conclusions: Mission: Control program allowed us to assesss the performance of our QC strategy and to design appropriate QC settings that are predicted to reduce the number of QC events and the number of unreliable patient results.

How to optimize the financial management of a diagnostic laboratory?

J. Kappelmayer, L. Székely, P. Antal-Szalmás

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

In Hungary there are two models for the management of a hospital laboratory. The first and quite widespread model is that a certain fixed budget is assigned by the hospital management for the laboratory, that should try to maintain all its costs within the proposed limit. The second is to ensure financial independence of the laboratory, which could be achieved both by privatization or by assuring financial independence for all hospital units including the laboratory within the state owned health care system. This latter possibility stimulates the laboratory management to quest new solutions.

In our Department we have been regularly analyzing fiscal achievements since our establishment 40 years ago and in the past 20 years we are enjoying a financial independence as all other units at the University of Debrecen. Our ways to improve service and maintain the financial balance of the laboratory were (i) to optimize reporting of the tests, (ii) to introduce or to terminate certain laboratory tests based on clinicians’ feed-back, (iii) to update methodology for professionally appropriate, but financially maintainable assays, and (iv) to raise the proportion of special laboratory tests that are carried out for remote (out of region) laboratories with much better financing circumstances than the level provided by the National Health Insurance Fund of Hungary.

A major proportion of laboratory spending are the personal costs of the workers, which considerably affect the financial balance. We found that in large multivalent laboratories, if the personal costs can be kept around 20% the laboratory can function with a positive balance, 30% personal costs usually mean a zero net balance, while 40% or higher values definitely lead to a negative financial balance. We are fully aware that personal costs can vary widely among the laboratories but the proportion should definitely be minimized by creating a maximally centralized service with the minimum number of satellite sites as well as introducing an intense automation in all fields involving clinical chemistry, hematology/immunology and molecular techniques, and applying a highly sophisticated laboratory software that permits the introduction of autovalidation.

National guideline on pre-analytical phase

A. Kocsis^1,3, J. Tóth^2,3, É. Ajzner^1,3

Jósa University Hospital, Central Laboratory, Szabolcs-Szatmár-Bereg County, Nyíregyháza, Hungary¹; University of Debrecen, Department of Laboratory Medicine, Debrecen, Hungary²; Working Group on the Extra-analytical phases of the Hungarian Society of Laboratory Medicine; Hungary³.

Working Group on the extra-analytical phases (WG-EA) of the Hungarian Society of Laboratory Medicine (HSLM) was set up in 2017 with the goal to improve the quality of the extra-analytical phases. The first activity of WG-EA was to develop a Hungarian national guideline for the pre-analytical phase (HU-G-PRE), which is in force in the period of 2017-2020.

The objective of this presentation is to highlight key messages and recommendations of HU-G-PRE. The guideline was developed by methods of evidence-based guideline development in consensus of laboratory professionals and general practitioners.

The guideline is based on systematic overview of the literature and the adaptation of the existing external guidelines and books on PRE practices and quality assurance in the period of 2002–16.

Ninety-five recommendations with indication of the level and strength of evidence were formulated for practices in laboratory test requesting, patient identification, sample preparation, transport, arrival and storage in the PRE phase. Each recommendation was followed by a short summary of the existing pieces of evidence as well as quality indicators recommended by EFLM Task and Finish Group on Performance specifications for the extra-analytical phases in the fields. In the presentation key messages and recommendations of the first HU-G-PRE will be discussed. In addition, activities of WG-EA to assist successful implementation of HU-G-PRE in Hungarian laboratories, such as benchmark data collection on the use of quality indicators in PRE phase, will be provided.

Patient Safety Improvement of Uniform Treatment of Alarm Results in the Laboratory Network

N. Szlatinszki¹, K. Barna T¹, D. Némethné B¹, I. Fejér¹, Zs. Csernák²

SYNLAB Laboratory, Dunaújváros¹, SYNLAB Hungary Ltd., Budapest², Hungary^1,2

In 2017 a working group has been launched in SYNLAB for consolidation of the critical results in the laboratory network in order to increase the patient safety. Out of the participants (19) 7 laboratories have microbiological specification. One of their goals was to review and develop the existing alert values and regulate the communication processes. Beside the central regulation the laboratories have to elaborate a local process for critical result management using a common template. In the laboratory network there is a uniform alert log diary where the communication conditions of the results are required to be documented within 1 hour. This makes it easy to use more indicators and compare them to each other and develop the processes within the network. In the laboratory at Dunaújváros, a total of 2384 alarm results’ communication was recorded, of which 2301 were successful (reliability 96%). In 19 cases they experienced deviation on the time frame. Repeated measurements of the alarm results can delay the diagnosis without having significant advantage. In the laboratory inspected, 280 alarm results were repeated, according to the recommendation of Clinical Laboratory Improvement Amendments regarding the total allowable error limit. Of these testings 158 were hematologic and 122 were chemical analysis. 7% of the repeated tests were unnecessary: among the hematologic results the platelet count varied (1%) from the recommendation and from the chemical results glucose (5%) showed deviation. These results suggest that repeated analysis for many automated tests should be stopped. Our data could serve as a catalyst for other laboratories to review the value of their repeat testing practices.

their repeat testing practices.

A novel urinary cystatin-C assay for monitoring kidney function in sepsis

¹P. Kustán,¹B. Szirmay, ¹Z. Horváth-Szalai, ^1,2D. Ragán, ¹A. Ludány, ¹A. Miseta, ²D. Mühl, ¹T. Kőszegi

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

²Department of Anaesthesiology and Intensive Therapy University of Pécs Medical School, Pécs, Hungary

Background: Early recognition of acute kidney injury (AKI), a severe complication of sepsis, is of utmost importance. Although urinary cystatin-C (u-CYSC) is reported to be a reliable marker of tubular dysfunction, up to now there is no commercially available automated u-CYSC test. Our aim was to adapt and validate a fully automated assay for routine u-CYSC measurements and to evaluate its potential clinical usefulness.

Materials and Methods: A particle enhanced immune turbidimetric assay (DiaSys) was developed on Cobas 8000/c502 analyzer. Spot urine samples from healthy individuals (n=117) and from septic patients (n=53) were analyzed. Septic patients were categorized as AKI (n=20) and non-AKI (n=33) patients. U-CYSC data were referred to urinary creatinine (u-CYSC/u-CREAT, mg/mol).

Results: The detection limit of our assay was 0.017 mg/L with an overall imprecision less than 5% of CV within the whole measuring range (0.0-1.0 mg/L). The assay time was 10 minutes. Upper reference limits for u-CYSC/u-CREAT were found to be <19.7 mg/mol for the adolescents and <12.4 mg/mol for adults. U-CYSC was significantly higher in septic patients compared to controls (p<0.005). U-CYSC/u-CREAT was higher in sepsis-AKI than in non-AKI groups (309.6 vs 63.5 mg/mol, p<0.005).

Conclusions: We developed a fast, sensitive and precise turbidimetric approach for automated u-CYSC determination. Our highly sensitive assay is ideal for routine u-CYSC measurements and might be a potential novel laboratory test in the management of acute kidney injury.

Fasting for quality: knowledge and practice of outpatients before phlebotomy regarding fasting state

I. Dinnyés

Petz Aladár County Teaching Hospital, Central Laboratory, Győr, Hungary.

A well-prepared patient before the laboratory testing is a key prerequisite of the correct test results. We studied the knowledge of our outpatients about the fasting requirements and their actual condition before phlebotomy. On the site of our phlebotomy unit 151 individuals (female: 88 (58.3%); male: 63 (41.7%)) were personally questioned between March and April 2018. 140 (92.7%) subjects claimed that they came with fasting, but actually, 109 (72.2%) subjects consumed the last meal at least 12 hours before, regardless of knowing or not the correct definition of the fasting state. 51 (33.8%) subjects gave the correct definition of the fasting state. 100 (66.2%) subjects did not know the exact time requirement from the last meal to the blood drawing. 37 (24.5%) patients did not consume any fluid in the morning, but 19 (12.6%) drank tea, coffee or milk just before the blood drawing. These are improper habits jeopardizing the preanalytical sample quality or making difficulty during or after the blood collection. The main source of information for preparing themselves before phlebotomy was the physician (92 (60.9%)) but 30 (19.9%) subjects were not informed at all about the proper preparation. Just 18 patients (12%) mentioned laboratory as the main source of information. The proportion of “ideally” prepared patients (well-informed, fasting and hydrated) was very small, 26 (17.2%). We conclude that our outpatients are not well-informed about the requirements of the fasting state. A good proportion of them is not properly prepared for phlebotomy. Laboratory should fill the gap/lack of information with proactive action towards patients and their physicians. Without this, although our test results will be analytically accurate, their overall effect could be harmful for the patients.

Serological diagnosis of Lyme borreliosis: uncertainty vs. inconsistency?

A. Zóka¹, E. Ujhelyi¹, M. Gönczi¹, V. Barbai¹, F. Bakó¹, Zs. Kienle², I. Vályi-Nagy¹

¹ Central Hospital of Southern Pest, National Institute of Hematology and Infectology, Budapest, Hungary

² National Public Health Institute, Budapest, Hungary

Although evidence-based guidelines provide us with clear algorithms, diagnostic skepticism remains popular even in the scientific literature. This may render feedback studies useful. We summarize our data of two-tier serology (ELISA: Biomedica Borrelia Recombinant Antigen IgM and IgG; Immunoblot: Mikrogen RecomLine IgM and IgG) generated in the Laboratory of Molecular Biology, National Institute of Hematology and Infectology, Central Hospital of Southern Pest in a one-year period. Between the 1st April 2017 and 1^st of April 2018, we received 648 test requests (IgG or IgM+IgG). ELISA screening was positive or borderline in 246 cases, among which 140 needed confirmation with immunoblot according to clinical data and international guidelines. Based on immunoblots, the numbers of positive, negative and borderline blot results were 83, 43 and 14 respectively. Thus, two-tier testing was conclusive in 634 cases (97,8%). 35% of confirmatory testing was indicated in cases with ‘uncertain’ early skin symptoms. We separated patients with chronic symptoms and a positive or borderline IgG immunoblot (n=40) in groups above (n=21) and below (n=19) the median test score. Seven (36,8%) were positive for any of the late antibodies (anti-p100 and anti-p18) within the lower range, while 19 (90,5%) within the upper range (Chi-square test p=0,0004). Optimizing the use of serology in cases with early skin symptoms and increasing specificity in the presence of chronic symptoms are both challenging. Antibody patterns may give a clue in certain cases. Correct timing of the tests, clear and well-documented indications for testing, and well-designed diagnostic panels including markers for alternative diagnoses (non-infectious ones as well) should precede further confirmatory or follow-up testing.

Iron deficiency and thrombocytosis

¹L. Sipos, ¹K. László, ²D. Fazekas, ¹V. Kellner

¹Synlab Laboratory Székesfehérvár, Hungary

²Synlab Budapest Diagnostic Center, Immunological Laboratory, Budapest, Hungary

Thrombocytosis (platelet count (PLT)>450 G/L) is observed in many reactive pathological states such as infections, inflammatory diseases, acute bleeding, iron deficiency (ID), malignancy. Thrombocytosis is caused by clonal disorders (e.g. essential thrombocythemia). The aim of this work was to investigate the relationship between ID and platelet counts. ID was diagnosed based on hemoglobin (Hb), mean cell volume (MCV), reticulocyte hemoglobin content (CHR), serum iron, ferritin (FERR) and soluble transferrin receptor (STFR) test results. Acute phase reaction was established on the basis of C-reactive protein (CRP). Complete blood counts were measured on Siemens ADVIA 2120i analyzer; CRP, serum iron and FERR concentrations on Beckman Coulter AU5800, STFR levels on Siemens BN II Nephelometer. Statistical correlations between PLT-CHR, PLT-FERR and PLT-STFR were analyzed based on data from 107 patients (76 with ID, 31 with normal iron status). Patients were classed into 7 groups for analysis as follows: normal iron status (A), all patients (B), all ID patients (C), ID patients with PLT<450 G/L (D), patients in group D with normal CRP levels (E), ID patients with PLT>450 G/L (F) and patients in group E with normal CRP levels (G). Correlations between PLT-CHR were found in A (p=0.025), C (p=0.024), and F groups (p=0.016). Statistical significance was also found between PLT-FERR in groups C (p=0.011), D (p=0.036) and F (p=0.001), while there was no correlation in E (p=0.731) and G (p=0.665) groups. Regarding PLT-STFR, significant correlation was only in group C (p=0.044). We conclude that the degree of thrombocytosis correlates more strongly with acute phase reaction than with iron deficiency in these cases.

Complement terminal pathway deficiency in a patient with recurrent meningitis

E. Szabó, D. Csuka, B. Takács, Z. Prohászka

Semmelweis University, 3^rd Department of Internal Medicine, Budapest, Hungary

The complement system is a key part of the innate immunity. The deficiency of terminal pathway components increases susceptibility to infections and their recurrence.

Medical history: A 22 years old female was hospitalized due to two episodes of meningococcal infection (at age of 15 due to meningitis, at age of 22 due to sepsis with serogroup C). Workup for the complement system identified the deficiency of classical and alternative pathways, whereas complement factors and regulators were in the reference range. Anti-FH and anti-C1q autoantibodies were negative, raising the possibility of terminal complement pathway deficiency. Level of complement component 8 (C8) in serum was repeatedly very low (16,5% and 18%, ref: 70-130%). Analysis was extended to family members (parents and 3 sisters) which excluded terminal pathway deficiency. These results raised the suspicion for a complex (including de novo variation) genetic trait behind the deficiency. Next-generation sequencing (NGS) found that the patient carries two heterozygous mutations in exon 3 and 9 of the C8B gene, both causing the generation of a premature stop codon of the complement C8 protein β-chain, and 3 additional rare, and multiple common variations. Detailed complement functional analysis and mutational screening of family members was very helpful to make conclusion on functionality of the identified variations.

The complex problem of identifying causative variations after NGS is increasing, and the example of our case highlights the necessity of detailed functional analysis and family screening for patients with rare diseases.

Occurrence of pre-analytical errors at BKM Hospital, Central Laboratory, Kalocsa

G. Tóth, A. Varga, E. Seres

Hospital of Bács-Kiskun County (BKM), Teaching Hospital of Szeged University, Faculty of Medicine, Kalocsa Szent Kereszt Hospital, Kalocsa, Hungary.

60-70% of clinical decision making is based on laboratory results, so it is extremely important to strictly follow protocols in the entire testing process - including pre-pre-analytical, pre-analytical, analytical, post-analytical and post-post-analytical phases. It is well-known that the errors made in the pre-analytical phase account for almost 60% of the total errors while post-analytical phase is responsible for only 25%. We aimed to examine the frequency of rejected samples due to pre-analytical errors in our laboratory in 2017 and the number results marked due to the presence of interfering factors disturbing the analytical phase. In 2017, 18.8% of all rejected samples were native (red cap) tubes; 17.3% citrate tubes; 33.9% Westergren tubes; 19.8% urine tubes; 0.3% stool containers; 4.9% EDTA tubes; 0.5% heparin syringes; 4.5% was immunochemistry (red cap) tubes. Out of the total number of tests, 0.08% were hemolyzed, 0.008% were lipemic samples and 0.005% were icteric samples. In many cases, sampling is not done in the laboratory, therefore we have also categorized the rejected samples and the hemolyzed samples based on the submitting departments, which provided – as far as the sampling aspects are concerned - an insight into the most problematic departments or other sampling points. Through the training of sampling staff in the areas concerned, we hope to improve the quality indicators in the future.

The effect of storage conditions on the stability of new psychoactive substances in urine

D. Hesszenberger¹, A. Lajtai², Á. Lakatos², M. Mayer³, A. Miseta²

University of Pécs, ¹Dept. Biochemistry and Medical Chemistry,

²Dept. Laboratory Medicine, ³Dept. Forensic Medicine, Pécs, Hungary.

The effects of the new psychoactive substances are similar to the already banned, conventional drugs, but their in vitro stability is less known. Urine samples from 18 drug abusers were collected and analyzed by HPLC method (baseline). The remaining samples were divided into 3 portions and were stored at room temperature (25 °C), in the refrigerator (4 °C), and in the freezer (-20 °C) for 21-280 days, then the measurement was repeated.

The control samples of N-ethyl-pentylone showed no significant difference compared to the samples in the freezer (p=0.13), as well as in the refrigerator (p=0.09) after 21 days, however, the degradation was significant (p=0.01) at room temperature. The parent compound of AB-FUBINACA had a decomposition rate of 47% at room temperature, while its metabolite was not detectable in a part of the urine samples regardless of the storage conditions. The concentration of N-ethyl-hexedrone was decreased by 40% during the 3 weeks of freezing, while it decreased by as much as 95% at room temperature. It turned out that 4-methyl-N-ethyl-norpentedrone is stable at -20 °C, as well as in the refrigerator; however almost a two-third decrease was observed at room temperature. The 4-Cl-PVP was not degraded in the freezer, while its concentration decreased by 42.8% at -4 °C, and by 28.6% at room temperature. In samples stored at room temperature for a longer period of time (5-9 months), a big part of the new psychoactive substances was nearly completely decomposed. Thus, we conclude that the storage exclusively at -20 °C is satisfactory for the majority of drugs.

Evaluation of serum indices recommended for Cobas c501 reagents

G. Kecskeméti¹, A. H. Shemirani², A.V. Oláh³

¹Gróf Tisza István Hospital, Berettyóújfalu, ²Central Laboratory, Erzsébet Hospital, Sátoraljaújhely, ³Department of Laboratory Medicine, Faculty of Medicine, University of Debrecen, Hungary

Background: Preanalytical errors are a major source of inaccuracy of the laboratory tests. Increased amounts of chromogens and particles may influence the results’ precision. Nowadays automated measurement of the most common interfering factors (hemolysis, icterus, lipemia) significantly reduces the number of preanalytical errors. Aim: The reliability of analyte-dependent limits of serum indices (SI) were tested for eight test parameters known to be affected significantly by hemolysis or icterus (CK, CK-MB, LDH, ALT, AST, Bil-direct, creatinine, and triglyceride). We evaluated the applicability of the recommended hemolysis (H, umol/L) and icterus (I, umol/L) limits of the tests.

Method: Effect of hemolysis and icterus were measured in a series of normal and pathological sera. First the serum samples were mixed with increasing volume of hemolyzed red blood cells, then other serum samples were mixed with icteric sera. The level of the analytes was determined simultaneously with serum indices on a Cobas c501 analyzer. The expected values of analytes were calculated from the original level in the sera and the volume of two components. If the difference between the measured and expected value of an analyte exceeded 10%, we considered SI as the cut-off value for significant interference. Results: For total bilirubin (BILT3) we accepted the recommended cut off of hemolysis (H:621). Our SI limits and the manufacturer’s cut-off values were similar for ALT (H:56), AST (H:26), CK (H:62), Bil-direct (H:16), creatinine (I:86), and triglyceride (I:171). In the case of the most hemolysis-sensitive tests (LDH, CK-MB) we slightly elevated the hemolysis index (from 12 to 19 for CK-MB, from 9 to 23 for LDH), because these results are important in critical conditions. Conclusion: Automated assessment of serum indices characterizes the quality of sera. It provides more objective evaluation of the sample and assists to obtain reliable results. In critical condition we may be more permissive, but we add a note, when significant interference is detected. We recommend that each laboratory should define their own SI cut-off values for the most sensitive tests.

Determination of ethylene glycol by high-performance liquid chromatography and comparison with the gold standard gas chromatography method

V. Lelovics, Á. Lakatos, M. Kuzma, A. Lajtai, A. Miseta

University of Pécs, Dept. of Laboratory Medicine,

Dept. of Forensic Medicine, Pécs, Hungary

Ethylene glycol is a common household toxic alcohol and often causes a life-threatening poisoning. Its detection and quantitative measurement is an important task of the clinical toxicology laboratory. In this study, we have developed and validated a HPLC based method which we can be easily carried out on weekends and at night shifts as well.

We spiked 6 different types of alcohol in normal serum and urine for standard solutions. The new method needs derivatizing the alcohols with benzoyl-chloride. We selected our test group from patients who were suspected to suffer from ethylene glycol poisoning.

We compared the traditional gas chromatography (with flame ionization detector) process with the new HPLC-UV measurement. Our technique’s sensitivity, specificity and precision met the clinical requirements and he results of the two procedures were equivalent. Thus, our new HPLC method is suitable for the determination of ethylene glycol and other toxic alcohols in emergency situations.

The detection of human cytomegalovirus reactivation could improve the treatment of ulcerative colitis

M. Petró¹, E. Halász¹, R. Myszoglád¹, I. Böjtös¹, K. Lőrinczy², J. Simon¹

¹Central Department of Laboratory Diagnostics, Medical Centre Hungarian Defence Forces, Budapest, Hungary.

²Department of Gastroenterology, Medical Centre Hungarian Defence Forces, Budapest, Hungary

Ulcerative colitis (UC) is usually treated with immunosuppressive agents. The human cytomegalovirus (CMV) can be reactivated under this type of management. The virus reactivation can induce inflammation in the intestinal mucosa, often resulting in severe lesions or ulcers, similar to UC relapse. Untreated CMV reactivation increases the risk of steroid-resistance and therapy-refractory complications. In case of severe colitis combined with CMV reactivation in the mucosa antiviral therapy should be initiated. Several methods are used for the detection of CMV, but only a few technics are suggested for the current diagnosis of virus reactivation. PCR is based on the direct detection of virus DNA in samples. In our study a real-time PCR method was used to measure the virus load in various samples, including biopsies. The viral load was monitored in order to find the correlation between the severity of UC and CMV DNA load. During our study 93 cases of UC were submitted for CMV PCR. Viral DNA was detected in 62 cases and 34 CMV positive patients were treated with an antiviral drug. In 25 cases the clinical symptoms were resulting in improvement. Our findings showed mainly high (>100) copies of viral DNA load in severe and mild-moderate UC. In addition, our observation was that the antiviral treatment was usually less efficient in cases when low (<100) virus DNA copies were found. In conclusion, early and accurate detection of CMV from inflamed colonic mucosa seems important in the proper treatment of severe ulcerative colitis.

Comparison of various troponin I tests and NT-proBNP in acute coronary syndrome patients

ML. Ősz¹, J. Bodnár¹, I. Gilányi¹, A. Lukács², B. Fodor^1,2

^1.BAZ County’s Central Hospital and University Teaching Hospital, Department of Laboratory Medicine, ^2.University of Miskolc, Faculty of Healthcare Studies

Introduction. The determination of troponin levels is essential in the diagnosing and management of acute coronary syndrome (ACS). NT-proBNP also has an important role in the stratification of left ventricular function and heart failure.

Aim. Comparison of serum troponin I results in hospitalized patients - due to acute cardiac events – measured by 2 tests of different sensitivities. Determination of reproducibility, CV% of troponin I values - around the cut off value - and ascertain the correlation with NT-proBNP results.

Patients and method. Troponin I levels were measured by Siemens Troponin I Ultra (TnIU) kit in parallel with Troponin I High Sensitive (TnIH) tests in 50 ACS patients. NT-pro-BNP tests were also performed with Siemens tests on Centaur XP instruments.

Results. The average troponin I concentration of the TnI U group was 43.88 ng/l (+/- 8.94 ng/l). It was 33,66 +/- 6,94 ng/l in the TnIH group respectively. The CV% value was under 10% in both groups. We observed a close correlation between TnIU and H results (Rho=0,467, p=0,001, Spearman correlation). Troponin I values showed a loose correlation with proBNP, but high TnIH values were obtained in patients with exceptionally high NT-proBNP values. This correlation was not observed in the TnIU tests.

Discussion. Determination of troponin I – e.g. around cut off – in acute coronary syndrome patients can be reproducibly performed with different sensitivity tests. In addition, Siemens high-sensitivity troponin I test correlates well with the highly elevated NT-proBNP value, which improves the global assessment of myocardial status.

Biodosimetric study of two patients with suspected radiation damage. A case-report.

G. Deli, K. Emődy, S. Papp, Á. Pataki and M. Mátyus,

Medical Centre, Hungarian Defence Forces, Budapest, Hungary

In our case report, two patients with suspected radiation injury is demonstrated. Blood samples were tested with cytokinesis blocked micronucleus (CBMN) and dicentric chromosome (DIC) assay. Methods: The CBMN and DIC assays are cytogenetic test methods. The blood is cultured in the presence of phytohaemagglutinin (PHA) for days in order to induce the lymphocytes to divide. Cell division is stopped at telophase in case of CBMN or at metaphase in case of DIC with cytochalasin-B and colcemid respectively. If the chromosomes are damaged due to ionizing radiation prior to the blood sampling, abnormal chromosomes occur in the metaphase cells. Acentric chromosome fragments and whole chromosomes that are unable to interact with the spindle lag behind at anaphase, and as a result they are not included in the main daughter nuclei, this results in micronucleus formation. The number of MN and DIC are counted in microscope. Evaluation: The results obtained with the CBMN showed an elevation compared to the reference value of our laboratory. We could not exclude radiation damage, so we performed the more complex but specific DIC test; we got the results few weeks later: microscopic processing did not reveal any DIC at all. Some “minutes” – spot like chromosome parts – were found in one of the two patients, but this does not indicate radiation exposure. In summary, no sign of radial damage was found in any of the patients.

Assessment of cell damage caused by ionizing radiation by various molecular biology methods

G. Deli, S. Papp, K. Emődy, Á. Pataki and M. Mátyus

Medical Centre, Hungarian Defence Forces, Budapest, Hungary

The extent of damage caused by ionizing radiation can be estimated from human blood by traditional microscopic examination based on formation of injured, dicentric chromosomes. This test is a very time consuming process that can be used only in limited rate in disaster situations.

The biochemistry and molecular biology methods available in our department provide fast and high throughput opportunities in detecting the effects of ionizing radiation on human body. These methods are PCR, chips, fluorescent probes, ELISA and western blot techniques. Their advantage is the high throughput, while the disadvantage is the limited time frame due to the fast repair mechanisms and the apoptosis of the damaged cells.

Nevertheless, the combined use of these methods can be suitable for the triage of a large crowd, detecting radiation exposure, or for the studies aimed at the effects of ionizing radiation at molecular level and in the search for radioprotective materials.

With these methods the radiation casualties can receive diagnosis and proper treatment faster during disasters thereby reducing the risk of developing malignant diseases.

In everyday life the soldiers of Hungarian Defence Forces have higher risk of exposition to ionizing radiation than civils. With these methods the radiation sensitivity of the soldiers deployed for mission can be determined and with the follow-up examination of the homecoming people with complaints in case of radiation injury the proper therapy can be selected.

Trimester-specific fructosamine reference intervals in healthy pregnant women

E. Pintér¹, K. László², B. Kávai¹, and J. Konderák¹

¹Synlab Budapest Diagnostic Center, Clinical Chemistry Laboratory, Budapest, Hungary, ²Synlab Laboratory Székesfehérvár, Hungary

Hemodilution and altered metabolic changes during pregnancy changes the behavior of serum proteins, including their glycosylation. Our aim was to determine trimester-specific reference intervals of fructosamine (FRA) for healthy pregnant women. The selection of 375 pregnant women was based upon their 1^st, 2^nd and 3^rd trimester. Samples were collected from 121, 127 and 127 persons at the 9,3 (± 2,04), 22,98 (±3.14) and 35.65 (± 3,42) gestational weeks, respectively. Diabetics, patients with impaired glucose tolerance and thyroid dysfunctions were excluded. Serum FRA, total protein and albumin were measured. The levels of FRA were determined by two different kinetic colorimetric tests, marked as “A and “B”. Non-parametric, and parametric percentile method was used to determine the reference range. Results: the 2,5-97.5^th percentage of FRA “A” testss were 166,58-288,95 μmol/l in the first trimester, 168-263μmol/l in the second trimester and 167-271μmol/l in the third trimester. On the other hand, these values of FRA “B” test were 194-269μmol/l in the first trimester, 180-233 umol/l in the second trimester and 171-220 μmol/l in the third trimester. Reference ranges of non-pregnant women given by the manufacturer were 161-351 μmol/l for test “A” and 205-285 μmol/l for test “B”. The expected FRA levels should have been decreased during pregnancy. According to our experience test “A” did not follow the reduction caused by pregnancy, while “B” test did. The FRA “A” test reference range was wider than that of the “B” test. In this study test “B” was more suitable for determining the FRA, than the test “A”. Conclusion: the review of the trimester-specific FRA reference ranges is inevitable for the accurate diabetic control of pregnant women.

Prevalence of myeloperoxidase deficiency in the hematological department of the Clinical Chemistry Laboratory of Synlab Diagnostic Center

E. Pintér, J. Konderák

Synlab Budapest Diagnostic Center, Clinical Chemistry Laboratory, Budapest, Hungary

Myeloperoxidase deficiency (MPO) results in insufficient microbial killing function, and before the appearance of modern hematological analyzers it was an extremely rarely diagnosed disease. Nowadays MPOD is usually visible on the scattergram and is a frequent laboratory diagnosis. The aim of this study was to determine the number of MPOD persons amongst the hematology samples in our laboratory to prevent queries. We determined retrospectively the number of MPOD patients in 151167 blood counts in 2017. Blood counts were measured with ADVIA 2120 hematology analyzer. Among the laboratory parameters, blood sedimentation rates, CRP, and blood glucose levels were determined to detect diabetes and inflammatory processes. Patients were followed for twelve months after their initial examination. Out of 114016 patients we found complete or partial MPO deficiency in 150 patients. We performed 494 tests in 150 patients within one year and amongst them 17 complete MPOD patients (9 women and 8 male, mean age: 52±19,98 and 57 ±19,2 years) were found, which means that the prevalence rate is 1/6707. In case of further 133 patients (79 women and 53 males mean age: 44,2 ±17,9 and 44,3 ± 14,73 years), partial MPOD was observed. Its prevalence is 1/857. Among patients with complete MPOD, a high CRP was found in 1 female patient, while the blood glucose concentration in each patient was in the reference range. Elevated blood sedimentation rate was detected in two females and in one male patient. From outpatients with partial MPO deficiency 16 patients had higher glucose level than the reference interval (12%). 16 patients had slightly elevated CRP (12%) and in 24 patients higher blood sedimentation rate was found (18%). Amongst the patients with replicate measurements of the inflammatory parameters there were no serious infections. MPOD does not mean therefore that all patients are susceptible to infections.

The effects of long-term cannabis smoke exposure in mice

K. Rusznák¹, K. Csekő^1,2, Zs. Varga¹, D. Csabai¹, Á. Bóna³, M. Mayer⁴, Zs. Kozma⁴, Zs. Helyes^1,2 and B. Czéh^{1,5 .}

¹Szentágothai Research Centr. Univ. Pécs, Pécs, Hungary; ²Dept. Pharmacology and Pharmacotherapy, Medical School. Univ. Pécs, Pécs, Hungary; ³Dept. Biochemistry and Medical Chemistry, Medical School. Univ. Pécs, Pécs, Hungary; ⁴Dept. Forensic Medicine, Medical School. Univ. Pécs, Pécs, Hungary; ⁵Dept Laboratory Medicine, Medical School. Univ. Pécs, Pécs, Hungary

Marijuana is a widely used recreational drug with increasing legalization worldwide for medical purposes. Our aim was to mimic real-life situations where young people smoke cannabis regularly to relax from everyday stress. Therefore, we exposed young adult male mice to daily stress and concomitant marijuana smoke for two months and investigated the consequences on physiology, behavior and adult hippocampal neurogenesis. Cannabinoid content of the smoke and urine samples was measured by HPLC and SFC-MS/MS.

Results: Both stress and cannabis exposure significantly reduced body weight gain. Cannabis smoke had no effect on pulmonary functions, but stress delayed the maturation of several lung functions. Neither stress, nor cannabis smoke affected the cognitive functioning of the animals. Results of the open field test revealed that cannabis had a mild anxiolytic effect and markedly increased self-grooming behavior. Stress blocked cell proliferation in the dentate gyrus, but cannabis had no effect on this parameter. Cannabis had a negative influence on the morphology and number of doublecortin-positive immature neurons. In summary, in this experimental design, we found that long-term exposure to cannabis smoke had either no effect or negative impact on various health-related measures. Finally, cannabis smoking could not alleviate any of the stress-induced effects.

Diagnostic accuracy of platelet count and mean platelet volume in patients with venous thromboembolism. A systematic review and meta-analysis

A.H. Shemirani¹,4, K.S. Zsóri², Z. Csiki³, Z. Bereczky⁴, S. Kovács⁵

Erzsébet Hospital ¹Central Laboratory, and ²Central Pharmacy, Sátoraljaújhely, Hungary; Debrecen University, ³Dep. Medicine, ⁴Div. Clinical Laboratory Science, Dep. Laboratory Medicine, and ⁵Dep. Research Methodology and Statistics, Debrecen, Hungary.

The purpose of this study was to evaluate the association between platelet count (PLT) and MPV and venous thromboembolism (VTE). We performed a quantitative synthesis on data from 18 studies in accordance with PRISMA guidelines. A random-effect meta-analysis was conducted for the assessment of heterogeneity using thrombosis place, type of analyzer, type of anticoagulant and incubation time of samples as covariates. A mixed-effect meta-regression was performed based on the subgroup for the whole samples using thrombosis place and method of measurement as moderators for MPV and PLT, respectively. The cumulative estimates and 95%CI of specificity, sensitivity, AUC and diagnostic odds ratio (DOR) for MPV were calculated using a random effect model. The quality assessments were evaluated according to the QUADAS-2. The primary outcome was the occurrence of VTE. Secondary outcomes included PLT and MPV. Patient with DVT is likely to have a higher value of MPV than control group (P<0.001). The presence of pulmonary embolism (PE) had no significant effect on the standardized mean difference of MPV between patients and controls. Patients are likely to have less PLT than the control group regarding all studies. However, subgroup analysis demonstrated that this effect was significant only for patients with PE (P<0.05). The summary receiver operating characteristic curve indicated that AUC was 0.745 (95%CI: 0.672-0.834). The DOR for MPV was 4.76 (95%CI: 2.3-9.85), with diagnostic accuracy of 0.66.

Electrophoretic analyses of perchloric acid soluble serum proteins in systemic inflammatory disorders

B. Szirmay¹, Cs. Páger^2,3, P. Kustán¹, E. Györgyi¹, T. Kőszegi^1,3, A. Ludány¹, F. Kilár^2,3, L. Makszin^2,3

¹Department of Laboratory Medicine, ²Institute of Bioanalysis, Faculty of Medicine, ³Szentágothai János Research Centre, University of Pécs, Hungary

The characterization of perchloric acid soluble proteins in healthy individuals and in patients with various diseases is of high importance. E.g., orosomucoid, which is an acute phase protein, can be detected in the supernatant of PCA treated sera even in healthy individuals. Recently, a perchloric acid precipitation method was worked out for the isolation and characterization of acid soluble serum proteins with low molecular mass. Striking differences were found when the amount and pattern of acid soluble proteins of healthy individuals were compared with those of patients suffering from systemic inflammatory diseases. The method has proven that in malignancies the amount of acid soluble proteins is increased, and characteristic changes in the PAGE protein patterns are obtained as well. The major goal of the research is to find and identify disease specific molecular patterns of perchloric acid soluble proteins in bacterial sepsis and in Crohn’s disease. The applied methods include one dimensional SDS gel electrophoresis, capillary zone electrophoresis and microchip electrophoresis. The Agilent 2100 Bioanalyzer microchip electrophoresis system was used to analyze labeled protein complexes with the High Sensitivity Protein 250 LabChip kit, applying some minor modifications. These chip electrophoretic methods are able to complete the conventional SDS-PAGE, with the advantage of better sensitivity, high speed and providing good quantification. Further plans include the analysis of the acid soluble proteins by mass spectrometry.

Thrombomodulin gene (THBD) polymorphisms play a protective role against transplant related mortality after hematopoietic stem cell transplantation (HSCT)

L. Varga¹, P. Kövy², N. Meggyesi², A. Bors², E. Torbágyi³, L. Gopcsa³, A. Tordai⁴, T. Masszi⁵, P. Reményi³, H. Andrikovics²

¹Hungarian National Blood Transfusion Service; ²Lab. Mol. Genet., Central Hospital of Southern Pest (DPC); ³Dept. Hematology and Stem Cell Transplantation, DPC; ⁴Dept. Pathophysiology, Semmelweis University (SE); ⁵ 3^rd Dept. Internal Medicine, SE, Budapest, Hungary

The endothelial membrane protein, thrombomodulin (THBD), is an important regulator of coagulation and innate immunity. Functional single-nucleotide polymorphisms (SNPs) within the THBD gene might contribute to abnormal reactivity during graft versus host disease (GvHD) after allogenic HSCT. We analyzed the impact of THBD SNPs (rs1962, rs1042579 and rs1042580) after HSCT [n=398; 217 sibling/181 HLA-matched donor; 240 myeloablative (MAC)/158 reduced intensity conditioning (RIC)]. SNPs were genotyped on LightCycler480.

Homozygosity for either THBD SNP influenced acute GvHD [8.5% (8/94) in homozygotes vs. 16.8% (51/303) in non-homozygotes, p=0.048] and transplant related mortality [16.0% (15/94) vs. 35.3% (107/303), p=0.002]. Relapse rate was not different. In homozygous patients, favorable overall survival was observed (at 48 months 60.5±5.2%. vs. 44.7±3.0%, p=0.02). The prognostic effect of THBD homozygosity was independent from other favorable factors like younger age at HSCT or sibling donor. The impact of the variants was more pronounced in MAC treatment (test for interaction: p=0.032). Our results suggest that recipient THBD gene polymorphisms rs1962, rs1042579 and rs1042580 may influence the development of acute GvHD and the overall survival in patients with myeloablative conditioning. Project was partly funded by NVKP_16 and NKFIH K104903 grants.

Actin is a promising urinary marker of sepsis-induced acute kidney injury

D. Ragán^1,2, P. Kustán^1,2, A. Ludány¹, D. Mühl², T. Kőszegi¹

¹Department of Laboratory Medicine, University of Pécs, Hungary

²Department of Anesthesiology and Intensive Therapy, University of Pécs, Hungary

Successful treatment following early diagnosis of sepsis is still a demanding challenge. The clinical relevance of various urinary proteins is not fully understood. Actin is a ubiquitous globular protein of 42 kDa. The so-called actin scavenger system binds and depolymerizes actin in the circulation during the physiological cell turnover, but its appearance in the urine is related to systemic diseases. Therefore, the main focus of our research is the quantitative analysis of actin in the urine of septic patients. Urine samples were taken from ophthalmological patients (n=12) as control and from ICU-patients (n=19) diagnosed with severe sepsis. Urinary actin concentrations (ng/ml) were determined with a Western blot/ECL (enhanced chemiluminescence) technique. A specific primary (Rabbit Anti-Human Actin, Sigma-Aldrich) and secondary antibody (Swine Anti-Rabbit Ig peroxidase labeled, Dako) were used during the immune reaction. A Femto Sensitivity Substrate and a digital CCD camera were applied for quantitative evaluation. A Mann-Whitney U test was used in the SPSS program for statistical analysis. Actin could not be detected with our method in the control samples. However, actin was present in every septic sample during follow-up. Urinary actin levels were even higher in septic patients with acute kidney injury (AKI, KDIGO classification) in contrast to the septic group without kidney injury (8.17 vs. 4.03 ng/ml, p<0.05). A sensitive and accurate method was developed for the measurement of urinary actin. Elevated urinary actin levels could indicate extensive cellular death, as well as severe kidney damage. Future studies may elucidate the importance of urinary actin regarding the diagnosis and prognosis of sepsis-induced AKI.

Measurement of Gamma-H2AX during radiotherapy of gynaecological tumours

G.Bekő¹, A.Ebegényi¹, É.Rozmarin¹, E.Zabolai¹, Sz.Déri², V.Drajkó²

Central Laboratory of Uzsoki Hospital, Budapest, Hungary

Clinical Oncoradiology of Uzsoki Hospital, Budapest, Hungary

It has become known in this decade, that if breaks occur in the DNA double helix, H₂A_X histones get rapidly phosphorylated at serine 139, aiding the DNA repair process. The phosphorylated form of H2Ax was named γ-H2Ax because it was first observed in cells exposed to γ- rays. IR-induced double-strand breaks (DSBs) cause the increase of γ- H2Ax level in human cells. The γ-H2Ax is formed throughout the whole cell cycle. It appears within minutes and reaches its maximum level after 30 minutes.

Our goal was to investigate the number and intensity of γ- H2Ax positive cells examining mononuclear cells in the peripheral blood of patients with gynaecological tumours undergoing radiotherapy.

35 patients were examined with stage II. and III. gynaecological tumour, between the age of 30 and 70. Their blood was taken 30 minutes after the 3^rd irradiation. The γ-H2Ax activity of their mononuclear cells was measured in fresh samples and also after one hour at 37°C incubation. The measurement was performed with the DNA Damage Kit from Becton Dickinson on the BD FACSCalibur^TMx analyzer.

In six cases there were more γ-H2Ax positive cells measured after one-hour incubation than in fresh samples. In three cases, the γ-H2Ax intensity decreased by less than 20% within one hour. In one case there were no γ-H2Ax positive cells found after radiotherapy.

The coherence was investigated between γ-H2Ax positive cells and the change of their intensity and the short- and long-term adverse reactions of the patients.

Orosomucoid in urine: a promising novel marker for monitoring systemic acute inflammation

¹P. Kustán,¹B. Szirmay, ¹Z. Horváth-Szalai, ^1,2D. Ragán, ¹A. Ludány, ¹A. Miseta, ³B. Németh, ²D. Mühl, ¹T. Kőszegi

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

²Department of Anaesthesiology and Intensive Therapy University of Pécs Medical School, Pécs, Hungary

³Department of Public Health Medicine, University of Pécs Medical School, Pécs, Hungary

Background: Monitoring systemic acute inflammation is essential for the early recognition of life-threatening complications as sepsis and acute kidney injury (AKI). Our aim was to study urinary orosomucoid (u-ORM) as a potential marker of inflammation.

Materials and Methods: Patients after cardiac surgery (n=38), patients after major abdominal surgery (n=20) and septic patients (n=60) were monitored in a 5-day follow-up study. U-ORM was measured by a novel automated turbidimetric assay and referred to urinary creatinine (u-ORM/u-CREAT, mg/mmol).

Results: After surgical interventions a rapid 10-fold elevation was found in u-ORM/u-CREAT values which decreased on the 5^th day after cardiac and even after abdominal surgery, if no complications developed. Moreover, u-ORM/u-CREAT values were about 10-times higher with non-decreasing tendency in septic patients compared to surgical cases (p<0.001). The diagnostic ability of u-ORM/u-CREAT for sepsis recognition was better than that of hs-CRP (AUC ROC: 0.954 vs 0.845). Dialyzed AKI patients showed extreme u-ORM values (52.2 (19.4-154.7) mg/mmol). U-ORM/u-CREAT correlated well with hs-CRP (p<0.001).

Conclusions: U-ORM seems to be a sensitive marker of inflammatory activation. Monitoring of u-ORM/u-CREAT might be able to reveal severe complications of inflammation and might help clinicians in rapid decision-making.

Evaluation of the analytical performance of Siemens high-sensitivity Troponin I immunoassay on Advia Centaur XPT platform

I. Fábián, I. Takács, É. Ajzner

Jósa András Teaching Hospital, Nyíregyháza, Hungary

According to the National Society of Cardiology the use of high sensitivity cardiac troponin (hs cTn) assays play a key role in the diagnosis and differential diagnosis of acute coronary syndrome (ACS).

We evaluated the analytical performance of a High-Sensitivity Troponin I (hs cTnI) chemiluminescent assay (Siemens, Germany) on Advia Centaur XPT platform (Siemens). We also analyzed if the assay fulfils high sensitivity criteria. Namely, if the functional sensitivity of hs cTnI is below the AMI diagnostic cut-off of 47,34 ng/L and if cTnI can be quantitated in more than 50% of healthy subjects.

The limit of blank and limit of quantitation values of the hs cTnI assay were determined by EP17 CLSI standard and resulted in good correlations with those provided by the manufacturer: 0,76 ng/L and 1,19 ng/L, respectively. Functional sensitivity of the hs cTnI assay was also measured by the above standard. cTnI concentration which can be measured with a minimum repeatability of CV≤ 10% was found at 4,12 ng/L. Repeatability and reproducibility of hs cTnI assay at a concentration level around the cut-off value were found 2% and 5%, respectively. cTnI values were found measurable in 41 out of 74 (55%) healthy subjects. cTnI values of patients with suspected ACS and patients with decreased renal function measured by the hs cTnI assay from Siemens correlated well with those measured by our laboratories’ former hs cTnI assay on Vidas (Biomérieux, France) providing R² of 0,89, and 0,93, respectively.

Siemens hs TnI assay showed a good analytical performance and met the criteria of high sensitivity in our studies.

Comparison of automated Siemens Advia Centaur procalcitonin assay with Vidas B.R.A.H.M.S procalcitonin assay

Zs. Kozma, I. Fábián, É. Ajzner

Jósa András Teaching Hospital, Nyíregyháza, Hungary

Procalcitonin (PCT) is a biomarker used in the diagnosis and management of bacterial infection and sepsis. Siemens Advia Centaur PCT Assay on Centaur XPT analyser (Siemens, Germany) (Siemens assay) and BRAHMS PCT Assay (Thermofisher Scientific, Germany) on Vidas analyser (Biomérieux, France) (Brahms assay) are both one step sandwich immunoassays for in vitro diagnostic use in the quantitative measurement of procalcitonin in human serum and plasma. The aim of this study was the evaluation and comparison of the analytical performance of these assays. Imprecision of the assays were assessed using low and high-level control materials provided by manufacturers and were found in the range given by manufacturers. Repeatability of the Siemens assay at low and high concentration were 0,01% and 1,08%, respectively. These values in the Brahms assay were 2,2% and 2,87% respectively. Reproducibility in Siemens and Brahms assays was detected as follows: 3,48% (low), 2,5% (high), 2,27% (low level), and 3,22% (high), respectively. The recovery of assayed control materials of the manufacturers showed better performance in Brahms assay (bias 0,88 at low concentration, -1,96 at high concentration) than in Siemens assay (bias -10,8 at low concentration, -2,88 at high concentration). PCT values of 27 patient samples (representing both normal and pathological values) were used in method correlation studies. Although the two assays differ in sample volume required, measurement range and analysis time, Siemens assay correlated well with the reference Brahms assay (R²=0,83). Siemens and Brahms assays correlated well and showed low imprecision and bias in our studies. Both assays can reliably used in diagnosis and follow-up of sepsis.

The importance of combined stool examinations in the investigation of intestinal diseases

D. Fazekas

Synlab Hungary, Budapest Diagnostic Center, Immunological Laboratory, Budapest, Hungary

Nowadays, the diseases related to the gastrointestinal tract are considered as endemic. However, establishing the exact diagnosis sometimes is a big challenge for the specialists. Patients may prefer the non-invasive laboratory examinations as the first step. The sensitivity and the specificity of diagnostic stool examinations are varying, therefore performing multiply examinations can increase these values. The aim of this study was to investigate the combined use of M2-PK, calprotectin, which are considered as new markers, and the fecal-occult-blood test, which is traditionally used for investigation of colon tumors. 788 cases, where all the three examinations were requested, have been statistically analyzed. Out of the 788 patients, there were 414 females (average age: 49 years, SD: 12.31) and 374 males (average age: 48 years, SD: 12.83). The indications of the investigations were screening, inflammatory disease-, and tumor-suspicion. 61.8% of examinations were negative for all parameters (232 males and 255 females), 1.4% were positive for all parameters (3 males and 8 females). At least one positive result was found at the rest of the cases. Considering the reference ranges, the results have been divided according to a score system. High scores have been found at 22 cases (4-5 points) and low scores at 279 cases (1-2-3 points). In the background of these positive results, different diseases may occur. With comparing the laboratory results and the clinical data, we are looking for the answer of what kind of diseases could be detected due to the different results of the three different laboratory examinations, and for the answer of how the normal range could be specified in case of inflammations and tumors. Considering the clinical data, beside the endoscopy, requesting these tests together can help establish the diagnosis.

Statistics of measurements processed on the newly introduced automated immunology analyzer in our laboratory and the evaluation of endocrinological requests patterns in the hospital

N. Farkas, M. Borsos, E. Kovács, A. M. Peti

Hospital of Siófok, Central Laboratory, Siófok, Hungary

Hormonal system secretions refer to the actual endocrine status of our body, which in vitro is measured using high sensitive - nano and pico measurement ranges - and high specific methods.

The purpose of our study was to compare the results of Roche Cobas e411 (E411) and Beckman Coulter UniCel DXI-600 (DXI-600) automated immunology analyzers, which use ECLIA and CLIA methods respectively, and to assess the endocrinological requests patterns, using data obtained from laboratory information system (LIS). Statistical analyzes were performed using IBM SPSS 21 and Microsoft Excel spreadsheet programs.

The LIS three years (2015-2017) statistics showed that on average 37.000 immunological tests were performed annually and the most common were: TSH, vitamin D, free T4, PSA, CEA and beta-cross laps. The comparison of the two automated immunology analyzers measuring methods showed a close correlation between PSA, estradiol, TSH, prolactin and free T4. Comparing the two different detecting methods, PSA reagents showed the best correlation (r=0.998, p<0.01). The interassay and intraassay coefficient of variations of immunological method on DXI-600 in case of TSH were 0.1-2.8% and <0.2% respectively. We observe that on DXI-600 the prolactin measurements had lower (r=0.986, p<0.01) values, while in case of estrogen they were higher (r=0.995, p<0.01), with strong coefficients of correlation.

Our investigations have shown that the DXI-600 measurement results showed good correlations with E411 therefore can be reliably used for endocrinological measurements and need closer cooperation with requesting doctors for more economical health care.

Tragic case of a drug abuser from the point of view of the toxicological laboratory

Á. Lakatos¹, A. Lajtai¹, M. Mayer², M. Kuzma², A. Miseta¹

University of Pécs, ¹Dept. of Laboratory Medicine, ²Dept. of Forensic Medicine, Pécs, Hungary.

In our toxicological laboratory unit, we examine the serum and urine samples of poisoned patients. We can identify a lot of medical and recreational drugs. Several times we perform quantitative analysis as well. Most of the cases are new but there are a few patients who return after some months or years, overdosing some drugs again accidentally or because of suicide trial. However, there was a patient (a woman from her 31 to her 37 years of age) who returned again and again for years because of overdosing drugs for different reasons (suicide trial or accidental). We made altogether 42 toxicological analyses connected to her between 2012 and 2018. She used alcohol, venlafaxine, alprazolam, quetiapine and some new psychoactive substances like pentedrone or α-PVP in different concentrations and variations during the seven years. Finally, she died in February this year not for overdosing the drugs, but for burning in a fire. Fortunately, this form of drug addiction is very extraordinary, but it may be illustrative to speak of her exceptional case.

Microparticles as potential biomarkers in chronic obstructive pulmonary disease

M. Tőkés-Füzesi¹, I. Ruzsics², P. Kustán¹, T. Molnár³, G.L. Kovács^1,4

University of Pécs, ¹Dept. of Laboratory Medicine, ²1st Dept. of Internal Medicine, ³Dept. of Anesthesiology and Intensive Therapy, ⁴ Szentágothai Research Centre, Pécs, Hungary.

Microparticles (MPs) are small shedding membrane vesicles released into the extracellular microenvironment from circulating blood and endothelial cells during cell activation or apoptosis mainly under inflammatory conditions. The role of endothelial microparticles (EMPs) in pathophysiology of chronic obstructive pulmonary disease (COPD) is relatively well-known. On the contrary, the release and function of MPs from other cellular origin (e.g. platelets, leukocytes, red blood cells) are not clearly evaluated in this patient group. We measured – in addition to the EMPs – MPs derived from other cells in the circulation of stable and exacerbated COPD patients. 19 healthy volunteers and 50 patients with COPD (34 with stable and 16 with exacerbated COPD) were enrolled in the study. Endothelial (CD31+, CD62E+), platelet derived (CD61+, CD41+, CD42a+, PAC1+), red blood cell derived (GlyA+), leukocyte derived (CD45+, CD13+, CD14+) and lymphocyte derived (CD56+) MPs were measured. MPs were separated from the cell free plasma after centrifugation of citrated blood. Flow cytometry was performed on Beckman Coulter FC500 analyzer. MP reference gate was set using 0.3-0.5-1μm microbeads with MP size gates of 0.5-1μm. Statistical analysis was done with SPSS 19 software. All the measured MPs were significantly (p<0.001) higher in COPD patients than in the controls. Furthermore, CD62E⁺, CD41+, CD42a+ and CD14+ MP values were significantly (p<0.001) elevated in exacerbated COPD compared to stable COPD. COPD is accompanied by increased numbers of various MPs in the systemic circulation, particularly platelet and monocyte derived MPs seem to be important in the process of exacerbation.

Urinary steroid profiles of women with granulosa cell tumour of the ovary

Zs. Preisz¹, F. Kilár¹, P.M. Gőcze², N. Farkas¹, A. Bufa¹

¹University of Pécs, Medical School, Institute of Bioanalysis, Pécs, Hungary

²University of Pécs, Medical School, Department of Obstetrics and Gynaecology, Pécs, Hungary

Granulosa cell tumour of the ovary (GCTs) is the most frequent sex cord stromal tumour and represents 5% of all primary ovarian cancers. Ovarian granulosa cell tumour is a malignant tumour with slow progression and in some cases this tumour is hormonally active.

The potential roles of androgen metabolism as co-factors in the development of GCTs were investigated. We obtained 24-hour urine samples from 15 patients with GCTs, and from 12 age-matched, healthy female subjects. The concentrations of 20 androgen, progesterone and corticoid metabolites in the urine samples of the two groups were quantitatively determined by gas chromatography-mass spectrometry with selected ion-monitoring (GC/MS/SIM).

Laboratory examination did not reveal elevated levels of serum tumour markers and hormones (CEA, CA-125, CA-15-3, CA-19-9, AFP, FSH, LH, P, E₂, T, A). The urinary concentrations of 16-OH-DHEA, PD, ∇5-PD, ∇5-PT and α-C were found significantly higher (p<0.05) in the patient group with GCTs compared to the control group.

The work was supported by ÁOK-KA-2015-16.

Kinetics of serum presepsin levels in septic patients

A. Marossy, B.B. Patai, Á. Csomós, B. Emődy-Kiss, J. Fent, J. Gonda, L. Keresztes, S. Lakatos, E. Takács, G. Zacher, J. Simon, M. Mátyus

Medical Centre of Hungarian Defence Forces, Budapest, Hungary

Presepsin, the soluble sub-type form of LPS receptor (sCD14st) was suggested to be a good biological marker for the early diagnosis and prognosis of bacterial sepsis.

In the present study, 25 adult patients (18 men and 7 women, mean age: 59 and 76 years, respectively) admitted to the emergency department with clinical signs of severe infections were involved. According to the clinical outcome, patients were divided into surviving and non-surviving groups.

Daily Presepsin concentrations were determined as far as it was possible or for at least 11 days. Changes of procalcitonin and C-reactive protein concentrations were also detected as part of routine follow-ups. The kinetics of serum Presepsin concentrations were compared between the surviving and non-surviving groups.

Our data suggest that if the concentration of Presepsin has a monotonous decrease and if its value becomes less than 500 ng/ml, the patient has a chance for survival and for recovery. If Presepsin concentration fluctuates around a relatively high level (1000 ng/ml) the prognosis is not too promising.

Considering that sepsis does not have homogeneous clinical picture, furthermore, patients usually have one or more other concomitant disease(s), therefore further data collection is needed in order to draw a solid conclusion on the prognostic value of the kinetics of Presepsin concentrations.

Determination of CD34+ absolute cell count by flow cytometry

M. Száraz-Széles, J.Kappelmayer, Z. Hevessy

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

The determination of CD34+ absolute cell count is an obligatory test during stem cell transplantation, which is applied in the treatment of malignant hematological disorders. This value is crucial in several steps during transplantation: (i) evaluating the success of the mobilization (ii) selecting the appropriate time for leukapheresis (iii) establishing the CD34+ cell count of the leukapheresis product and the stem cell concentrate to be administered for the patients.

The method is based on using fluorescently tagged antibodies and known concentration of beads. The analysis is carried out by the single platform technique using the ISHAGE protocol.

In the past year we performed CD34 enumeration from 837 samples, derived from 115 patients during peripheral stem cell transplantation. Overall 339 peripheral blood samples, 201 leukapheresis products and 297 stem cell concentrates were examined.

The analysis needs to be done immediately but at latest within 24 hours. This test is carried out in an ‘on call system’ each day including weekends. During regular working hours the turnaround time of the assay is 90 minutes. Since the CD34+ cell count is of utmost importance in clinical decision making, we handle each case as an emergency test in the Flow Cytometry lab. The test has an external quality control; the UK-NEQAS CD34 enumeration program, where 12 samples are analyzed annually and their results are reported online.

Cerebrospinal fluid case studies – exceptions that prove the rule

D. Bálint-Nagy¹, É. Érckövi¹, A. Egri²

¹Kiskunhalas Laboratory, SYNLAB Hungary Ltd., Hungary and ²Semmelweis Hospital, Kiskunhalas, Hungary

The timely identification of the underlying etiology of cases with meningitis represents a severe challenge in clinical practice. It can be generally stated that the sooner the cause is identified the better the chances are for the patient, and a delay in the initiation of therapy is associated with a significantly worse prognosis. Our study presents two exceptions. Our first case is a 48-year-old female patient admitted to the Emergency Department with headache, nausea, vomiting and fatigue. Her past medical history was remarkable for COPD and bronchial asthma. The other case is a 44-year-old male patient who was transferred to the Emergency Dept. in a confused state. No relevant information could be obtained from the patient himself. The female patient had been treated for a purulent meningitis one month before the current presentation. She had previously undergone a surgery for nasal polyp, with a tampon left in the right maxillary sinus for more than two weeks. On her current emergency admission, the laboratory findings and the clinical picture were suggestive of tuberculous meningitis. On the basis of leukocytosis, elevated C-reactive protein levels, and the clinical picture, meningitis was suspected in the male patient, and microbiological culture was performed. The CSF culture of the female patient was positive for Nocardia species. The CSF and blood culture of the male patient proved positive for Listeria monocytogenes. In our female patient, the pathogen was revealed several months after the onset of the first symptoms; however, the patient could be discharged a couple of days after starting the targeted therapy, and she is still well and works years after. In the case of the young male patient with Listeria on the other hand, although the pathogen was identified within a few days, the patient was beyond help.

Fecal calprotectin as a biomarker in the differential diagnosis of bowel diseases

J. Nevelős¹, A. Berbécsné P. ¹, J. Nagy¹, I. Guthy², L. Szegedi³, É. Ajzner¹

Jósa András University Hospital, ¹Central Laboratory, ²Department of Paediatrics and ³Internal Medicine, Nyíregyháza, Hungary

Fecal calprotectin is an important biomarker in differential diagnosis of organic (ulcerative colitis and Crohn’s disease) and functional bowel diseases (BD). The goal of this retrospective study was to evaluate the diagnostic sensitivity of calprotectin assay in patients admitted to our institution in 2017 with suspected BD. Clinical data and fecal calprotectin concentrations measured by Calprolab enzyme-linked immunosorbent quantitative assay (ELISA) of 731 anonymized patients with gastrointestinal symptoms were included in the study. Inflammatory bowel disease (IBD) representing organic BD group and irritable bowel syndrome (IBS) representing functional BD group were classified in 214 and 80 patients, respectively. Other gastrointestinal diseases were diagnosed in 139 patients. In the remaining 220 patients BD was excluded (non-BD group). Our findings correlated well with previous observations, fecal calprotectin concentrations were significantly higher in IBD group (p<0,05) compared to non-BD and other BD groups. In addition, highest levels were found in untreated IBD patients. 200-250 mg/kg and 150 mg/kg calprotectin concentrations are suggested as key thresholds in the literature for discriminating active IBDs from all BDs and non-BDs for the decision of the need of colonoscopy. Using the above thresholds to discriminate untreated IBDs from non-BDs, receiver operator curve analyses resulted in a sensitivity of 96%, and a negative predictive value (NPV) of 100% while specificities were found between 95-99,5%. Calprotectin assay measured by Calprolab ELISA was found to have a very good diagnostic sensitivity with 100% NPV for the differential diagnosis of acute IBDs.

Invasive non-typhoidal Salmonella infections in Jósa András Teaching Hospital in 2014-2018

M. Bacskai¹, A. Farkas², K. Papp¹, M. Vámos¹

Central Laboratory¹, Department of Infectology² Jósa András Teaching Hospital, Nyíregyháza, Hungary

Non-typhoidal salmonellosis is the second most commonly reported foodborne disease in Europe. While most infections are mild, some cause more severe disease such as bacteraemia or meningitis in approximately 5-6% of patients. The elderly and the immunocompromised are at particular risk for bacteremia. The objective of this study was to characterize clinical cases detected with invasive non-typhoidal salmonellosis in our institution during the period of January 2014 and March 2018. Fifteen episodes of invasive non-typhoid Salmonella (NTS) infections were identified in this period. Male to female ratio was 7:8. Average age of patients were 68. The following pieces of information were collected from the patients’medical records: underlying disease(s), clinical presentation, immunosuppressive therapy, bacteriological characteristics of the disease, antibiotic susceptibility of isolates, treatment and clinical outcome. Gastoenteritis was detected in 14 cases, immunosuppressive therapy was applied in 5 patients, and predisposing underlying disease could be detected in 10 patients. The verification of Salmonella species was performed in the National Center for Epidemiology. Salmonella Enteritidis, Salmonella Typhimurium, monophasic Salmonella Typhimurium, and other Salmonella enterica were identified in 7, 2, 2 and 4 isolates, respectively. 13 isolates were found susceptible to every antimicrobial agent that were tested. One strain was found resistant to ampicillin, and another one to ciprofloxacin. No multiresistant strains were detected. Stool testings were positive for Salmonella infection in four episodes. Mortality rate was 13,3%. Inappropriate antibiotic treatment or lack of antibiotics were found associated with fatal outcome.

Agreement between the results of anti-dsDNA antibody measurements with a conventional ELISA, a chemiluminescent method and the Crithidia luciliae immunofluorescence test

TG. Szabó, Zs. Pósa, E. Barabás, Zs. Beleznay

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

The detection of serum anti-dsDNA antibodies might support the diagnosis of SLE and serves as an aid for monitoring. Our laboratory has recently switched from an ELISA-based assay (Orgentec) to CLIA (Inova). We assessed the results obtained by the two different techniques and related to those obtained by the Crithidia luciliae immunofluorescence test (CLIFT, Inova). We tested the sera of 41 patients exhibiting ANA pattern compatible with dsDNA.

Numeric ELISA and CLIA results correlated statistically significantly (p<0.001, r=0.71). However, Bland-Altman analysis revealed that the good agreement between results confined to lower values range [<100 U/ml]. Defining specificity and sensitivity by CLIFT results (after the exclusion of 5 patients with ambiguous pattern), manufacturer defined cutoff values (20 U/ml for ELISA and 27 U/ml for CLIA) already ensured a fair sensitivity for both tests. The best Youden statistic was found at 40 U/ml ELISA cut-off values (sensitivity and specificity values were 0,71 and 0,75, respectively) and 30 U/ml CLIA values (sensitivity and specificity values were 0,71 and 0,33, respectively).

The low specificity of CLIA indicates a high rate of positive results that should be considered when CLIA test results are interpreted.

Qualitative detection and identification of Alpha-1 Antitrypsin deficiency from dried blood samples by gel-isoelectric focusing & genotyping abnormal phenotypes

M. Valyon¹, A. Gál², M. J. Molnar², K. Tafferner¹, I. Horváth¹

¹National Korányi Institute of Pulmonology, Budapest, Hungary

²Semmelweis University, Institute of Genomic Medicine and Rare Disorders, Budapest, Hungary.

The tendency of developing chronic obstructive pulmonary disease (COPD) is significantly genetically determined. The gene for Alpha-1 Antitrypsin (A1AT) is coded at a single locus on chromosome 14 and A1AT is mainly produced in hepatic cells and by the immune system. The background of the genetic errors typically influences the serine protease inhibitor (SERPINA). The polymorphic gene presents different allels from normal MM leading to PiZZ gene mutation. A1AT deficiency is a rare disease, the incidence is not known exactly. Despite the fact that the World Health Organization (WHO) recommends that all COPD patients at least once should be screened in life, it is usually not examined.

Therefore the COPD patients in our study were screened.

288 dried blood spot samples have been phenotyped by performing Isoelectric focusing method (sebia). MM normal, and MS, MZ pathological controls were used. 32 (10,8) samples showed abnormal results. Phenotype MS: 13, M0: 2, MZ: 9, MZ Pratt: 1.

Genotyping has been completed in 9 cases until now. In 6 cases mutation was not confirmed by the use of serine protease inhibitor SERPINA 1 gene sequence analysis. In one case, in the form of heterozygous 739C>T c (p. Arg247Cys) mutation (PiF phenotype) has been identified, and in two cases (c) 863A>T (Glu288Val) (rsl17580) mutation (S-allele) has been detected.

This study shows that alpha-1 antitrypsin deficiency can be underestimated and the screening of this genetical disorder is of value in COPD care.

Wegener’s granulomatosis (case riport)

A. Komáromi, I. Dinnyés, N. Szabó, Z. Sipák

Petz Aladár County Hospital, Győr, Hungary

Wegener’s granulomatosis is a necrotizing inflammation of small and medium size vessels with granuloma formation. It is a rare multisystem autoimmune disease of unknown etiology (10 cases per year per 1 million inhabitants). It is a very heterogeneous disease in respect of severity and clinical manifestation. While it can be a rapidly progressive disease with fatal ending, there are forms limited only to one organ. Diagnosis is supported by the positivity of antineutrophil cytoplasmic antibodies and the presence of the typical histological findings.

In this study we present 2 cases. First is a middle-aged woman with joint complaints, who developed a vasculitis necrotisans that affected multiple organs in a short time with fatal ending. The other patient is a young man with high fever, two episodes of hemoptysis and productive cough. He developed vasculitis necrotisans affecting the lungs and kidneys. Thanks to early diagnosis and early therapy, his treatment is currently under way. Hopefully he will be in remission as soon as possible.

In both cases the working team of Central Laboratory in Győr Hospital had a prominent role in finding the final diagnosis.

The impact of sample preparation on direct antiglobulin test

K. Buzer, A. Izsó, J. Szakács, Sz. Szakony

St. Imre Teaching Hospital, Budapest, Hungary

Background: The Direct Antiglobulin Test (DAT) is used to detect immunoglobulin or complement bound in vivo to red blood cell (RBC) membranes by the use of anti-human globulin to form a visible agglutination reaction. In screening of DAT we use polyspecific reagent and microcolumn gel card (Diagnostic Grifols, Barcelona, Spain).

Methods: When semi-automatic instrument (Diana) was changed to automatic (Wadiana®) in our laboratory, the proportion of DAT positive results has increased significantly. When exploring the causes of the deviation, different sample preparation was found. According to the manufacturer’s recommendations, the anticoagulated sample should be centrifuged between 1600 and 2000 g for 5 minutes. We used 3000-3600 g for 10 minutes. Our aim was to find the appropriate sample preparation which significantly reduces the occurrence of the false DAT positive results.

Results: The DAT positive samples (centrifuged at 3000-3600 g for 10 minutes and measured on Wadiana®) were homogenized and centrifuged again at 1600, 1800 and 2000 g for 5 minutes and tested on the same instrument. 15.2%, 13.5%, and 45% of the samples remained positive, which were centrifuged at 1600, 1800 and 2000 g. The difference between the original and re-centrifuged samples was statistically significant in all three cases (p<0.05) but the largest decrease was achieved by 1800 g.

Conclusions: We could significantly reduce the false positive DAT results by selecting the appropriate sample preparation.

Comparison of chemiluminescent and conventional ELISA techniques in autoantibody detection

Zs. Csizmadia, K. Böröcz, V. Telek, V. Varga, T. Berki

Department of Immunology and Biotechnology, Clinical Centre, University of Pécs, Pécs, Hungary

Because of the presence of high amounts of interfering antibodies, it is important to choose the most accurate immunoserological assay for autoantibody detection to support the diagnosis of autoimmune diseases. The chemiluminescent immunoassay (CIA) is a recent alternative to conventional ELISA tests, allowing highly sensitive, more specific, and less time-consuming parallel autoantibody measurements.

We compared our well-established ELISA tests with the new CIA, where antigens are immobilized on magnetic microbeads instead of polystyrene plate wells, enabling a several fold higher surface to give a more sensitive detection of antibody titers. For simple test comparison samples were chosen from our routine diagnostic laboratory serum-bank (n>500), while for clinical confirmation of the CIA anti-dsDNA antibody measurements, we used sera from patients (n>80) with established SLE clinical diagnosis.

There was a high concordance between results obtained by ELISA and CIA: anti-dsDNA 75%, anti-Cardiolipin 90%, anti-B2GP 82%, anti-MPO 98%, anti-PR3 91%, anti-tTG 86%. Comparing anti-dsDNA measurements, the CIA laboratory data showed a better correlation with the clinical history of the patients (p-value<0.0001, alpha=0.05).

In summary, CIA enables fast, parallel autoantibody measurements with continuous loading of samples, without need of pre-dilution, and repeated measurement of standards. Although ELISA tests and the new CIA method showed high agreement, further clinical evidence based confirmation of the CIA test is needed for the full implementation of the test, and method changes.

Development of a combined high throughput and cost-effective indirect ELISA for MMR vaccine efficacy screening

K. Böröcz, Zs. Csizmadia, V. Varga, V. Telek, T. Berki, P. Németh

Department of Immunology and Biotechnology, Clinical Centre, University of Pécs, Pécs, Hungary

Nowadays recent measles virus epidemics together with an increasing number of rubella cases raise the question of waning immunity to the MMR vaccine. Consequently, there is a need for continuous monitoring of immunological protection against infection at population level. For such monitoring to be feasible, a cost-effective, reliable and high-throughput assay is necessary. Herein we describe the development of an ELISA protocol for efficacy-assessment of the MMR vaccine, combined with the practical testing of the newly-developed assay. A serum bank of anonymous patient sera was established (N>3000 samples). Cohorts were based on measles immunization schedules and/or changes in vaccine components. Our ELISA was performed by using Siemens BEP 2000 Advance System. Data were confirmed using commercially available kits, anti-measles nucleocapsid monoclonal antibody-based (anti-N) sandwich ELISA and indirect immunofluorescence (IIF). We established the operational protocol of a standardized, feasible and cost-saving assay. The results obtained are in high agreement with the confirmatory methods (based on the correspondence analysis of both IIF and anti-N sandwich ELISA; p-value<0.0001 with alpha=0.05) and reflect measles vaccination history in Hungary. Based on the measurement of 1985 sera, the highest ratio of low/questionable antibody level samples was detected in cluster ‘1978-1987’ (~25.4%), followed by cluster ‘1969-1977’ (~15.4%).

Our assay is suitable for assessment of anti-MMR immunity in a large cohort of subjects. The assay is standard and cost-effective, allows high-throughput screening and has superior signal-to-noise ratio.

In vitro inhibition of blood catalase enzyme activity

T. Nagy, Sz. Matastik, D. Molnár-Berczeli,

University of Debrecen Deptment of Medical Imaging Faculty of Medicine Debrecen, Hungary.

The enzyme catalase decomposes hydrogen peroxide into oxygen and water. Acatalasemia is a disorder marked by congenital absence of catalase. It is a homozygous mutant condition with enzyme activity of 1-8%. Hypocatalasemia is the heterozygous condition with about 50 % activity of the normal blood catalase.

In this study we studied whether other blood components could also consume hydrogen peroxide. For the inhibition of human blood catalase, we added for 1 mL of blood 107 μL of 1 mol/L sodium azide, 200 μL of 5 mol/L potassium cyanide and 90 μL of 2 mol/L 3-amini-1,2,4-triazole. We measured the activity of baseline and inhibited catalase with a spectrophotometric assay. This assay uses ammonium molybdate and its yellow complex formed with hydrogen peroxide is measured at 405 nm.

The initial blood catalase activity was 92-24.5 MU/L. After sodium azide inhibition the blood catalase activity decreased to 0,4-9,6 MU/L (1-14 %), after potassium cyanide to 0,1-12,8 MU/L (1-24 %) and after 3-amino-1,2,4-triazole to 1,6-33,2 MU/L (3,2-18,1 %) (n=18). The variation of the temperature, concentration, reaction time did not change catalase activity significantly.

We suggest that other blood components such as peroxidase, glutathione peroxidase, hemoglobin, peroxiredoxin could contribute to the consumption of hydrogen peroxide. Based on these results we plan to test the effects of these inhibitors on human blood samples from patients with catalase deficiency.

Fructosamine measurement in diabetes mellitus: establishment of reference ranges and evaluation of glycemic control effectiveness by two commercial laboratory tests

B. Kávai, E. Pintér, J. Konderák

Synlab Budapest Diagnostic Center, Clinical Chemistry Laboratory, Budapest, Hungary

Anaysis of serum fructosamine (FA) is an important test for glycemic control in diabetes mellitus (DM), reflecting the average glucose level of the preceding 2-3 weeks. The role of FA is more significant when hemoglobin A1C (A1C) is not measurable. The aims of this study were to establish a reference range that is more applicable to our own population than the factory’s specified and to evaluate the reliability of the test for diabetic patients. Methods: 408 serum samples from nondiabetic adults (males: 295, females:113; age: 9-69 years; A1C%: 4,1-6,0) and 120 samples of diabetic patients (males:61, females:59; age: 20-86 years; A1C: ≥6.5%) were analyzed on Beckman-Coulter platform with reagents manufactured by Sentinel (A) and Roche (B). From the results of both FA tests, reference ranges were calculated and Receiver Operating Characteristic (ROC) test were performed. Results: Reference ranges at test A and B were 178.2-303.9 and 212.8-295.2 μmol/L respectively, with no significant difference between males and females (p=0.853). Correlation coefficients were 0.539 and 0.757 between A1C and FA of test A and B respectively. Results obtained with ROC test: area under the curve: 0.788, 0.864; sensitivity: 70.8, 73.3; specificity: 73.3, 80.5; cutoffs: 252 and 280 μmol/L for the test A and B respectively. Conclusions: Our reference ranges and manufacturers’ reference ranges in μmol/L differed significantly at test A: 178.2-303.9 versus 161-351 in females, 118-282 in males, and less at test B: 212.8-295,2 versus 205-285. The results obtained of ROC, and the correlation with A1C also demonstrate the better performance of the test B. We suggest that reference ranges for the local populations should be used with FA test, especially for the glycemic control in pregnancy.

Most common allergen specific IgE antibodies in a Hungarian population

K. Lakatos, É. Imreh

Semmelweis University, Institute of Laboratory Medicine, Budapest, Hungary

The prevalence of allergic diseases is increasing worldwide. Currently, approximately 40% of the global population is sensitized to some environmental proteins, based on the detectable plasma levels of allergen specific IgE.

We determined the prevalence of the most common allergen specific IgE antibodies and their level (RAST category) present in a population living in the central region of Hungary.

Allergen specific IgE antibodies were assessed by an immunoblot method between February 2017 and April 2018, using the Polycheck Hungary Food 20 and Hungary Inhalation 20 panels.

1395 inhalation and 2989 nutritive panels including 20 – 20 specific IgE tests were assayed, as requested by clinicians. Individual inhalation and nutritive allergens were positive on average in 6.8% and 0.97% of patients, respectively. The most common perennial inhalation and nutritive allergens included ragweed, 6 grass mix, D. pteronyssinus, and D. farinae; and banana, peanut, and apple, with prevalence ranging between 14.6-12.9% and 2.21-1.41% of the patients based on panel positivity, respectively. IgE antibody levels were the highest in patients with positivity to feathers mix (4.0 RAST category on average), D. pteronyssinus and D. farinae (3.74 and 3.75 respectively) among inhalation allergens; and casein (3.88) and codfish (3.44) among nutritive allergens.

In conclusion, sensitization to ragweed was detectable in nearly 15% of our patient population. Although twice as many nutrition panels were requested by clinicians as inhalation panels, the occurrence of sensitization to nutritive allergens seems to be markedly lower than to inhalation allergens.

New etiology of bite wound infections in the era of MALDI-TOF mass spectrometry: Moryella indoligenes

T. Nagy¹, A. Shemesh², L. P. Szabó^2,3, G. Ádám^2,3, J. Gonda¹, Zs. Biró¹, V. K. Márkus¹, I. Szekula¹, R. Vass¹, A. Méri¹, J. Simon¹

¹Medical Centre, Hungarian Defence Forces, Central Dept. of Laboratory Diagnostics, Budapest, Hungary

²Medical Centre, Hungarian Defence Forces, Dept. of Emergency, Budapest, Hungary

³Medical Centre, Hungarian Defence Forces, Dept. of Orthopedics and Traumatology, Budapest, Hungary

Bite injuries are most commonly caused by dogs, cats and humans, respectively. Approximately 5–20% of dog bites and 30–60% of cat bites are infected. The rate of infection as a consequence of human bite falls in the range of 10% to 30%. Microorganisms which cause bite infections usually originate from the mouth, sometimes from the skin of the victim or from the environment. The microbiology of bite wound infections in humans is often polymicrobial, with a broad mixture of aerobic and anaerobic microorganisms. Anaerobes were isolated from more than two thirds of human and animal bite wound infections, especially from those associated with abscess formation. Our presentation shows an overview of the current knowledge about bacterial agents of dog, cat and human bites infections, including a case study of cat bite hand wound infection caused by Pasteurella multocida, Bacteroides fragilis, pigmented Prevotella sp. and Moryella indoligenes, all detected by Bruker MALDI-TOF MS. The Moryella genus was named in 2007 by Carlier et al., and contains only one species, Moryella indoligenes. These organisms are Gram-positive, anaerobic, non-spore forming, rod-shaped bacteria with pointed ends. For the detection of Moryella indoligenes modern molecular techniques were succesfully used not only in typical polymicrobial infections such as buttock, intra-abdominal and polymicrobial thigh abscesses and recurrent diabetic foot ulcers infections but also in infections where the involvement of anaerobic bacteria was not taken into account, including a routine culture of sputum. Increased awareness of the importance of anaerobic organisms as a cause of wound bites infections may contribute to the increase of their isolation and detection in human samples in clinical microbiology laboratories. In conclusion, MALDI-TOF MS was proved to be a rapid, inexpensive and reliable method for improving the detection of anaerobic bacteria, such as Moryella indoligenes, in the human samples.

Monitoring of drug level and anti-drug antibody production during vedolizumab therapy in patients with inflammatory bowel disease

G. Nagy¹, É. Török¹, K. Palatka², Z. Kébel², P. Antal-Szalmás¹

University of Debrecen, Department of Laboratory Medicine¹, Department of Gastroenterology², Debrecen, Hungary.

Integrin α4β7 is expressed on gut-specific lymphocytes and plays a pivotal role in their migration to the intestine. Vedolizumab (VDZ, trade name Entyvio), a humanized monoclonal IgG1 antibody to the α4β7 integrin blocks their adhesion to the gut vascular endothelium. In 2014 Entyvio was approved for patients with ulcerative colitis (UC) and Crohn’s disease (CD) in Hungary.

Our aim was to find and evaluate laboratory tests capable of measuring vedolizumab and anti-vedolizumab antibody (AVA) levels.

After overviewing the available methods, LISA-TRACKER Duo Vedolizumab ELISA kit (ref: LTV 005, TheraDiag, Croissy-Beaubourg, France) was chosen. Seventeen samples of 15 patients (9 UC/6 CD) were analyzed. Mean VDZ levels were 18.2 μg/mL and 7.4 μg/mL in patients with UC and CD, respectively (p=0.242). Drug levels were significantly higher in patients on concomitant immune modulating therapy (16.9 μg/mL vs 3.9 μg/mL, p=0.033). There was no significant correlation between drug concentrations and CRP or disease activity scores. Anti-vedolizumab antibody was not detected in any of the patients (0/15) in accordance with the approximately 4% AVA positivity reported in vedolizumab immunogenicity studies (p=0.430). Five patients had drug levels under the measuring range of the test (<2 μg/mL) suggesting the possibility of having low affinity anti-drug antibodies not detected by the bridging ELISA method used.

In conclusion, LISA-TRACKER Duo Vedolizumab ELISA kit seems to be appropriate to monitor drug and anti-drug antibody levels in patients with inflammatory bowel disease. However, insensitivity of the ELISA methods for detecting low affinity anti-drug antibody may limit its use to determine immunogenicity of vedolizumab.

Diagnostic importance of serum alkaline phosphatase electrophoresis in various oncological cases

F. Schmidt, M. Csobán, I. Csípő, E. Enyed, J. Kappelmayer

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

The diagnostics of oncological diseases strongly depends on laboratory tests, dominantly on tumor marker determinations. Several other rare laboratory tests may also contribute to early detection of metastasis, like electrophoresis of serum alkaline phosphatase (ALP). The serum samples were electrophoresed on alkaline buffered agarose gels on Sebia’s Hydrasys instruments. Each sample was applied in duplicate with lectin deposited anodally from the point of application. We report three cases with the rare finding of a macro alkaline phosphatase (macroALP), alpha 1 isoenzyme ALP and Regan isoenzyme of ALP in various suspected or confirmed oncological cases. The macroALP was detected in the electrophoretogram of a 57-year-old alcoholic patient suffering from liver disease with suspected ulcerative colitis or ventricular tumor. The majority of the ALP activity in the patient’s serum sample was localized near the application zone in samples with or without 56°C heat processing. Alpha 1 isoenzyme was detected in a 69-year-old patient diagnosed with gastric tumor that metastatized into the liver. This isoenzyme is negative in the healthy population and may be a useful marker for the diagnosis of liver metastases. Initial studies show that alpha 1 fast liver fraction of ALP, detected by electrophoresis, can be considered as one of the best known tests for the detection of liver metastases. The Regan isoenzyme was detected in a 28-year-old female patient with an anamnesis of recent rapid weight loss and fatigue. Pregnancy and viral hepatic infections were excluded. Abdominal and thoracic CT scans were negative. Placental and Regan isoenzymes are products of the same structural gene, with small differences due to post-genetic modification and have been identified in association with various tumours. The electrophoresis showed a heat resistant isoenzyme located between the intestinal and bone fractions. Further gynecological investigations were performed (CA125, HE4) with no evidence of ovarial tumor.

Our findings demonstrate that several isoenzymes of alkaline phosphatase are in association with various tumors. Moreover, some of these isoenzymes show high sensitivity and specificity for the detection of liver metastases during cancer.

The importance of early diagnoses of adrenal tumour. Case reports

R. Pócs¹, E. Takács², E. Sárváry³, G. Bekő¹

¹Uzsoki Hospital, Central Laboratory, Budapest, Hungary

²Uzsoki Hospital, Department of Endocrinology, Budapest, Hungary

³Semmelweis University, Dep. of Transplantation, Budapest, Hungary

The diagnosis of adrenal tumour is based on laboratory and imaging tests. Lab tests can detect hypercortisolism using 24-hour urine sample, midnight plasma and salivary cortisol levels, low-dose dexamethasone (DST). An early diagnosis of pheochromocytoma can be established by measuring catecholamines and their metabolites in plasma and urine (selective metanephrine measurement) as well as chromogranin A in serum.

The first case is a 32–year-old man. In his case 19x14mm structure was described in the left adrenal gland by abdominal computer tomography (CT), although laboratory examination started just 5 years later. By this time symptoms of Cushing syndrome have occurred. He has been operated in 2018 when the left adrenal gland and the spleen have been removed.

In the second case a 47-year-old woman had untreatable hypertonia (210/120 mmHg) since 2000. After 17 years she was examined with suspected pheochromocytoma. Here also surrenal process was described in the adrenal gland. Elevated metanephrine (5272 μg/24h), normetanephrine (17411 μg/24h), 3-methoxytyramine (1276 μg/24h) and chromogranin A (2416,6 ng/mL) concentrations were measured. Unfortunately, the patient has died unexpectedly in stroke before the proposed operation.

With these two cases I would like to emphasize the importance of early laboratory analyses, hormone protocols, which can influence significantly the patient’s outcome.

Unexpected laboratory result – case presentation

T. Nagy¹, N. Szász², F. Zsigmond ² R. Vass¹, A. Nagy¹, G. Pesti¹, J. Gonda¹, J. Simon¹

¹ Medical Centre Hungarian Defence Forces, Central Dept. of Laboratory Diagnostics, Budapest, Hungary

² Medical Centre Hungarian Defence Forces, Dept. of Gastroenterology, Budapest, Hungary

A 59-year-old male patient presented with symptoms of bloody diarrhea in 2012. The clinical examination started with hematology, chemistry laboratory tests, and stool microbiology. After the exclusion of infectious colitis, the diagnosis was confirmed by colonoscopy. Clinical picture, macroscopic view of the colon and histopathology results showed the diagnosis of ulcerative colitis (UC). The patient received immunosuppressive therapy for more than 1.5 year. The only side effect was myopathy. Remission could not be reached by conventional therapy (azathioprine, mesalamine), therefore biological treatment was applied. At the beginning, infliximab infusion seemed to be an effective choice, but after the second induction therapy the symptoms recurred. Then a therapy change for adalimumab was considered unsuccessfully. Prior to performing colectomy, the last therapeutic option was participating in a clinical trial testing a new biological treatment. At the screening period stool microbiology tests were required. Immunochromatographic test revealed positive Giardia antigen reaction. Microscopic examination confirmed Giardia cysts in the stool. After metronidazole treatment two negative results were obtained. The patient felt better, his symptoms became more moderate, however, UC was still active at endoscopy control.

The objectives of the study were to present the laboratory diagnosis of Giardia intestinalis infection and to highlight the importance of suspecting infection in the clinical practice, especially in inflammatory bowel disease in immunocompromised hosts.

The role of an EQA provider in handling laboratory errors

P.M. Molnár, E. Sárkány

QualiCont In Vitro Diagnostic Quality Control Nonprofit Ltd., Szeged, Hungary

Just as in the process of a routine laboratory, in the process of External Quality Assessment (EQA) pre-analytical, analytical and post-analytical phases can be defined. Typically, those activities of EQA mean the processes of input/pre-analytical phases, which may occur in the post-analytical phase of the routine laboratory practice.

The errors from the point of view of that phase of the whole EQA process in which the errors of an EQA provider and an EQA participant can be determined, was analyzed by QualiCont in 2012 for the first time. The errors of EQA participants can be identified mostly in the post-analytical phase. The ratio of the pre-analytical errors is much lower and the ratio of the analytical errors is not significantly different from the literary data which can be found concerning the laboratory errors.

QualiCont has been accredited since March 2014, but this fact did not change this ratio significantly. The percentages of post-analytical errors of laboratory errors are as follows: 2014 - 92%, 2015 - 93.8%, 2016 - 89.8% and 2017 - 90.4%. Therefore, EQA providers should develop and apply a process for handling wrongly reported results, which requires much work. Everyone’s interest is that errors in EQA schemes be reduced in some way.

The aim of QualiCont’s software development, focusing on problems of laboratories, is to help their success in the external quality assessment with applying the latest available technologies.

Among others, the software specifications include the implementation of such technical solutions that may reduce the administrative errors of laboratories (e.g. unit conversion function). The new interface will provide easier overview and handling for the user labs and further support for quick detection and development of quality issues. QualiCont’s new online interactive management system, the I-QC Next, will be presented within the quality management section of the present Congress.

The role of natriuretic peptide testing in heart failure classification based on the latest European Society of Cardiology heart failure guideline

J. Gonda, M. Szabó, N. Nyolczas, V. Galasz, J. Simon

Medical Centre Hungarian Defence Forces, Central Dept. of Laboratory Diagnostics, Budapest, Hungary.

The latest heart failure guideline (2016) of ESC (European Society of Cardiology) defines the most novel classification of heart failure (HF) based on the ejection fraction (EF). The guideline introduces the HF with mid-range EF (HFmrEF) (40%≤EF<50%). The HF with reduced EF (HFrEF) (EF<40%), HF with preserved EF (HFpEF) (EF≥50%) groups were already existing. In the diagnosis of HFmrEF and HFpEF natriuretic peptide tests play a crucial role. The aim of our study was to study the role of laboratory diagnostics in the novel cardiology decision-making algorithm. The study was conducted on 361 patients with heart failure symptoms (mean age 63.1±15.1 years, 65.4 % male, left ventricular EF: 42.4±14.9 %, NT-proBNP: 2851.92±5119.4 pg/mL) presented in the Dept. of Cardiology, Medical Centre Hungarian Defence Forces (Budapest, Hungary) between Dec 1, 2013 and Nov 30, 2015. 48% of patients had at least one follow-up NT-proBNP testing between Oct 1, 2016 and Dec 31, 2017. The echocardiography showed EF<40% in 46.8%, 40%≤EF<50% in 14.1%, EF≥50% in 39.1% of patients. Based both on the NT-proBNP (>125 pg/mL) and echocardiography criteria only 86.4% of patients were proved to have HF. 54.1% of patients had HFrEF, 12.8% HFmrEF and 33.1% HFpEF. The NT-proBNP level of 17 out of 172 patients having a follow-up NT-proBNP result decreased below the criteria level. Based on our data, in a real-life population of HF patients the proportion of patients with HFmrEF is approximately half of that of HFpEF and a quarter of that of HFrEF class. If NT-proBNP is evaluated along with HF signs and symptoms and EF value in the decision-making, the valid proportion of HFpEF is substantially lower than it has been known in epidemiological studies so far.

Evaluation of the vitamin D status in patients with breast cancer

D.B. Némethné¹, K.T. Barna¹, B. Pánczél², V. Szendrényi²

¹SYNLAB Laboratory of Dunaújváros, Dunaújváros, ²Szent Rókus Hospital of Baja, Baja, Hungary

One of the risk factors of breast cancer is vitamin D deficiency, which plays a role in the development of the disease, increases the chance for higher grades and worsens its prognosis. The aim of this work was to compare 25(OH)D levels with the histological type of tumor (benign or malignant), tumor Grade and the hormone sensitivity of the cancer. Materials and methods: Vitamin 25(OH)D levels were determined in samples of 188 female patients with breast cancer (age: 57.8 ± 16.8 years) on Cobas e411 analyzer (Roche). Results: Insufficient 25(OH)D levels were detected in 86% of patients, while 14% of patients had normal 25(OH)D supply. 25(OH)D deficiency was significantly more frequent than normal 25(OH)D level, independently of tumor type (p<0.01). In case of estrogen receptor positive (ER+) and progesterone receptor positive (PR+) tumors, 25(OH)D deficiency was detected significantly more frequent (p<0.01), than optimal vitamin supply. Similar phenomenon was observed in invasive ductal cancer (p<0.01). Insufficient 25(OH)D levels were measured in 94% of triple negative tumors (p=0.004). Comparing 25(OH)D levels of ER+ and ER- tumors there was no significant difference between the two groups. Likewise, PR+ and PR- cases were not different regarding 25(OH)D deficiency (p=0.08 was in both comparison). Similar observation was found if 25(OH)D levels of triple negatives cases were compared to those of the whole population (p=0.09). Conclusions: 25(OH)D deficiency was more frequent in malignant breast tumors compared with benign ones. Sufficient 25(OH)D supply is associated with better hormone sensitivity. Regular 25(OH)D level measurements and vitamin D supplementation are recommended in groups with high risk of tumors.

Resolving violations of the Landsteiner’s law in ABO blood group typing

L. Varga, Zs. Nemes Nagy, A. Mosonyiné Kőszegi, A. Szilvási, I. Zsigmond Soós, H. Andrikovics

Hungarian National Blood Transfusion Service, Budapest, Hungary

The ABO transferase gene polymorphisms determine two blood group antigens and their combinations create four blood groups (A, B, AB and O). According to the Landsteiner’s law: “if the agglutinogen is present on the red blood cell surface, the corresponding agglutinin must be absent from the plasma.” Discrepancies are mostly caused by patient’s health conditions. Weaker reaction’s strength or unexpected extra reactions are the most commonly observed discrepancies. Molecular genetic tests may help in resolving contradictions. 1. The expression of ABO antigens may decrease in oncohematologic diseases or the level of ABO antibodies may waste due to immunodeficiency. Case 1.: A and B antigens were not detectable with serological methods in a patient with acute myeloid leukemia. In the patient’s sera, anti-B antibody level was low. Molecular genetic test identified O¹A genotype. 2. Missing weak or mixed field agglutination of A and B antigens may also refer to inherited subgroup properties. Case 2. and 3.: Weak expression of the A antigen detected with serological methods without any hematological disease in previous medical history. ABO O¹A^x, or O¹A^w04/w11 genotypes were confirmed by molecular genetics. In summary, deviations from the Landsteiner’s law need to be thoroughly evaluated. Genotyping is an effective supplementary method for blood group serological testing. In cases with unclear serological results, patients in need of blood transfusion must be transfused only with group O washed red blood cell concentrates. The altered immunohematological status may also call attention to the onset of an illness. The evaluation of donors’ ABO group is essential as it has an impact on the suitability of blood donation.

First experiences using a newly installed fully automated hematology analyser system: the Sysmex-XN-series

G. Kiss, M. Tőkés-Füzesi, O. Ács, A. Miseta

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

Our laboratory had the opportunity to install a fully automated hematology analyser system, supported by GINOP-2.3.3-15-2016-00012. The system consists of 3 analytical modules, a slidemaker stainer and a digital cell morphology analyser, all linked together with a transportation unit. One of the 3 analytical modules is in addition capable of optical red cell, reticulocyte and fluorescent platelet measurements, one module is in addition capable of body fluid analysis. With the use of slidemaker stainer and cell image analysis, reduced turnaround time is not the only benefit, it also ensures a standardised quality of the blood films. The whole process from tube registration through rack management to validation of results is automated and controlled by an information processing unit (extended IPU). This laboratory workflow support software controls the complete order management and ensures that each individual tube is fully traceable. The technical and biomedical validation process applies a hematology rule set that can be customised to the user’s requirements. These rules can be pre-analytical, biomedical, or can be triggered based on flags coming from the analyser. By using the rules, rerun and reflex measurements, smear-check is triggered and done automatically. Validation process is eased by using 2 concepts, one for red cells and another for platelets in special cases, when interpreting correct results is difficult or nearly impossible.

Based on our first experiences, we wish to demonstrate the advantages of using this hematological system via problematic samples and patient cases. We would also like to interpret the difficulties of the integration of the IPU rule set to our laboratory’s workflow, and patient population.

New higher education vocational laboratory assistant’s training system in Hungary

B. Fodor^1,2, I. Gilányi ², E. Kiss-Tóth ¹

¹University of Miskolc, Faculty of Health Care Studies, Miskolc, Hungary

²BAZ County’ Central Hospital and University Teaching Hospital, Department of Laboratory Medicine, Hungary

The increasing – medical, biotechnological, technical, IT, etc. - activity of highly qualified laboratory staff is appreciated in clinical laboratories. However, the gradual and postgraduate training opportunities are very limited in Hungary and the education system is very confusing for clinical laboratory assistants. The professional quality of the most OKJ-type trainings is often inadequate. Since 2015, the Faculty of Health Care Studies at the University of Miskolc implemented an educational framework to promote a new – academic type – assistant training system. The curriculum was prepared for a two-year higher education vocational training. When compiling the curriculum, we have been striving to integrate the professional material to a high level, so the last half year of the educational time is a 6-week practical training, which can be completed in the leading laboratories of Hungary. The authors recommend the nationwide extension of this type of laboratory assistant educational system.

Relationship of pro- and anti-inflammatory ractors with vascular stiffness in chronic hemodialysis patients

A. Peti¹, T. Gyimesi², O. Lakatos³, B. Sági^2,4, E. Sulyok⁵, B. Csiky^2,4

¹Hospital of Siófok, Central Laboratory, Siófok, ²Nephrological Center and 2^nd Dep. of Medicine, Medical School (MS), Univ. of Pécs (UP), ³Dep. of Pediatrics, MS, UP, ⁴FMC Dialysis Center Pécs, ⁵Doctoral School of Health Sciences, UP, Hungary

The study tries to relate pro-inflammatory and protective factors in patients with chronic renal failure (CRF) with vascular stiffness (VS) measurement parameters.

The patient group included 96 patients receiving regular hemodialysis treatment and the control group consisted of 20 patients without major renal, cardiovascular or metabolic morbidities. Carotid-femoral VS parameters were determined by using applanation tonometry and in addition to routine laboratory tests fetuin-A, α-Klotho, TNF-α and TGF-β₁ were determined by ELISA method; hsCRP and 25(OH)D vitamin by automated immunoassay. Statistical analyses were performed by using IBM SPSS 21.

Comparing the HD patients’ laboratory findings to those of controls, we observed significantly increased serum levels of pro-inflammatory biomarkers while anti-inflammatory biomarkers were significantly decreased. Multiple linear regression analyses showed association between clinical and laboratory parameters with the studied inflammatory biomarkers but not with VS. After three-year follow-up we observed that low levels of vitamin D, α-Klotho and fetuin-A were associated with adverse cardiovascular events, while all-cause mortality was related to higher hsCRP and lower vitamin D.

We think that in case of CRF patients these findings (i) could provide additional information on their accelerated arterial stiffness pathomechanism and (ii) could demonstrate the direct influence of these inflammatory biomarkers on major outcome measures.

Novel color at the point-of-care test’s palette: procalcitonin

I. Kiss, I. Földesi

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

Procalcitonin (PCT) is a well acknowledged biomarker of inflammation and has been used to guide antibiotic therapy for decades. The quantitative serum PCT tests applied in routine practice give correct results, but they require sophisticated laboratory environment and equipment. This raises the need for alternative tests, which can be performed directly at the bedside: the Point-of-Care Tests (POCT). In this study we compared the accuracy of two Point-of-Care PCT tests: ABSOGEN™ PCT (Bumyoungbio, Inc., Suwon, Korea) and Biosynex PCT test (Biosynex, Strasbourg Cedex, France), when compared to the results determined by our routinely used chemiluminescence (Roche, Mannheim, Germany) method. Both POC tests are able to give qualitative results from whole blood making them ideal for bedside use. In addition, the ABSOGEN™ test could also give semi-quantitative result if an appropriate reader is available. Previous studies have evaluated both POC tests separately and both have shown high sensitivity and specificity. On the basis of the serum PCT results and the anticoagulated sample (e.g. EDTA whole blood for hematology) availability we selected and analyzed the data of parallel measurements in 50 patients. The serum and whole blood tests were compared to the results measured by quantitative immunoassay. Our preliminary data suggest that both Point-of-Care Tests could be very useful in the emergency setting to reduce the unnecessary and more expensive quantitative test requests in case of negative PCT-POCT results.

Diagnosis of hereditary red cell membrane defects

Z.A. Mezei¹, Zs. Hevessy¹, J. Kappelmayer¹, E. Marián²

¹University of Debrecen, Faculty of Medicine, Department of Laboratory Medicine, Debrecen, Hungary, ²András Jósa Szabolcs-Szatmár-Bereg County Hospital and University Teaching Hospital, Nyíregyháza, Hungary

Hereditary spherocytosis is the most frequent red cell membrane disorder associated with hemolytic anemia, while hereditary stomatocytosis is less frequent. In Northern Europe the prevalence of red cell membrane disorders is 2-3/10.000. Autosomal dominant pattern is observed in 75% of the disorders. The clinical symptoms are highly variable, about 5% of the patients have very severe hemolytic anemia.

The flow cytometry-based eosin-5-maleimide (EMA) test is part of our laboratory workflow since 2012. Values below the reference interval (0.90-1.10) indicate spherocytosis, while elevated values are characteristic of overhydrated stomatocytosis.

Of the 112 EMA tests performed since 2012, 41 patients displayed values that indicated spherocytosis and 7 patients showed values indicating overhydrated stomatocytosis. The diagnostic workflow is demonstrated through the case presentation of an eight-year-old child investigated at our laboratory. She requires blood transfusion every 6 weeks. Her hematology parameters were inconclusive and reticulocytosis was not present. The osmotic fragility test was negative. Cryohemolysis test result was under the reference interval. The EMA was 1.26, above the reference interval. The examination of the peripheral blood smear revealed the presence of a large number of stomatocytes. Based on these results, the diagnosis of overhydrated stomatocytosis was concluded.

Hereditary red cell membrane defects have a broad range of clinical severity, but it is important to recognize and correctly diagnose these cases.

Association of macro-TSH with non-thyroid autoimmune diseases

K. Koller¹, E. Fata¹, Z. Lőcsei¹, E. Toldy^2,3

Department of Internal Medicine¹ and Central Laboratory² of Markusovszky University Teaching Hospital, Szombathely, Institute of Diagnostics, Faculty of Health Science, University of Pécs³

Introduction: The incidence of subclinical hypothyroidism in the general population is relative high (4-20%). It depends not only on age, gender but also the applied laboratory method. Recently Hattori et al. called attention to the presence of the macro-TSH, which is a biological inactive form. Commercial immunoassay systems variably recognize macro-TSH next to the TSH form which is biologically active. Aim: To analyze our TSH assay’s (ECLIA, Roche) macro-TSH sensitivity considering anti-thyrotropin autoantibodies (TPO-Ab), other autoimmune diseases and thyroid status. Method: 292 blood samples (212 female, 80 male, age: 50,8±18,4 year) taken from patients with suspicion of thyroid disease were screened for macro-TSH. Non-thyroid autoimmune disease was found in 81 cases (16 systemic, 18 organ-specific autoimmune diseases and 47 rheumatoid arthritis: RA). 116 TPO-Ab negative patients, who were euthyroid and not suffering from autoimmune disease created our control group. We measured the TSH level in native samples and after polyethylene glycol (PEG) adsorption. The macro-TSH values were calculated from the precipitable TSH ratio. We also measured free T4 and TPO-Ab levels in all cases. Results: 67 cases (23%) were found TPO-Ab positive, from these patients 15 also had non-thyroid autoimmune diseases. In 99 cases (34%) hypothyroidism was found (81 subclinical, 18 manifest). In manifest hypothyroidism cases with positive TPO-Ab results, significantly higher macro-TSH prevalence was found than in TPO-Ab negative cases (53±13% vs. 31±22%, p<0,01). In subclinical hypothyroidism, almost the same macro-TSH prevalence was found in TPO-Ab positive (59±13% n=36) and TPO-Ab negative (55±14% n=45) groups. The relevant macro-TSH limit was the precipitable TSH ratio 62%. According to this, macro-TSH occurred in 6% of the euthyroid and in 43% of the subclinical hypothyroidism cases. Calculating with lower TSH limits by the PEG method, 43 of 81 subclinical cases were classified to be euthyroid. Conclusions: We recommend to measure macro-TSH in patients with subclinical hypothyroidism independently of TPO-Ab status, because the result may change the therapeutic strategy.

Catecholamine measurements in the serum of drug users

¹G. Far, ²A. Lakatos, ²A. Lajtai, ²A. Miseta

University of Pécs, Faculty of Health Sciences, Hungary ¹, University of Pécs, Medical School, Department of Laboratory Medicine, Hungary²,

Recently, there has been an increase in the variety of new recreational drugs called “new psychoactive substances”. Some of them are still uncontrolled, others are already illegal drugs. These drugs alter the catecholamine balance in the central nervous system. We aimed to study whether stimulants cause detectable changes in serum catecholamine concentrations. We analyzed 9 serum samples of stimulant drug-users, and 6 control samples. The drugs were identified by Shimadzu Prominence TOX.I.S II HPLC-DAD equipment. The catecholamine measurements were performed on the Shimadzu HPLC system with ANTEC EC detector by the HPLC kit of Chromsystems. We found that the level of all measured catecholamines (epinephrine, norepinephrine and dopamine) was significantly higher (p<0,05) in the serum of drug users than in the control serums.