Leiden University Medical Center, Department of Clinical ... On-call shifts last for one week (24/7). The clinical chemist on call is in-house during office hours and ...... the Laboratory of Bel- grade's University Zvezdara Medical Center . Siemens.
Posters P01 - Accreditation and laboratory management P01-01 Increasing profitability of a specialized medical biochemistry laboratory Buljanovic V (1), Patajac H (2), Petrovecki M (3) (1) General Country Hospital Našice, Specialized Medical Biochemistry Laboratory, Našice, Croatia (2) Adris Group d.d., Adris Group d.d., Rovinj, Croatia (3) Dubrava University Hospital, School of Medicine, University of Rijeka, Rijeka, Croatia, Zagreb, Croatia Corresponding author: [emailprotected]
Introduction: Laboratory's profitability is expressed as the operating profit, which is the final result of profit and loss account. Operating profit may be increased by organizational changes, i.e. by monitoring all laboratory expenses and processes. Methods: The annual profitability of the specialized Medical Biochemistry Laboratory at General County Hospital in Našice, Croatia, in the study period 20072010 was evaluated using the profit and loss account, which included three basic elements: revenue, expenses, and their difference. Revenues were realized from reimbursements for laboratory tests performed, and expenses included all expenditures required for the realization of the annual revenue. Results: The Laboratory’s operating profit in 2007 was HRK 719,926, and the operating margin was 12%, (profit expressed as percentage), showing that the laboratory made profit and that, after the deduction of all operating expenses per 100 units of revenue, it retained 12 units of profit from performing its basic operating activities. In 2008, the operating profit increased to HRK 3,415,269 and operating margin increased to 39%, which is a 4.7-fold increase in comparison with the previous year. In 2009 and 2010, operating profits were HRK 3,049,245 and HRK 3,149,922, respectively, and the operating margin was 36%. Conclusion: The analysis of the operating business performance of the specialized Medical Biochemistry Laboratory at General County Hospital in Našice showed that laboratory’s profitability could be inBiochemia Medica 2012;22(3):A54-A204
A54
creased and maintained only by implementing organizational changes, without any technological investments. This finding could help other laboratories in Croatia to increase their profitability.
P01-02 Six sigma in clinical laboratory: analytical process management – focus on reduction of response time Ribeiro R, Sousa MJ, Sousa JG, Sousa G Centro Medicina Laboratorial DR. Germano de Sousa, Quality, Lisbon, Portugal Corresponding author: [emailprotected]
The philosophy of Six Sigma quality can be seen as a strategy that achieves perfection in products and processes. This holistic view was a new tool for the Clinical Laboratory, which in the context of business competitiveness concerns the relentless pursuit of perfect products by establishing an organizational advantage in various aspects. The product of the clinical laboratory is the result of quality from the service until the delivery of the report of the results. From the standpoint of the user, there are two perceived characteristics: accurate and unambiguous results, and the time of delivery. The Six Sigma project developed in Centro Medicina Laboratorial Dr. Germano de Sousa can bring great benefits to the analytical process, in the management of the analytical process of clinical laboratory, focusing on response time, and maintaining the quality of analytical results. According to the studies of reproducibility and repeatability, along with sigma metric as an index of performance we want to standardize the analytical repetitions performed per parameter to confirm the results, thus reducing the process time and reducing costs as the cost of using / technical occupation, additional expense of reagents and possible repeated harvesting by insufficient amount of biological sample. The use of a Six Sigma strategy in the clinical laboratory, not just being used as a metric for performance analysis, but in the context of case management provides a stand-
2nd European Joint Congress of EFLM and UEMS
ardization of a complex and multidisciplinary, aligns the goals of quality and costs, focusing on customers and the health system´s financial organization.
P01-03 Development of the balanced scorecard of a software program for the management of quality indicators De La Torre Fernandez J, Soriano Bueno G, Cazalla Martin F, Navajas Luque F, Rodriguez Peña F, Guzman Gonzalez A Hospital Comarcal De La Axarquia, Velez-Malaga, Laboratory, Malaga, Spain Corresponding author: [emailprotected]
Background: Indicators related to laboratory processes represented in a balanced scorecard (BCS) can be obtained in an automated, fast and effective way through the Omnium software, for the continuous improvement of the quality. Materials and methods: (17) indicators are calculated monthly from variables (70) registered daily in the tab of each patient of the laboratory computer system and indicate a resulted incidence. Areas, processes, fields and aggregators are defined in the BCS module of Omnium. The computer application Omnium (Roche) acts as DataWareHouse selecting, filtering and transforming data from Omega (Roche) and generating results from each indicator with its goal classified by colors. A browser displays the BCS in aggregators, fields, processes, areas and indicators, and a BCS tree graphically represents it to obtain the requested information, to study the evolution. Results: 17 indicators are used: 6 of quality of the application, 4 of extraction, 4 of processing and transport, 1 of reception, 1of validation time and 1 of response time. The objective of each preanalytical indicator is < 0.3%, alarm indicator between 0.8 and 1.3, and danger indicator >1.3%. In the period JanuaryMay of 2012 all our indicators were below the target except “Samples not received”, which value was 1.35.
Conclusions: With "Omnium", we obtain indicators of monthly automated extraction framed in a BCS organized in variables, indicators, areas, processes and fields. Staff knows their monthly indicators, evolution and goals for the following month. The browser and the BCS tree allow a better monitoring of the indicators evolution.
P01-04 Artificial urine – possibility or a good try Culej J (1), Nikolac N (2), Krajačić-Karas G (1), Šturm D (1) (1) Sestre Milosrdnice University Hospital, Department of transfusiology and haemostasis - Clinic for tumors, Zagreb, Croatia (2) Sestre Milosrdnice University Hospital, Clinical Institute of Chemistry, Zagreb, Croatia Corresponding author: [emailprotected]
Background: Based to our knowledge, external quality control assessment for urinalysis in most schemes is organized through sending pictures of urine sediment into laboratories. Although useful in testing of element recognition, this approach doesn’t cover the whole process of urine sediment preparation. The aim of our study was to prepare artificial, low infective sample in order to test whole process of urine sediment analysis. Materials and methods: We suspended small amount of human blood, epithelial cells from buccal mucosa and baker's yeast in saline fluid. This mixture was distributed into 13 samples and given for sediment analysis to a 13 professionals into 4 laboratories on the same day. Results: As we expected, most of professionals managed to recognize all elements added into saline. Overall agreement for erythrocytes was 13/13, for epithelial cells 12/13, for leukocytes 10/13 and for yeasts 9/13. Relatively low agreement on yeast recognition might be due to growth of cells in samples that were examined last and also because of similarity of these elements to erythrocytes. Raters that didn’t find any leukocytes are from laboratory that reported smaller amount of urine used for Biochemia Medica 2012;22(3):A54-A204
A55
2nd European Joint Congress of EFLM and UEMS
analysis. Some of raters reported artefacts and salts that were not present in the samples. Also, they were confused with morphology of yeasts and complained about morphology of erythrocytes. Conclusion: This approach has potential to develop into quality control of the total process of urine sediment analysis. However, because the transport conditions could influence composition of samples it could be applied locally.
P01-05 Accreditation according to standard UNE-EN-ISO 15189: 2007. Evaluation of a program in biochemistry Cazalla Martin F, Soriano Bueno G, De La Torre Fernandez J, Navajas Luque F, Sanchez-Montes Moreno S, De La Torre Navarrete J Hospital Comarcal De La Axarquia, Velez-Malaga, Laboratory, Malaga, Spain
lished three groups: 60 U/mL): of these, 18 were anti-TG positive (> 280 U/mL) and 16 negative. In addition, 11 (3.16%) were anti-TPO negative and antiTG positive. In reviewing the medical history of patients with anti-TPO negative and high anti-TG, we observed that 100% had thyroid disease: 18.18% autoimmune thyroid disease inherited by the mother, 63.64% euthyroid autoimmune thyroiditis and 18.18% subclinical hypothyroidism. Conclusions: According to the results, the quantification of anti-TG rises by 32.3% the diagnostic yield of childhood thyroid diseases (34 positive anti-TPO positive+11 only positive anti-TG) which would justify the joint determination of both antibodies in the population study.
P07-11 Testosterone quantification in human serum using liquid chromatography with tandem mass spectrometry Depoorter M, Djuidjé C, Delongie K, Wallemacq P, Gruson D, Haufroid V
male hypogonadism as well as female hyperandrogenism. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) is one of the most specific and sensitive technique available in clinical laboratories. The objective of our work was to develop and validate a LC-MS/MS method for testosterone measurement in serum and process to its comparison with immunoassay method. Materials and methods: The analysis was performed on an Agilent 6460 Triple Quad LC-MS/MS spectrometer with electrospray ionization (ESI). A liquid/liquid extraction (LLE) was used for sample preparation. The calibration curve ranged from 0.025 ng/mL to 50 ng/mL. The method comparison was performed with 80 samples (65 males and 15 females) using the DxI® immunoassay (Beckman Coulter). Results: The calibration curves showed good linearity with a mean R² coefficient of 0.9986 (ANOVA P-value: 2.45E-08). The limit of quantification (LOQ) of the method was set at 0.01 ng/mL. Passing Bablok regression analysis showed slopes of 1.07 and 0.58 in males and females, respectively. Bland Altman plots revealed mean differences of -0.01 and -0.11 ng/mL for males and females, respectively. The method showed accurate results according to external quality control samples (UKNEQAS). The chromatographic conditions also showed a clear separation between testosterone, aldosterone, androstenedione, 17-OH-progesterone and 11-deoxycortisol allowing a simultaneous measurement of those analytes. Conclusion: Our LC-MS/MS method is sensitive enough to measure testosterone levels in female samples with a good accuracy. The bias observed compared to immunoassays is more important in female than in male samples.
Saint-Luc Hospital, Catholic university of Louvain, Department of clinical chemistry, Brussel, Belgium Corresponding author: [emailprotected]
Background: Evaluation of testosterone levels is important for the diagnostic and follow-up of Biochemia Medica 2012;22(3):A54-A204
A103
2nd European Joint Congress of EFLM and UEMS
P07-12 Pheochromocytoma biochemical diagnosis
are increased just 2 or 2.5 times the upper limit of the RR, it should be excluded causes of false positive results.
Ferreira M (1), Titonel S (2), Rego M (3), Reguengo H (3) (1) Hospital de Santo António - Centro Hospitalar do Porto, Endocrinology, Diabetes and Metabolism, Porto, Portugal (2) Hospital de Santo António - Centro Hospitalar do Porto, Clinical Pathology, Porto, Portugal (3) Hospital de Santo António - Centro Hospitalar do Porto, Clinical Chemistry, Porto, Portugal Corresponding author: [emailprotected]
Background: Pheochromocytoma is an adrenal tumor which produces catecholamines. The measurement of catecholamines and/or its metabolites in blood or urine are essential to diagnosis. It isn’t clear from which value the diagnosis should be considered. This work compares levels of catecholamines or its metabolites in urine among patients with and without pheochromocytoma. Materials and methods: Selection of patients with pheochromocytoma diagnosed from January/1990 to December/2011, followed in Centro Hospitalar do Porto, and patients with false positive tests. Demographical, clinical and analytical data were collected and analysed in SPSS 20.0. Results 22 patients had pheochromocytoma (50% were men), with 47.8 ± 16.6 years-old (17-70). Nineteen patients had elevated normetanephrine or metanephrine (the other 3 patients had high levels of norepinephrine, dopamine or VMA). Twenty patients had at least one increased metabolite 3 times above the upper limit (UL) of the reference range (RR). Nineteen patients (86.4%) had at least one increased metabolite 4 times above the UL of the RR. Among patients without pheochromocytoma, sometimes one metabolite was increased (mostly norepinephrine, normetanephrine or VMA), but it was always below 2.5 times the UL of the RR. In 17 (77%) patients, the metabolites levels weren’t above 2 times the UL of the RR. Conclusions: In the authors’ laboratory, the pheochromocytoma biochemical diagnosis must be considered when there is an increased metabolite above 4 times the UL of the RR. When the levels Biochemia Medica 2012;22(3):A54-A204
A104
P07-13 Comparison of the results of HPLC methods for determination of methanephrine and normethanephrine from urine and blood plasma considering the diagnosis of tumor pheochromocytoma Brabcová Vránková A, Widimský Jr. J, Zelinka T, Škrha J 3rd Internal Department, First Faculty of Medicine and General Teaching Hospital, Charles University in Prague, Prague, Czech Republic Corresponding author: [emailprotected]
Background: Determination of catecholamines and their O-methyl metabolites normetanephrine (NMN) and metanephrine (MN), in biological fluids plays an important role in the diagnosis of pheochromocytoma (PHEO) - chromaffin cells tumor. The aim of the study was to compare a HPLC with electrochemical detection method (HPLC-ED) for the determination of methanephrine and normethanephrine in blood plasma and a HPLC with fluorescence detection method (HPLC-FLD) for the determination of the same analytes in conjugated form from urine. Both methods interpret the results in relation to the presence of PHEO. The ability of methods to distinguish the patients with and without PHEO has been proved. Materials and methods: Determination of metanephrines from plasma (HPLC-ED) and from urine (HPLC-FLD). Standards: NMN ((±) normetanephrine hydrochloride), MN ((±) metanephrine hydrochloride) and HMBA (4-hydroxy-3-methoxybenzylamine-hydrochloride) used as internal standard (Sigma-Aldrich, St.Louis, USA). Chromatography: HPLC system Agilent 1100 (Agilent Technologies, Wilmington, USA). Metanephrines from plasma are determinated in free form, for this reason is possible to directly start by solid phase ex-
2nd European Joint Congress of EFLM and UEMS
traction (SPE) without necessity of hydrolysis. Urine sample is necessary to pass through an acid hydrolysis. During this procedure, releasing of conjugated metanephrines from a bond is happened. Hydrolysis is folloved by SPE. Eluted samples are applied onto a HPLC reversed phase column. Results: The sensitivity and specificity of methods in tumor diagnosis has been calculated. It has been shown that the sensitivity of both methods has reached 100%, the specificity of methods is lower (94% for the HPLC-ED method and 80% for the HPLC-FLD). Conclusions: The sensitivity shows excellent ability of both methods to recognize the patients with PHEO. Weaker specificity mainly of the HPLC-FLD method rarely admits false-positive results. Acknowledgments: Financial support from the project 5302-28002PVK and MPO-Tip FR-TI4/331.
P08 – Evaluation of analytical systems
Results: Carry over: 0.465% for RBC, 0.117% for WBC 0,19%, for EPI and 0.058% for BACT. Withinrun imprecision of cell counts expressed as CV% (mean cell count/μl) was found for RBC 6.86%, for WBC 6.97%, for EPI 35%. Between run imprecision was carried out in 30 replicates of two urine control at different concentrations for RBC, WBC, EPI, BACT, and CAST. The mean and CV% was calculated. Passing-Bablok regression were determined for RBC: y = -0.0321 + 1.0383x, for WBC: y = -0.2990 + 1.0499x, for EPI y = 0.2285 + 1.0191x, for SRC y = -0.2161 + 1.6129x, for CAST y = 0.0 + 4.2666x, respectively. Diagnostic accuracy of Sysmex UF1000i showed the following results: 93.2% for RBC, 97.2% for WBC, 92.6% for EPI, 97% for SRC and 60.0% for PC. (Further results: Sensitivity data are: RBC: 96.4%, WBC: 98.0%, EPI: 96.7%, SRC: 60.0%, PC: 79.6%. Specificity data are: RBC: 90.5%, WBC: 95.1%, EPI: 89.7%, SRC: 98.2%, PC: 56.9%.) Conclusion: Sysmex UF 1000i urine analyzer eliminated manual sample preparation. It has proven good precision for analyzing cellular elements.
P08-01 Evaluation of the Sysmex UF-1000i urinanalyzer Pinter E, Toth J, Konderák J
P08-02 Evaluating performance of Cobas c311 and Cobas c501 in the emergency laboratory setting
Synlab Hungary Ltd, Medical Biochemical Department, Budapest, Hungary
Lapić I, Lukić I, Kutnjak V, Fressl Juroš G, Rogić D
Corresponding author: [emailprotected]
Clinical Hospital Center Zagreb, Clinical Institute of Laboratory Diagnostics, Zagreb, Croatia
Background: The aim of this study was to compare the results of Sysmex UF 1000i analyzer for red blood cells (RBC), white blood cells (WBC), epithel cells (EPI), small round cells (SRC) and pathological cast against manual microscopy of uncentrifuged urine specimens using Fuchs-Rosenthal cell counting chamber.
Corresponding author: [emailprotected]
Materials and methods: Sample size: 500 outpatient urines. Carryover, precision, Passing&Bablok regression, Pearson correlation, Receiver Operating Curves (ROC) and diagnostic accuracy were tested.
Materials and methods: Validation included assessment of within-day (N = 30) and between-day imprecision (N = 30), inaccuracy (bias) (N = 30), linearity and method comparison with the hitherto used analyzer Olympus AU2700 (N = 30). Statistical
Background: The aim of our study was to perform the analytical validation of the automated biochemistry analyzers Cobas c311 and c501 (Roche Diagnostics, Germany), for the purposes of the emergency laboratory.
Biochemia Medica 2012;22(3):A54-A204
A105
2nd European Joint Congress of EFLM and UEMS
analysis was performed and the results were judged by comparing with the specifications according to Westgard. Results: Statistical analysis fulfilled almost all required criteria. Within-day and between-day imprecision, evaluated at two levels, for all analytes on both analyzers revealed acceptable coefficients of variation (CV < 5%). Bias for calcium, glucose, total proteins and urea on Cobas c311 was beyond the range of desirable specifications while on Cobas c501 only bias for chlorides exceeded the recommended value. For all analytes, the range of linearity defined by the manufacturer was proved. According to the Passing-Bablok regression analysis, the results of the comparison study showed statistically negligible deviations except for direct bilirubin and CRP for which proportional error was identified. Moreover, method comparison yielded high coefficients of correlation (r > 0.95) for all analytes determined on both analyzers. Conclusion: Cobas analyzers show acceptable precision and accuracy for all but a few analytes for which slight adjustment of the instrument factor is required, meet the established requirements and specifications and therefore can be implemented in routine laboratory work. However, the performance of Cobas c311 does not meet the turnaround time for the high throughput emergency laboratory requirements.
P08-03 Analytical performance of the OC-Sensor DIANA immunochemical faecal occult blood test Halloran S, Pearson S, Piggott C, Young M NHS Bowel Cancer Screening Programme, Southern Hub, Guildford, United Kingdom Corresponding author: [emailprotected]
Background: The automated analyzer, OC-Sensor DIANA (Eiken Chemical Co., Ltd., Japan), will be used in the ‘FIT for Follow-Up’ study. The study will Biochemia Medica 2012;22(3):A54-A204
A106
investigate the three-year programme sensitivity of an annual faecal immunochemical test (FIT) for haemoglobin (Hb) for the detection of advanced adenomas or colorectal cancer in patients diagnosed with intermediate risk polyps through the NHS Bowel Cancer Screening Programme. Materials and methods: The analytical performance of DIANA was evaluated using buffered Hb solutions and compared with that reported in 2009. Results: The Clinical and Laboratory Standards Institute (CLSI) five-day imprecision protocol demonstrated good within-assay and total imprecision at concentrations > 159 ng Hb/mL buffer (CV < 1.7% and < 3.4% respectively). Within-assay and total imprecision at 43 ng/mL was poor (CV 11.5%). The manufacturers do not recommend the use of quantitative results < 50 ng/mL. The QC materials containing average concentrations of 84, 152 and 658 ng/mL gave within-assay CVs of < 1.3% and for control material with average concentrations of 153 and 655 ng/mL it gave day-to-day CVs of < 2.6%. The assay was linear between 45 and 930 ng/mL supporting the manufacturer’s claimed measuring range of 50–1000 ng/mL. The regression equations from 2011 (y = 1.03x-4.99) and 2009 (y = 1.06x+8.54) were similar. The analyzer's sample carry-over effect in both evaluations was well within acceptable limits for interaction at < 0.5%. Conclusion: Analyzer performance was consistent with manufacturer’s claims. ‘Baseline’ performance will be compared with future evaluations performed at regular intervals throughout the course of the ‘FIT for Follow-Up’ study.
2nd European Joint Congress of EFLM and UEMS
P08-04 Evaluation and validation of the Cobas 6000 analyzer series modules c501: the urine analytes Šimičević L, Cigula Kurajica V, Vogrinc Ž, Sertić J Clinical Hospital Center Zagreb, Clinical Institute of Laboratory Diagnosis, Zagreb, Croatia Corresponding author: [emailprotected]
Background: The aim of study was to assess the analytical performance of the Cobas 6000 analyzer series modules c501 (Roche Diagnostics) and to validate it in our routine set of urine analytes. Materials and methods: We investigated 12 routine urine analytes tests (amylase, glucose, urea, creatinine, urate, total proteins, sodium, potassium, chloride, calcium, phosphate, magnesium) on two modules c501 (R1+R2). The evaluation protocol consisted of imprecision: within-run (10 sequential runs) and between-run (10 consecutive working days, 2 sequential runs) with commercial controls (Liquicheck, Biorad; Precinorm PUC/Precipath PUC, Roche), inaccuracy (N = 20), and method comparison (routine urine samples, N = 30) vs. Beckman Coulter AU640. Results: All analytes on both modules have within-run imprecision < 3%, except phosphate and magnesium (R2). For all analytes between-run imprecision was < 5%. All analytes fulfilled quality requirements for imprecision and for total error. A quality requirement for inaccuracy was met by all analytes on both modules with exception of urate on R1 and amylase on R1+R2. The correlation with comparison method showed no difference between methods for glucose, amylase, urea, sodium, phosphate, magnesium on R2 and for potassium, amylase, urea on R1. Constant difference was observed for five analytes on R1, and three on R2. Proportional difference was found for five analytes on R1, and two on R2. Two tests need further investigation, chloride on R1, and creatinine on both modules, due to significant deviation from linearity.
Conclusions: Cobas 6000 analyzer showed optimal analytical performance with mostly fulfilled quality control requirements and acceptable method comparisons for urine analytes.
P08-05 Differences between bromcresol green and bromcresol purple measured albumin Garcia Moreira V, Martinez Gago M, Fernandez Leivas A, Bachiller Sister M, Laborda Gonzalez B, Fernandez Rodriguez E Hospital de Cabueñes, Servicio de Analisis Clinicos, Gijon, Spain Corresponding author: [emailprotected]
Background: Usually, albumin is measured by techniques employing dye-binding assays: bromcresol green (BCG) and bromcresol purple (BCP). In management of an individual patient with albumin results from different laboratories, the appreciation of the methodology used and the ability to convert results between methods will be helpful. Material and methods: 85 plasma and 80 serum samples were analyzed by BCP(DimensionRX- Siemens) and BCG (Advia2400-Siemens) methods. Of these, 75 serum samples were also run on capillary zone electrophoresis (CZE). Significant differences were determined by Student paired-t and ANOVA tests, using MedCalc. Significance was set at P < 0.05. Passing-Bablok regression, Bland-Altman plots and Pearson correlation were used to examine the relationship between methods. Results: Correlation AlbBCG and AlbBCP was r = 0.948 (P < 0.01), but the mean difference was large 6.42g/L (CI 95%mean: 5.88-6.96) (P < 0.01). The regression equation was AlbBCG = 0.784 AlbBCP + 12.58. Bland-Altman plots shows greater bias at lower albumin concentrations. For the normal concentration group (G1; AlbBCP ≥ 35 g/L), mild hypoalbuminemia (G2; 30-35 g/L), moderate hypoalbuminemia (G3; 20-29 g/L) and severe hypoalbuminemia (G4; ≤ 20 g/L), mean differences were 3.07 g/L, 6.33 g/L, 7.50 g/L and 9.92 g/L respectively (ANOVA; P < 0.01). Using the Newman-Keuls Biochemia Medica 2012;22(3):A54-A204
A107
2nd European Joint Congress of EFLM and UEMS
post-hoc test, differences were found between G1 vs. G2, G3 and G4 as well as between G4 versus G2 and G3 (P < 0.05). Also, a positive bias was observed between BCG/CZE (mean 3.54 g/L) and good correlation between CZE/BCP with a mean difference < 1g/L. Conclusion: Albumin results from BCP and BCG methods may result in unacceptable differences and clinical confusion. The BCP method is superior method to evaluate the serum albumin levels, due to BCP albumin is more specific than BCG.
P08-06 Sodium measurements in urine by the patient at home: primary limitations overcome
and flame photometry, respectively). Accordingly, the software algorithm converting electropherograms to sodium concentrations was adapted. The Multireader was then compared to flame photometry in a set of new experiments: none of 14 results (range: 70-302 mmol/L) showed significant bias. Multireader CVs were 2.2% and 3.1% at a level of 108 and 313 mmol/L, respectively. The new algorithm therefore shows an evident improvement in analytical performance. Despite several innovations, ease of use remained underdeveloped. Conclusions: Although analytical CVs are considerably higher, the POC method seems clinically equivalent to flame photometry. The novel system is definitely promising, but its ease of use has to be improved.
Schenk P, Romijn F, Kolb R, Cobbaert C
P08-07
Leiden University Medical Center, Department of Clinical Chemistry, Leiden, Netherlands
Evaluation and validation of the Cobas 6000 analyzer modules c501: the routine serum analytes
Corresponding author: [emailprotected]
Šimičević L, Cigula Kurajica V, Vogrinc Ž, Sertić J Background: It might be useful if hypertensive patients can self-monitor their sodium intake by assessing sodium excretion in urine at home. Materials and methods: We evaluated a novel system, consisting of disposable syringes + LabChips and the Medimate Multireader, intended for POC sodium measurements in human urine, using moving boundary and capillary zone electrophoresis and conductivity detection. The system was compared to the Roche Modular ISE (standard laboratory method) and IL 943 flame photometer (gold standard), and precision was determined. Ease of use was evaluated by two experienced laboratory technicians. Results: We compared the Multireader to both laboratory methods for 38 different 24-hour urine specimens (range: 33-195 mmol/L): > 5% of results were outside the allowable total error interval (+/28.8%). Multireader CVs were 6.6% and 16.4% at a level of 156 and 52 mmol/L sodium, respectively (with CVs in the 1-2% and 0.1-0.2% range for ISE Biochemia Medica 2012;22(3):A54-A204
A108
Clinical Hospital Center Zagreb, Clinical Institute of Laboratory Diagnostics, Zagreb, Croatia Corresponding author: [emailprotected]
Background: The aim of the study was to assess the analytical performance of the Cobas 6000 analyzer series modules c501 (Roche Diagnostics) and to validate it in routine set of serum analytes. Materials and methods: We investigated complete routine serum tests for 36 analytes and preliminary present the results for cholesterol, HDL, LDL, triglycerides, uric acid, iron, and UIBC on two modules c501 (R1+R2) plus ACE (R1), and cooper (R2). The evaluation protocol consisted of imprecision: within-run (10 sequential runs) and betweenrun (10 consecutive working days, 2 sequential runs) with commercial controls (PreciControl ClinChem Multi 1/2, Roche; HumAsy Control 2/3, Randox; ACE controls N/H, Bühlmann), inaccuracy
2nd European Joint Congress of EFLM and UEMS
(N = 20), and method comparison (routine serum samples, N = 30) vs. Beckman Coulter AU640.
mance when multiple methods are validated simultaneously.
Results: The majority of analytes on the assigned modules have a within-run imprecision < 2%. For all analytes, except cooper and UIBC, the between-run imprecision was < 3%. Quality requirements for imprecision and total error were met by all analytes on the assigned modules, excluding cooper. Quality requirement for inaccuracy were fulfilled by all analytes with the exception of HDL (R2). The correlation with comparison method showed no difference between methods for cholesterol, LDL, cooper, uric acid, and ACE on the assigned modules. Triglycerides and HDL showed some difference on both modules. UIBC and triglycerides on R1, due to significant deviation from linearity need further investigation.
Materials and methods: Data were collected from method validation study performed on Roche Cobas 6000 analyzer c501 (Roche, Germany) (published in Biochemia Medica 2011;21(2):18290). TE is calculated according to equation: TE = Bias + σCV where σ stands for desired confidence level (σ=2-6). This equation is the basis for MEDx chart where each method is presented as operating point defined by Bias on y-axis and CV on xaxis. Several criteria for σ value are presented on the chart according to which method performance is classified. If imprecision and inaccuracy are expressed as percentage of TE and such adjustment of x and y axis scaling is made than all operating points can be presented on the same chart called normalized MEDx chart.
Conclusions: These preliminary results showed that Cobas 6000 analyzer has optimal analytical performance with mostly fulfilled quality control requirements and acceptable method comparisons for our routine serum analytes.
P08-08 Normalized MEDx chart – useful tool for quick assessment of multiple method performance
Results: Validation of 30 analytical methods performance at Roche Cobas 6000 biochemistry analyzer are presented graphically as normalized MEDx chart. Conclusion: Normalized MEDx chart is useful tool for comprehensive presentation of validation data and quick overall judgment of analyzer performance according to recommended quality specifications. Chart is easy to create and even easier to interpret as opposed to comparing numerous figures and analyzing large tables.
Šupak Smolčić V (1), Bilić-Zulle L (1,2) (1) Rijeka Clinical Hospital Center, Clinical Institute of Laboratory Diagnostics, Rijeka, Croatia (2) Rijeka University School of Medicine, Department of Medical Informatics, Rijeka, Croatia Corresponding author: [emailprotected]
Background: Method validation includes determination of inaccuracy (bias) and imprecision (CV) which represent systemic and random error respectively, and which are basic parameters for calculation of total error (TE). Each method is evaluated regarding its recommended quality specification. Aim of this presentation is to introduce normalized MEDx chart, a simple graphical method for quick assessment of analytical method perfor-
P08-09 Evaluation of haematological analyzer Cell Dyn Ruby Šega M, Perbil Lazič K, Lokar L University Clinical Centre Maribor, Centre of Transfusion Medicine, Maribor, Slovenia Corresponding author: [emailprotected]
Background: The goal of evaluation was to estimate the working of new haematological analyzer CELL-DYN Ruby. The aim of measurements of parameters WBC, RBC, HGB, HCT, MCV, MCH, and PLT Biochemia Medica 2012;22(3):A54-A204
A109
2nd European Joint Congress of EFLM and UEMS
as recommended by CLSI was to determine the "Long-term imprecision" and "bias" and provide an assessment of the acceptability of a new analyzer. Materials and methods: We have used commercial controls to evaluate „Long-term imprecision’’ and determined standard deviation and coefficient of variation through statistical analysis of the results. To compare two methods the testing was performed on the extinguishers Cell Dyn 1700 and Cell Dyn Ruby within three hours. Every day the testing of series 8-10 samples was performed in the sequence 1,2,3,4,5,6,7,8 and duplicates in the sequence 8,7,6,5,4,3,2,1 ever. Using statistical analysis, we have compared the comparability of methods and calculated power connections with a correlation factor. Results: In estimating analytical variability, we have determined the maximum coefficient of variation of platelets from 2% to 5%. In assessing the comparison of methods Cell Dyn 1700 and Cell Dyn Ruby, we have found out that both methods are comparable. Correlation coefficients for WBC, RBC, HGB, HCT and PLT were between 0.9773 and 0.9976, for MCV and MCH they were between 0.9476 and 0.9988. Conclusions: Based on our results, we can conclude that the haematological analyzer Cell Dyn Ruby is suitable for determination of complete blood counts in the hemostasiological laboratory.
P08-10 Overestimation of albumin (bromcresol green method): influence of acute-phase serum globulins Garcia Moreira V, Martinez Gago M, Laborda Gonzalez B, Fernandez Leivas A, Llorente Torres A, Fernandez Rodriguez E Hospital de Cabueñes, Servicio de Analisis Clinicos, Gijon, Spain Corresponding author: [emailprotected]
Background: As the most specific method for albumin measurement (immunonephelometry) is Biochemia Medica 2012;22(3):A54-A204
A110
expensive, most laboratories use dye-binding methods, being the two most common the bromcresol green (AlbBCG) and bromcresol purple (AlbBCP). AlbBCG and AlbBCP often yield discordant results, with a mean difference about 5 g/L. The objective of this study was to evaluate the influence of serum globulin on discrepancies albumin results measuring by BCG and BCP methods. Material and methods: Concentrations of serum albumin, globulins (alpha1-, alpha2-, beta and gamma) and C-Reactive Protein (CRP) were analyzed in 75 serum specimens by BCG and BCP and capillary zone electrophoresis (CZE) and nephelometry respectively. Comparisons were performed using Student t-test for paired data, Pearson correlation and Bland-Altman plot, using MedCalc software. Significance was set at P < 0.05. Results: Correlation between AlbBCP-AlbBCG was 0.976 (P < 0.01) but the mean difference was 4.53g/L. Between AlbBCG- AlbCZE mean difference was 3.54g/L. AlbBCP is in good agreement AlbCZE estimation with a mean difference of 0.97g/L. Differences between AlbBCG and AlbBCP become more evident with lower albumin concentration. AlbBCG assay bias shown a good correlation with alpha1-globulin concentrations (r = 0.746; P < 0.01); a moderate (r = 0.633; P < 0.01) and weak correlation (r = 0.575; P < 0.01) was observed with CRP and alpha2-globulin, respectively; finally, we found no correlation with beta-globulin (r = 0.151; P = 0.228), and a poor correlation with gamma-globulin (r = -0.272; P = 0.02). Conclusion: BCG method is not absolutely specific for albumin. Serum acute phase globulins (alpha1 and alpha2) contribute to the overestimation of albumin concentration by BCG assay, with the greatest effects observed for alpha1-globulin.
2nd European Joint Congress of EFLM and UEMS
P08-11 Comparison of sodium values between emergency and routine biochemical laboratory Fernandez Leivas A, Garcia Moreira V, Martinez Gago M, Marcos Corona L, Garcia Alonso S, Fernandez Rodriguez E Hospital de Cabueñes, Servicio de Analisis Clinicos, Gijon, Spain
centages were changed to a 26.1% of false negatives and 3% of false positives. Conclusions: Contrary to the initial suspicion, we found more false negatives than false positives, 13% and 8.9% respectively. So, the application of the linear regression line, while reducing the false positive rate (3%), doubles the number of false negatives (26.1%). The linear regression increased by 7.1% the total number of differing values, there by not suggest their application in clinical practice.
Corresponding author: [emailprotected]
Introduction: In our hospital, the determination of ions is performed by indirect potentiometry: Dimension RxL-Max (Siemens) at the emergency laboratory and Advia2400(Siemens) in the routine laboratory. Suspecting a downward trend in the determination of sodium(Na) in the ER laboratory, we process the same samples on both analyzers, using the Advia2400 as a reference method. Material and methods: On 3 different days, 158 serum samples were processed in parallel in both analyzers. Statistical analysis of the results was performed using MedCalc® software, using the Kolmogorov-Smirnov test, Pearson correlation and linear regression. Statistical significance was set at P 0.98 except for calcium (r = 0.884), sodium (r = 0.837) and chloride (r = 0.881). TAT for the Cobas c311 was on the average 11% longer then Olympus 480. Conclusion: Cobas c311 biochemistry analyzer is acceptable for emergency purposes, however the overall TAT is slightly longer than recommended.
P08-14 Comparison of Emerald and CELL-DYN 1800 for white blood cells and granulocytes in oncology patients Mittel K, Supak Smolcic V, Dvornik S Clinical Hospital Center Rijeka, Rijeka, Croatia, Clinical Department of Laboratory Diagnostics, Rijeka, Croatia Corresponding author: [emailprotected]
Background: The absolute number of the white blood cells (WBC) and neutrophil granulocytes (GRAN) in peripheral blood of patients undergoing chemotherapy or radiotherapy is an important indicator of their immunodeficiency. Capillary blood sampling from finger is an option in case of frequent blood controls and damaged blood vessels. Hematology analyzers Abbot CELL-DYN Emerald and CELL-DYN 1800 are good choices because of the small sample volume needed for analyses (9.8 and 30 uL respectively). The aim of the study was to compare the performance of
2nd European Joint Congress of EFLM and UEMS
CELL-DYN Emerald and CELL-DYN 1800 for WBC and GRAN measurement. Materials and methods: Capillary blood samples of 193 patients were analyzed on CELL-DYN Emerald and CELL-DYN 1800 for WBC and GRAN. Blood was collected from finger using BD Microtainer® tube with K2-EDTA anticoagulant. Passing-Bablok regression was used to compare results from two analyzers. Results: Correlation coefficient for both parameters is r = 0.98, P < 0.001. Intercept for WBC is 0.31, 95%Cl 0.15-0.48 and slope is 0.94, 95%Cl 0.92-0.97. For GRAN intercept is 0.07, 95%Cl -0.01-0.13 and slope is 0.94, 95%Cl 0.92-0.96. Results indicated small constant and proportional error for WBC and small proportional error for GRAN. Conclusions: Despite the small constant and proportional error for WBC and small proportional error for GRAN, both analyzers can be used to access WBC and GRAN count in capillary blood samples. CELL-DYN Emerald needs very small sample volume for analysis and thus it is very useful for capillary blood samples from oncology patients.
Healthcare Diagnostics Inc., New York, USA) hematology analyzers can replace the manual reference method in daily clinical onset. Materials and methods: One hundred thirty three (N = 133) non-CSF body fluid samples were prospectively studied, including 74 ascitic fluids (55.6%), 49 pleural fluids (36.8%), 3 pericardial fluids (2.3%), 4 synovial fluids (3.0%) and 3 drainage liquids (2.3%). Each fluid was analyzed by two experienced cytologists and the two above mentioned hematology analyzers. Results and conclusions: Our results indicate that in serous fluids – ascitic, pleural and pericardial – is possible to use either Sysmex XE-5000 or Siemens ADVIA 2120i to perform total cell count. However, cell differentiation using these approaches lacks both enough sensibility and specificity and therefore they should not be used. Finally, the automated analysis did not provide accurate results for other body fluids.
P09 – Haematology 1 P09-01
P08-15 Body fluids automated cell count: a comparison study between Sysmex XE-5000 and Siemens ADVIA 2120i Rego M, Titonel S Centro Hospitalar do Porto, Serviço de Química Clínica, Porto, Portugal
Verification of CD34+ stem cell analysis according to ISHAGE protocol on Beckton Dickinson FACSCanto Sokolić I, Šiftar Z, Kardum Paro M, Flegar Meštrić Z C.H."Merkur", Department of Clinical Chemistry and Laboratory Medicine, Zagreb, Croatia Corresponding author: [emailprotected]
Corresponding author: [emailprotected]
Introduction: Body fluid cytology, including total cell count and differentiation, continues to be a time-consuming task in modern clinical laboratories. The evolution of hematological analyzers, with new approaches to the counting methodology, has brought new possibilities in biological fluids cytology. The aim of this study is to assess whether Sysmex XE-5000 (Sysmex Corporation, Kobe, Japan) and Siemens ADVIA 2120i (Siemens
Background: Implementation of new analytical equipment into routine work requires verification procedure. We have verified flow cytometer Beckton Dickinson FACSCanto II for ISHAGE protocol (Sutherland et al, 1996, J Hematotherapy, 5:213226) stem cell (CD34+) analysis according to CLSI EP-A2-User Verification of Performance for Precision and Trueness; Approved Guideline-2nd ed.,Vol.25,No.17.,2005. in concordance with ISO 15189. Biochemia Medica 2012;22(3):A54-A204
A113
2nd European Joint Congress of EFLM and UEMS
Materials and methods: Analysis was performed on peripheral blood samples, fresh leukapheresates of peripheral blood and BC StemCell Control Kit samples. Acquisition data were analyzed using BD FACSDiva 6.0 software. Verification included assessment of random error check based upon triplicate measurement of commercial samples five days in a row, assessment of measurement uncertainty, conformation of linearity, comparison of results from reference analyzer and analysis of external quality control samples. Results: Performance characteristics: within-laboratory precision for Stem Cell Low (11.6 x 106 / L) was 9.2%, for Stem Cell High (31.5 x 106 /L) 8.0%. We confirmed linearity on peripheral blood sample 0-97.2 x 106 /L. Measurement uncertainty for Stem Cell Low was 28.4% and Stem Cell High 19.2%. Comparison of results with Beckman Coulter Cytomix FC500 was satisfactory (Passing Bablock, Y = -0.01117 + 1.0112x, P > 0.10). UK NEQAS for Immunophenotyping, Stem Cell enumeration scheme results were within target values (median ± 25 percentile) for CD34+ absolute and relative count. Conclusion: According to our verification procedure, BD FACS Canto II is cytometer of choice for performing CD 34+ stem cell absolute count analysis.
P09-02 Copper deficiencies simulating a myelodysplastic syndrome Gallego A (1), Vera J (2), Peral E (3), Martín J (1), Bermudo C (1), Fábregas M (2) (1) Hospital Universitario Virgen Macarena, Bioquímica Clínica, Sevilla, Spain (2) Hospital Universitario Virgen Macarena, Hematología, Sevilla, Spain (3) Hospital Universitario Virgen Macarena, Medicina Interna, Sevilla, Spain Corresponding author: [emailprotected]
Introduction: Copper is an essential trace element present in a number of metalloproteins and Biochemia Medica 2012;22(3):A54-A204
A114
appears to be required for the absorption and utilization of iron. Copper deficiency has been described in malnourished children, in both infants and adults receiving prolonged enteral or parenteral alimentation, and following gastric resection or bariatric gastric reduction surgery. It is already known that cupper deficiency could resemble a myelodysplastic syndrome with anemia, neutropenia and trombocytopenia. Case report: Patient 32 years male with spastic tetraplegia after severe TBI caused by a traffic accident in 2009. It was decided to place jejunostomy in June 2010. In June 2011 a normocytic anemia was detected without signs of bleeding according both clinically and by the diagnostic tests performed and treated according to protocol. In January 2012 was readmitted for persistent leucopenia and anemia in transfusion range. It was performed bone marrow aspirate, being found a bicytopenia, dysplastic features, reactive bone and good representation of the three series. Values copper and ceruloplasmin levels were requested, being respectively: 36 mcg/dL (Normal being 65165) and 12 mg/dL (Normal 20-40). Parenteral alimentation was maintained with supplements of copper, recovering values cupperhemic to enter the normal range and resolving the bicitopeny and symptoms secondary to it. Conclusion: In patients with unexplained refractory cytopenias, dysplastic features and comorbidities that may trigger malnutrition such as parenteral alimentation, you should order determination of copper and ceruloplasmin levels before diagnosis of myelodysplastic syndrome.
2nd European Joint Congress of EFLM and UEMS
P09-03
P09-04
Multiple myeloma: a case report
Which value can be used as a positivity threshold for Large Unstained Cells on Advia to make slides?
Cachapuz I (1), Branco A (2), Paiva A (2), Magalhaes C (1), Costa M (2) (1) Matosinhos Local Health Unit , Clinical Pathology Service, Oporto, Portugal (2) Matosinhos Local Health Unit, Nephrology Service, Oporto, Portugal Corresponding author: [emailprotected]
Introduction: The multiple myeloma is a neoplasm of B cells, associated to the presence of a monoclonal protein in serum and/or urine. It is clinically manifested by different symptoms. Case report: 58 year-old woman, with depression, obesity and hypertension, presented at the ER (Emergency Room) of Pedro Hispano Hospital on the 13th January 2012 due to nonoliguric acute kidney injury (creatinine: 7.7 mg/dL), anemia ( Hb: 7.1 mg/dL ),calcium 8.2 mg/dL, proteinuria (7 g/ dia). Renal echography: normal. Blood transfusion was made. Declared having anemia for 6 months, under study, and she had already made an upper digestive endoscopic and colonoscopy both without changes. Three months before hospitalization: 10 kg loss, asthenia, anorexia, nausea, pain in the left costal margin. At the ER, abdominal CT scan revealed lytic lesions. The day after, the patient made a serum proteinogram: beta peak. The serum protein electrophoresis (SPE) revealed the presence of a monoclonal component lambda (4.54 g/L). Since there was no recovery of kidney function, haemodialysis was started. A myelogram was performed and revealed pathological plasma cells. The cytogenetic laboratory didn’t detect IGH_MMSET fusion, nor 17p13 deletion (gene TP53), at the bone marrow. She started chemotherapy with Bortezomib and Dexametazona on the 20th January 2012, and was treated with high cutoff filters without success in the recovery of kidney function. Conclusion: This clinical case underlines the importance of the clinical-laboratory interaction for the correct and timely diagnosis of haemato-onchological pathologies. SPE is still an important auxiliary diagnostic tool.
László K, Patonai Z, Kellner V, Fekete M Synlab Hungary Ltd., Laboratory of Székesfehérvár, Székesfehérvár, Hungary Corresponding author: [emailprotected]
Introduction: The Advia 2120i hematological system uses peroxidase staining for white blood cells differential testing. Peroxidase-negative cells, large lymphocytes, plasma cells, blasts are counted to large unstained cells (LUC) category. The manufacturer recommends the normal range for LUC < 4%. If LUC > 4%, it is suggested to make a slide and analyse morphology of cells by microscopy. The aim of this study was to find a real positivity threshold for LUC, where the results by microscopy give additional information to the clinician beyond the mere numerical data. Materials and methods: ROC curve analysis was performed to obtain the appropriate results. Blood samples of 350 patients were examined by automated Advia 2120i and microscopy. Three analyses were performed and results were considered to be positive as follows, ”1”: if blasts or any number of reactive/granulated lymphocytes were found by microscopy; ”2”: if blasts or a few (4-6) or more reactive/granulated lymphocytes-, and ”3”: if blasts or several (> 7) reactive/granulated lymphocytes were counted by microscopy. Results: In analysis ”1”, the positivity threshold for LUC > 4% was observed, the true negative rate (TNR; specificity): 74.76%; the true positive rate (TPR, sensitivity): 88.81%. High number of false positive results (FPR) were detected: 25.24%. In case of ”2” the positivity threshold for LUC > 5% was found, TNR: 83.27%, TPR: 66.25%, FPR: 16.73%. In analysis ”3” the positivity threshold for LUC > 6%, TNR: 94.08%, TPR: 56.45% and FPR: 5.92% were obtained. Conclusion: Overall, even LUC > 6% was found to be reliable positivity threshold to request blood smears. Biochemia Medica 2012;22(3):A54-A204
A115
2nd European Joint Congress of EFLM and UEMS
P09-05 Role of P-selectin glycoprotein ligand-1 (PSGL-1) during G-CSF treatment in a mouse model Miszti Blasius K (1), Felszeghy S (2), Hevessy Z (1), Kappelmayer J (1) (1) Medical and Health Science Centre, University of Debrecen, Department of Laboratory Medicine, Debrecen, Hungary (2) Medical and Health Science Centre, University of Debrecen, Department of Anatomy, Debrecen, Hungary Corresponding author: [emailprotected]
Background: The effect of G-CSF (Neupogen) was investigated in wild-type (WT) and PSGL-1 knockout (KO) mice to establish the role of this mucin in myeloid cell mobilization. G-CSF activates tissue proteases that cleave several adhesion molecules, thus enhances the mobilization of myeloid cells and haemopoietic stem cells. Materials and methods: WT and KO mice (12-16 week old, 20-25 gram body weight, N = 15) were treated with a single dose of 250 mg/kg cyclophosphamide to induce cytopenia, subsequently mice received 7.8 µg/kg G-CSF twice a day for 4 days. Retro-orbital blood samples were drawn to determine leukocyte counts by Siemens Advia 120 analyser.
zation from the bone marrow and delayed extravasation in the peripheral vessels in PSGL-1-/animals.
P09-06 Usefulness of red cell indices in differentiating microcitic anemias Pavela J, Fijačko M, Dobrošević B, Paradinović K, Šerić V Clinical Hospital Centre Osijek, Department of Clinical Laboratory Diagnostics, Osijek, Croatia Corresponding author: [emailprotected]
Backgound: Iron deficiency anemia (IDA) and beta-thalassemia trait (β-TT ) are the most common forms of microcytic anemia. This study was conducted to compare the validity of various discrimination indices in differentiating β-TT from IDA by calculating their sensitivity, specificity and Youden's index. Materials and methods: A total of 146 patients (69 IDA and 77 β-TT) with microcytic anemia were involved in this study. We calculate six discrimination indices i.e. RBC count, Mentzer indeks (MI), Shine & Lal (S&L), Srivastava (SI), England & Fraser (E&F) and Red cell Distribution Width Indeks (RDWI). The number of correctly identified cases were determined and sensitivity, specifity, positive and negative predictive value and Youden's index of each discrimination index were calculated.
Results: Neutrophil granulocyte count increased upon completion of G-CSF treatment but were significantly different in WT and KO mice (28.3 G/L vs. 47.7 G/L), while the monocyte counts were 2.0 G/L and 4.1 G/L. Four days after the last G-CSF treatment, both strains displayed considerably reduced neutrophil (1.8 G/L and 9.8 G/L) and monocyte (0.4 G/L and 1.6 G/L) counts, the values always being higher in KO animals. Contrary, eosinophil granulocyte values became elevated only at this sampling time, being 0.5 G/L (WT) and 1.2 G/L (KO). There was a similar difference in atypical cell counts always being higher in KO animals.
Results: The percentage of correctly diagnosed is highest for E&F and RDWI (86%) folowed by MI (79%) index. None of the discrimination index showed 100% sensitivity and specificity. Youden's index wich includes both, sensitivity and specificity is in descending order RDWI > E&F >MI > RBC >SL >SI.
Conclusions: The lack of PSGL-1 results in higher mature and precursor myeloid cell release after GCSF treatment with delayed effects on eosinophils. The differences are caused by both faster mobili-
Conclusion: RDWI, England-Fraser and Mentzer index are the most reliable formulae in discrimination between iron deficiency anemia and betathalassemia trait.
Biochemia Medica 2012;22(3):A54-A204
A116
2nd European Joint Congress of EFLM and UEMS
P09-07 A method of preparing fresh blood samples for Croatian haematology external quality assessment Nazor A (1), Flegar-Mestric Z (1), Hećimović A (2), Šiftar Z (1), Hundrić Hašpl Ž (2) (1) University Hospital Merkur, Institute of Clinical Chemistry and Laboratory Medicine, Zagreb, Croatia (2) Croatian Institute for Transfusion Medicine, Blood bank, Zagreb, Croatia
Conclusion: The home-made control material used in Croatian EQA scheme shows applicability to all haematological analyzers and provides a long term, retrospective assessment of laboratory performance and comparability of results on national level.
P09-08 Differential count in healthy newborns Paradinović K (1), Milić M (1), Drmić S (2), Šerić V (1)
Corresponding author: [emailprotected]
Background: Croatian EQA involves participation in the National External Quality Assurance Scheme organized under the auspices of the Croatian Society of Medical Biochemists by processing of unknown samples 3 times per annum. The suitability of home-made control material for complete blood count was evaluated. Materials and methods: The Croatian Institute for Transfusion Medicine prepared the homemade control material for complete blood count. A single portion of fresh blood is used given by voluntary donor having given consent to its intending use. A minor portion of the blood is tested for Hepatitis B and C as well as HIV and TP and was nonreactive for all these markers. After 30 minutes mixing in a way without making foam, 1.5 ml K2EDTA molarity 1 mol/L is added and the mixing was continued next 2-5 minutes. The blood is distributed into suitable 7.5 x 12 mm sterile tubes, capped and stored at +2 °C to +8 °C. The samples were analysed for homogeneity and stability according to the ISO 13528 requirements. Results: The home made-samples are delivered within 24 hours throughout Croatia in 200 medical biochemistry laboratories and are analyzed on 37 different types of analyzers from 11 different manufacturers. Assessment against consensus values and the percent allowed difference score is introduced for evaluation of obtained results. The high degree of inter-laboratory comparability is approved.
(1) University Medical Center Osijek, Department of Clinical Laboratory Diagnostics, Osijek, Croatia (2) General Hospital of Vinkovci, Department for Biochemistry, Vinkovci, Croatia Corresponding author: [emailprotected]
Background: An accurate differential count is important in the diagnosis and monitoring of various clinical states. Our aim was to compare WBC differential morphology flags from haematology analyzer and differential blood count from microscopic slide examination in healthy newborns. Materials and methods: CBC count was determined on haematology analyzer Sysmex XE2100. Differential was performed by examination of peripheral blood smear using light microscope. Slides were stained by May Grunwald Giemsa. We analysed 252 blood samples from healthy newborns aged 1-3 days. Results: We compared flags for immature gran and left shift with nonsegmented neutrofiles, metamyelocytes, myelocytes. Average for nonsegmented neutrofiles was 6%. Compatible values (flags=pos, microscope=poz; flags=neg, microscope=neg) were in 105 cases. Noncompatibile values (flags=neg, microscope=pos) were in 11 cases and (flags=pos, microscope=neg) were in 140 cases. Conclusions: Comparing the results, we noticed that the haematology analyser is more analytically sensitive. Microscopic slide examination should bee done in all samples flaged by haematology analayzer. Biochemia Medica 2012;22(3):A54-A204
A117
2nd European Joint Congress of EFLM and UEMS
P09-09
P09-10
The effect of the cellularity of the bone marrow aspirate sample for cytogenetic cultures
Verification of the Body Fluid Application on the Sysmex XE-5000 Hematology Analyzer
Tegg E (1), Raik E (2) (1) University of Tasmania, Pathology, Hobart, Australia (2) Royal North Shore Hospital, Haematology, Sydney, Australia Corresponding author: [emailprotected]
Background: The role of cytogenetics in the diagnosis and prognosis of haematological malignancies is well recognised. The contribution made by the quality of the sample given to a cytogenetics laboratory to successfully provide diagnostic results is not. Materials and methods: This was a retrospective control study made after a change to the bone marrow aspiration technique. The test group consisted of the first 65 patients who underwent a bone marrow biopsy requiring cytogenetic analysis after this change. The control group consisted of 65 patients who had undergone a bone marrow biopsy for cytogenetic analysis prior to this change. The parameters measured were disease type, morphological cellularity of the aspirated marrow, cell count of the cytogenetic sample, cytogenetic failure rate, number of metaphases obtained and the banding resolution achieved. Results: Results were analysed using Microsoft Excel student t-test. The cellularity of the cytogenetic sample was significantly increased (1.23 x 109/L compared to 0.59 x 109/L; P < 0.001). The culture failure rate fell significantly from 15% to 2%. (P < 0.01). The optimal number of metaphases (20 metaphases per patient) was achieved in 75% of cases compared to 50% in the control group (P < 0.04). The banding resolution was significantly increased in the test group compared to the control group (P < 0.04). Conclusions: Information in the literature is sparse when assessing the effect of BM cellularity on the ability to obtain a successful cytogenetic result. This study showed the positive effect of increasing the cellularity of the bone marrow aspirate sample for the successful cytogenetic analysis. Biochemia Medica 2012;22(3):A54-A204
A118
Šiftar Z (1), Kardum-Paro M (1), Nazor A (1), Sokolić I (1), Kardum Skelin I (2), Flegar Meštrić Z (1) (1) Merkur University Hospital, Institute of Clinical Chemistry and Laboratory Medicine, Zagreb, Croatia (2) Merkur University Hospital, Institute of Clinical Cytology and Cytogenetics, Zagreb, Croatia Corresponding author: [emailprotected]
Background: Aim of this study is to investigate the performance of BF application on the Sysmex XE-5000 hematology analyzer in differential diagnosis of exudates and transudates. Materials and methods: The XE-5000 BF application provides white blood cell (BF-WBC) and total nucleated cell (BF-TC), as well as erythrocyte (BFERC) count. BF-WBC is further subcategorized in mononuclear and polymorphonuclear cells, BFMN and BF-PMN. Performance of application was evaluated according to CLSI H56-A guidelines. Comparison to reference methods: microscopic cell counting in Neubauer chamber and differential on MGG stained smears after citospin preparation were done. Additionally, measurement of cells on XS2100 analyzer was included in comparative study. Automated cell counting was performed in Institute of Clinical Chemistry and Laboratory Medicine, whilst the microscopic in Institute of Clinical Cytology and Cytogenetics. It was done on various fluids (pleural effusion and ascites, dialysis fluid within peritoneal dialysis treatment) specimens. For correlation calculation Passing and Bablock regression analysis is used. Results: Repeatability of BF-WBC measurements was performed on 3 concentration levels. Coefficients of variation (CVs) were 21, 14 and 7.5, and met manufacturer specifications. BF-WBC linearity was proven for measurement range of 3 to 3420. Achieved coefficients of correlation with microscopic and measured counts on Sysmex XE 2100 for 35 specimens were 0.92 and 0.98, respectively. Obtained coefficient of 0.89 was found in compari-
2nd European Joint Congress of EFLM and UEMS
son of automated and microscopic differential on 23 samples. Conclusions: Evaluation of BF application on Sysmex XE-5000 showed significant improvement in the ability of automated hematology analyzers regarding body fluid cell count analysis.
P09-11 Integration of point-of-care hematology analyzers in the hospital and laboratory information system Baršić I (1), Brkljačić V (1), Šrenger V (2), Majdenić K (3), Stojmilović S (3), Sertić J (1)
cal area network with LIS, further integrated into HIS. According to predefined criteria, LIS performes autovalidation and results are forwarded to HIS. In this way, results are available within a few seconds. Conclusion: Integration of point-of-care devices in the hospital and laboratory information system allows the storage of patient results with electronic medical patient records and easier access to results when they are needed again. It also gives us the ability to have better control over reagent consumption and over results obtained from these devices.
P09-12
(1) University Hospital Center Zagreb, Clinical Institute of Laboratory Diagnostics, Zagreb, Croatia (2) University Hospital Center Zagreb, Center for Informatics, Zagreb, Croatia (3) IN2 Ltd, Health sector, Zagreb, Croatia
Blood loss from laboratory tests in adult hospitalized patients
Corresponding author: [emailprotected]
Eskisehir Osmangazi University, The Medical School, Medical Biochemistry, Eskisehir, Turkey
Background: In 2000, our Institute started with implementing point-of-care testing at sites where it was important to decrease TAT, either for critical care patients or in order to increase efficiency and patients turnaround time. University Hospital Center Zagreb has 14 blood gas analyzers (GEM Premier 3000,Rapidlab 1265), 6 hematology analyzers (Sysmex pocH-100i), 7 coagulation monitoring system analyzers (CoaguChek XS, thromboelastometry ROTEM, aggregometry Multiplate® ) and 105 glucometers (Accu Chek Inform II). Results from these devices are used to assess the current status of patients and often are not entered in the medical record, thus remaining unaccounted for in the final patient report with diagnostic and therapeutic procedures. Therefore, we initiated integration of point-of-care devices in the hospital and laboratory information system (HIS, LIS), starting with hematology analyzers. Materials and methods: Sysmex pocH-100i is an automated hematology analyzer that provides 19-parameter complete blood counts with threepart leucocyte differential. It is connected via a lo-
Alatas O, Kocaturk E, Bekmez M, Bayrak C, Harmansa A
Corresponding author: [emailprotected]
Introduction: Blood loss for laboratory tests can be important especially for inpatients. The aim of this study was to determine approximate blood loss of inpatients during their stay in hospital and whether this loss contributes to changes in hemoglobin levels. Materials and methods: All patients hospitalized for eight or nine days in twenty five clinics of University Hospital of Eskisehir Osmangazi University in 2011 were reviewed retrospectively. We estimated blood loss by multiplying the number and approximate volumes of sampling tubes that used for laboratory analysis of 1792 adult inpatients. Moreover, the first and last sample hemoglobin levels of hospitalized patients were compared. Results: The mean blood drawn from patients was estimated 35.27 mL. The highest blood volume was observed in patients from Chest Disease Clinic. Total tube number used for 1792 patient was 22,926, and the red top tube was consisted of 36% Biochemia Medica 2012;22(3):A54-A204
A119
2nd European Joint Congress of EFLM and UEMS
of all tubes. Among the eight clinics which have more than fifty patients, Chest Disease Clinic was used the highest number of tubes. The first day median level of hemoglobin was reduced from 12.7 g/dL to 11.7 g/dL in eight days hospitalized patients and from 12.5 g/dL to 11.7 g/dL in nine days hospitalized patients. Results: Blood loss from laboratory diagnostic testing is highly associated with changes in hemoglobin levels for inpatients and may contribute to anemia especially in high risk group patients. For this reason, unnecessary test requirements should be prevented and minimum test repetition time should take into consideration in order to reduce blood loss arise from laboratory diagnostic testing.
P10 – Haematology 2
mean baseline MPV was greater than that in those without a primary outcome (9.1 ± 0.6 vs. 8.9 ± 0.7 fl, P < 0.05), PDW was less (13.4 ± 1.2 vs. 13.8 ± 1.1 %). In patients with adverse outcomes fibrinogen (6.3 ± 0.4 vs. 4.6 ± 0.5 g/L, P = 0.02), D-dimers (660 ± 24.1 vs. 382.2 ± 13.0 ng/mL, P = 0.001), BNP (103.5 ± 7.9 ng/mL vs. 61.6 ± 9.3, P < 0.001), von Willebrand factor (vWf) (186.2 ± 14.3 vs. 160.4 ± 20.1%, P = 0.03), spontaneous and induced (ADF, adrenalin) platelet aggregation (P ≤ 0.05) were greater compare to patients without. The criteria for 6 months adverse events were MPV > 9.0 fL, fibrinogen > 6.4 g/L, Ddimer > 640 ng/mL, BNP > 110 ng/mL, vWf > 180%. Conclusion: MPV can be used as a risk biomarker in prognosticating the 6 months outcomes for unstable angina. Patients adverse outcomes in unstable angina have increased level of fibrinogen, Ddimer, BNP, vWf, spontaneous and induced platelet aggregation
P10-01 State of plasma and platelets hemostasis factors in patients with unstable angina Markava I (1), Miadzvedzeva A (2), Helis L (1), Kaljadka M (3), Russkikh I (3) (1) RPSC Cardiology, Laboratory of heart surgery, Minsk, Belarus (2) RPSC Cardiology, 2nd cardiological department, Minsk, Belarus (3) RPSC Cardiology, Clinical laboratory, Minsk, Belarus
P10-02 Evaluation of platelet parameters for differential diagnosis of thrombocytosis Peran N, Reškov Ž, Meniga D, Pamuković-Jaram D, Vesna S General hospital Šibenik, Laboratory of Biochemistry and Hematology, Šibenik, Croatia
Corresponding author: [emailprotected]
Corresponding author: [emailprotected]
Methods and results: We studied 100 patients defined clinically as unstable angina (M/F = 60/40, 61 ± 12.2 years). Analyzed blood tests (platelet count, MPV- mean platelet volume, PDW – platelet distribution width), cardiac markers (CK-MB, myoglobin, troponin), biochemical analysis (C-reactive protein, BNP), coagulation blood tests, platelet aggregation test were taken at admission. Statistical analysis for the study was conducted using Statistica 6.0 (StatSoft. Inc,USA). The combined primary end point (all-cause mortality, nonfatal myocardial infarction, recurrent UA, urgent percutaneous coronary intervention or coronary artery bypass grafting) at 6 months occurred in 36 (36%). In these patients, the
Background: There are two types of thrombocytosis: primary and secondary. Primary thrombocytosis (PT) is caused by a chronic myeloproliferative disorders (CMPD) and secondary thrombocytosis is called reactive thrombocytosis (RT) since it is associated with inflammatory states. Differential diagnosis of thrombocytosis is not always obvious. In automated blood cell analyzers various platelet parameters can now be measured. In this study, we analyzed whether 6 platelet parameters can be used for the differentitation of PT from RT.
Biochemia Medica 2012;22(3):A54-A204
A120
Materials and metods: The platelet counts, mean platelet volume (MPV), plateletcrit (PCT), platelet distribution width (PDW), mean platelet mass
2nd European Joint Congress of EFLM and UEMS
(MPM), mean platelet component concentration (MPC) and large platelets (LPLT) were studied in 40 patients with RT, 18 patients with PT and 60 normal control. Platelet parameters were measured by ADVIA 2120 (Bayer Diagnostics, USA). Significance was determined using the Mann-Whitney test. Results: Patients with CMPD had significantly higher MPV, PDW, L-PLT, PCT, MPM than those with reactive thromboytosis. There was no significance difference in MPC between patients with PT and RT, but in both groups MPC was significantly lower than in control group. Also, there was no difference in MPM between patients with primary thrombocytosis and control group. Conclusion: The platelet parameters are useful for the differential diagnosis of thrombocytosis. High MPV, PDW, and L-PLT with high platelet counts suggest primary thrombocytosis.
P10-03 Evaluation of in-house normal pool plasma for partial thromboplastin time mixing study on an ACL TOP Costa P, Reis J, Silva C, Simão L, Diamantino I, Valido F
samples with prolonged PTT (ratio > 1.20) were properly mixed in equal volume with the normal pool plasmas and evaluated for PTT. Correlation was evaluated by pearson’s correlation factor. Mean and standard deviation (SD) were analyzed for all groups. T-Test was performed for group comparison. Results: Mean ± SD for the mixing tests of the three groups as ratio: In-house – 1.16 ± 0.13, IL – 1.23 ± 0.17, Stago – 1.14 ± 0.16. T-Test analysis between groups: In-house vs. IL – 0.0052, In-house vs. Stago – 0.4765, IL vs. Stago – 0.0016. All groups showed a good pearson correlation: In-house vs. IL – 0.7683, In-house vs. Stago – 0.8883, IL vs. Stago – 0.8349. Conclusions: A critical evaluation of T-Test and correlation between in-house normal pool indicates great similarity of results compared to those obtained with the commercial plasma pools tested, thus suggesting that a careful selection of donors allows preparing in-house normal pool plasma that fits the quality demands of routine laboratory mixing PTT tests.
P10-04
IPOC FG EPE, Clinical Pathology, Coimbra, Portugal
Comparison of two methods for measuring FVIII levels in hemophilia A patients
Corresponding author: [emailprotected]
Miloš M (1), Coen Herak D (1), Zupančić-Šalek S (2), Zadro R (1)
Background: Normal pool plasma plays a critical role on the safe outcome on testing prolonged partial thromboplastin time (PTT). Mixing study for qualitative inhibitor detection is commonly used on routine laboratory testing, allowing detection or exclusion of an inhibitor as differentiation from primary factor deficiency.
(1) University hospital centre Zagreb, Department of laboratory diagnostics, Zagreb, Croatia (2) University hospital centre Zagreb, Department of medicine, Zagreb, Croatia
Materials and methods: In-house normal pool plasma (In-house) was obtained from 50 healthy donors immediately after collection and centrifugation (3000 rpm; 10 minutes), properly mixed and stored at - 60 °C. Commercial normal pool plasma from ILTM Normal Control® (IL) and StagoTM Pool Norm® (Stago) were used for result comparison. 78
Corresponding author: [emailprotected]
Background: Correct determination of FVIII activity is essential for the assessment of severity of hemophilia A, as well as for patient-tailored treatment strategy. The most commonly performed assay for measuring FVIII activity is one-stage clotting assay. Several groups of authors have reported discrepancies between one-stage and chromogenic assay for FVIII determination. Biochemia Medica 2012;22(3):A54-A204
A121
2nd European Joint Congress of EFLM and UEMS
Materials and methods: We have compared the one-stage and chromogenic FVIII assay (Siemens, Marburg, Germany) in 100 hemophilia A patients (56 severe and 44 non-severe) and 101 healthy male subjects. Results: Good correlation between assays was obtained in healthy subjects as well as in severe and non-severe group of patients (coefficients of correlation 0.607; 0.904 and 0.875, respectively), but statisticaly significant difference was found in all groups. According to Bland & Altman the mean differencies between assays in healthy subjects, severe and non-severe patients were 17.6%, 47.8% and 61.7%, respectively. Usually, the results with chromogenic assay were lower than results with one-stage clotting assay. Similar correlation with clinical parameters (age at first joint bleed, number of joints with hemophilic arthropathy, number of annual joint bleeds and annual FVIII consumption) was found: coefficients of correlation between 0.568 and 0.688 for one-stage and between 0.521 and 0.619 for chromogenic assay. Conclusions: Two assays mostly show good correlation but clinically important discrepancies could be observed. It is recommended that all hemophilia centres have available both types of assay. Chromogenic assay should be used in the cases of normal aPTT and one-stage FVIII activity with presence of personal or familiy history of bleeding.
P10-05 Design and application of lupus anticoagulant diagnostic algorithm Banys V, Cimoš R Vilnius University Faculty of Medicine, Department of Physiology, Biochemistry, Microbiology and Laboratory Medicine, Vilnius, Lithuania Corresponding author: [emailprotected]
Background: Lupus anticoagulant (LA) diagnostics is highly complicated. Laboratory’s responsibility is to select appropriate assays as recomBiochemia Medica 2012;22(3):A54-A204
A122
mended by certain published guidelines. Results must be interpreted properly, which is not an effortless and easy task. Aim of study was to design LA diagnostics algorithm, implement it in routine work of local laboratory, evaluate difference of diagnostic capabilities between single assays (aPTT, dRVVT) and complex of methods (assays plus indexes) used in new LA algorithm. Materials and methods: In total 68 persons were selected, 40 relatively healthy for calculation of dRVVT reference values, and 28 suspected to have positive LA. aPTT, dRVVT screen and confirm, mixing studies of aPTT and dRVVT screen were performed. dRVVT normalization ratio, Rosner index (RI) of aPTT, dRVVT screen, correction index (CI) of dRVVT were calculated. Results: Plasmas with suspected LA gave different numbers of positive results when different interpretative breakpoints were used: aPTT RI >15% was determined in 10.7%, dRVVT screen RI > 1517.9% and dRVVT CI > 10-75% of patients. Conclusions: Narrower than manufacturer’s dRVVT reference intervals resulted in more positive LA results, most probably meaning greater number of false positives. No statistically significant difference in sex dependant dRVVT reference values was found. LA algorithm gave much less positive LA results than merely results of single assays. Despite using different reference values LA algorithm gave the same number of positive LA results and positives were detected in the same specimen.
2nd European Joint Congress of EFLM and UEMS
P10-06 Relationship between homocysteine level and MTHFR haplotype in patients with thromophilia Beletic A (1), Djordjevic V (2), Canic I (1), Mirkovic D (3), Radojkovic D (2), Majkic-Singh N (3) (1) Clinical Center of Serbia, Belgrade, Serbia, Center for Medical Biochemistry, Belgrade, Serbia (2) Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Serbia, Laboratory for Molecular Biology, Belgrade, Serbia (3) Clinical Center of Serbia, Belgrade and Faculty of Pharmacy, University of Belgrade, Serbia, Center for Medical Biochemistry, Belgrade, Serbia Corresponding author: [emailprotected]
Background: Increased homocysteine (Hcy) concentration is associated with increased risk for venous and arterial thrombosis. Reduced methylentetrahidrofolate reductase (MTHFR) activity, due to the presence of C677T and A1298C polymorphisms, is one of the causes of hyperhomocysteinema. In a group of patients with thrombophilia, Hcy leveles were compared between MTHFR haplotypes. Materials and methods: Study enrolled 30 patients (12 men, 18 women). Hcy was measured using HPLC technique with fluorescent detection. PCR amplification and reverse allele-specific oligonucleotide hybridization were employed for MTHFR haplotype determination. Statistical analyses included Kruskal-Wallis and Mann Whitney U tests. Results: The following distribution of MTHFR haplotypes was observed: 677CC/1298AA was present in 4 (median Hcy = 10.8 μM), 677CC/1298AC in 7 (median Hcy = 11.7 μM), 677CC/1298CC in 3 (median Hcy = 9.7 μM), 677CT/1298AA in 4 (median Hcy = 10.4 μM), 677CT/1298AC in 8 (median Hcy = 11.2 μM) and 677TT/1298AA in 4 patients (median Hcy = 14.6 μM). No statistically significant difference was observed between Hcy concentration corresponding to the detected haplotypes. Conclusions: In a group of patients with thrombophilia, no significant difference in Hcy concentra-
tion, related to MTHFR haplotype, was detected. This finding further implies that the Hcy measurement has greater clinical importance than MTHFR genotyping, but such assumption has to be tested in larger studies.
P10-07 Validation of Hemoclot® Thrombin Inhibitors assay for the new oral anticoagulant dabigatran Lilja J, Leinonen J, Saarela E, Lassila R, Joutsi-Korhonen L HUSLAB Laboratory Services, Helsinki University Central Hospital, Coagulation Disorders Unit, Clinical Chemistry and Hematology, Helsinki, Finland Corresponding author: [emailprotected]
Background: The direct thrombin inhibitor dabigatran does not require laboratory monitoring routinely. However, under special circumstances (e.g. renal insufficiency, bleeding complication, thrombosis, major surgery) testing may be crucial. Routine coagulation assays prothrombin time (PT), INR and activated partial thromboplastin time (APTT) are of limited value. We validated a commercial assay based on diluted thrombin time (TT). Materials and methods: Hemoclot® Thrombin Inhibitors test with control samples (Hyphen, Aniara) was evaluated for repeatability and linearity. Patient results (N = 30) were compared with Owren PT (Nycotest PT, Medinor), APTT (Actin FSL, Siemens Healthcare Diagnostics) and TT (Thrombin Reagent, Siemens). Coagulation analyser BCS XP (Siemens) was used. Results: Intra-assay repeatability (CV%) tested with patient samples was 7.2%. Inter-assay repeatabilities with control samples were 10.1% (at 130 µg/L) and 7.9% (at 290 µg/L). The method was linear within the concentration range 50-470 µg/L (R = 0.997). Correlation between Hemoclot® and APTT was moderate (R2 = 0.77), however there was considerable variation in APTT results, likely reflecting the clinical diversity of the patients. Even Biochemia Medica 2012;22(3):A54-A204
A123
2nd European Joint Congress of EFLM and UEMS
at the highest concentration (490 µg/L), APTT was prolonged only to 62 s (reference values 23-33 s). Correlation with PT was poor (R2 = 0.23) expectedly. TT exceeded the measurement range (> 140 s) already at low concentrations (< 50 µg/L). Conclusions: Hemoclot® Thrombin Inhibitors demonstrated acceptable repeatability and linearity and seems suitable for dabigatran assessment. Here, APTT poorly estimated dabigatran concentration. Interpretation of the results depends on the clinical situation and timing. Yet, the cut-off values or safety limits in different clinical conditions remain unestablished.
P10-08 Indicators of activation of the coagulation system in children with Henoch-Schönlein purpura
Results: The results of coagulation tests expressed as median (95% confidence interval (95% CI), interquartile range (IQR)) were: PV ratio 0.96 (0.87-1.02, 0.85-1.04); APTV (sec) 26.7 (24.6-28.3, 24.0-28.5); fibrinogen (g/L) 3.7 (3.2-4.6, 3.1-4.8), TT (sec) 16.5 (16.1-17.6, 15.9-17.9), D-dimer (mg/L) 5.3 (1.8-7.7, 1.68.1). This study showed no alterations of PT, APTT and TT tests in HSP patients, fibrinogen was increased in 6/21 patients while D-dimer levels were increased in all 21 patients. In patients grouped according to the number of organs involved (2, 3 and 4) D-dimer concentrations were as follows: 2 organs involved: 2.9 mg/L (IQR = 1.4-7.5); 3 organs involved: 7.0 (IQR = 2.9-9.0) and 4 organs involved: 6.5 (IQR = 3.9-9.0), but there were no significant differences in D-dimer concentrations between groups. Conclusion: Increased D-dimer concentrations in all 21 paediatric patients with HSP suggest that activation of coagulation including hyperfibrinolysis secondary to the endothelial damage to be a typical feature of HSP.
Obuljen J (1), Kapović A (2), Linarić I (1), Margetić S (3), Magdić B (1), Žižić V (1) (1) Children's Hospital Zagreb, Department of Laboratory Diagnostics, Zagreb, Croatia (2) Children's Hospital Zagreb, Department of Pulmology, Alergology and Rheumatology, Zagreb, Croatia (3) Children's Hospital Zagreb, Department of Laboratory Diagnostics, Zagreb, Croatia
P10-09 Pre-analytical routines in coagulation testing: are guidelines followed? Kristoffersen A (1), Stavelin A (2), Vannes S (1), Berge Kristensen GB (3)
Corresponding author: [emailprotected]
Background: Henoch-Schönlein purpura (HSP) is the most common vasculitis of childhood, affecting skin, joints, gastrointestinal tract and kidneys with clinical manifestations of bleeding tendency. The aim of this study was to evaluate global coagulation tests and D-dimer concentration as laboratory signs of activated coagulation system at first presentation of HSP. Materials and methods: The study included 21 paediatric patients with HSP, median age 6 (range 3-16). Global coagulation tests PT, APTT, TT, fibrinogen were determined with coagulometric methods (Sysmex CA-560 analyzer, Siemens, Germany). D-dimer concentration was measured with microparticle enzyme immunoassay (Axsis Shield Diagnostics Ltd., USA) on Axsym analyzer (Abbott, USA). Biochemia Medica 2012;22(3):A54-A204
A124
(1) Laboratory of Clinical Biochemistry, Haukeland University Hospital, Bergen, Norway (2) Norwegian Quality Improvement of Primary Care Laboratories (NOKLUS), Department of Public Health and Primary Health Care, University of Bergen, Bergen, Norway (3) Norwegian Clinical Chemistry EQA Programme, Norwegian Clinical Chemistry EQA Programme, Bergen, Norway Corresponding author: [emailprotected]
Background: It has been documented that about 70% of errors in laboratory medicine occur in the preanalytical phase. The aim of this survey was to study preanalytical conditions of routine hemostasis testing in Norwegian laboratories, and compare them with current guidelines. Materials and methods: Hospital laboratories in Norway (N = 69) were invited to fill in a web-based
2nd European Joint Congress of EFLM and UEMS
questionnaire regarding preanalytical routines for routine hemostasis testing. The first part focused on instruments and reagents for the different coagulation analyses used, and the second part focused on routines regarding venipuncture (needle gauge, citrate concentration, use of stasis, filling volume), handling of the sample before analysis, as well as routines regarding detection and handling of sample clot, high/low hematocrit, hemolysis, bilirubinemia or lipemia. The third part focused on storage and stability and the handling of samples from primary care or other hospital laboratories. Results: 57 of 69 laboratories responded. There was good agreement regarding needle gauge, temperature in the centrifuge and type of glass used for sample collection (3.2% Na-citrate), all more or less following the CLSI guidelines (H21-A5). However, large differences in practice were seen amongst the participants regarding centrifugation speed and duration, accepted fill volumes of the collection tubes accepted and accepted sample material (fresh or frozen plasma or citrated blood) and stability of the blood samples. In addition, there were few routines for detection of clot, pathological hematocrit, hemolysis, bilirubinemia and lipemia.
are poorly described. This study aimed to characterize and develop an algorithm to manage the donors with polycythaemia. Matrials and methods: Between November 2009 and November 2011 blood donors with repeated B-Hb above the WHO limit for polycythaemia vera (10.2 and 11.5 mmol/L/ 16.5 and 18.5 g/dL for women and men, respectively) were offered consultations by a hospital-based haematologist. At consultation haematocrit, MCV, P-erythropoietin, P-ferritin, B-Hb, platelet- and leukocyte count, JAK2 V617 and JAK2 exon12 analysis were performed, in addition to other routine parameters. Results: Among 46 donors with repeated high B-Hb, 39 had a history of smoking, which may contribute to polyglobulia. Two had PV, 5 had severe hypertension, one of them because of kidney artery stenosis, and two had diabetes mellitus. Ten donors were deferred and of the 36 donors that were not deferred, 30 donated again before May 2012, where the B-Hb was significantly lower.
Conclusions: Wide variation is seen in preanalytical routines in hemostasis testing, often not according to the CLSI guideline.
Conclusion: Thus, we found a high morbidity among these donors, and recommend JAK2 V617 and JAK2 exon12 screening and clinical investigations for donors with concurrent high B-Hb, high haematocrit and iron deficiency. Also we recommend these donors to reduce smoking to reduce the risk of thrombosis in general.
P10-10
P10-11
Characterization of blood donors with high haemoglobin concentration Magnussen K (1), Hasselbalch H (2), Ullum H (3), Weis Bjerrum O (2) (1) Copenhagen University Hospital, Rigshospitalet / Hvidovre Hospital, Clinical Immunology / Blood Centre, Copenhagen, Denmark (2) Copenhagen University Hospital, Roskilde Hospital, Haematology, Copenhagen, Denmark (3) Copenhagen University Hospital, Rigshospitalet, Clinical Immunology / Blood Centre, Copenhagen, Denmark Corresponding author: [emailprotected]
Background: The prevalence and causes of high haemoglobin concentration among blood donors
Blood donors with thalassemia trait in a blood bank outside the thalassemia belt Magnussen K Copenhagen University Hospital, Rigshospitalet / Hvidovre Hospital, Clinical Immunology / Blood Centre, Copenhagen, Denmark Corresponding author: [emailprotected]
Background: By far the most frequent reason for Danish blood donors to have a haemoglobin level below the level of acceptance for blood donation is iron deficiency. Hence in all low haemoglobinBiochemia Medica 2012;22(3):A54-A204
A125
2nd European Joint Congress of EFLM and UEMS
donors ferritin is measured. If haemoglobin remains low by the next visit or if the low haemoglobin cannot be explained by low ferritin, further tests are done. Five donors with mean cell volume (MCV) ≤ 78 fL, not explained by iron deficiency are described. Materials and methods: Samples from donors with unexplained microcytaemia, were sent to Centre for Haemoglobinopathies, where Hb-electrophoresis and nucleic acid investigations were done. Summary: With increasing globalisation donors heterozygous for thalassemia may turn up even in Nordic blood banks. Their haemoglobin level will be in the low normal area, often below the lower limit for donation, except for the α3,7 deletion where the haemoglobin level often is normal. Heterozygous thalassemia is rarely of clinical consequence, except in pregnancy where genetic counselling may be warranted. Provided that the haemoglobin level is above the limit for acceptance, people with thalassemia trait may become blood donors.
P10-12 Temporary impact of blood donation on physical performance in moderately physically active men Magnussen K (1), Ziegler A (2), Grand J (2), Helge J (2) (1) Copenhagen University Hospital, Rigshospitalet / Hvidovre Hospital, Clinical Immunology / Blood Centre, Copenhagen, Denmark (2) University of Copenhagen, Faculty of Health Sciences, Copenhagen, Denmark Corresponding author: [emailprotected]
Background: Donation of blood negatively affects maximal oxygen consumption (VO2max) and endurance, and donors ask about the duration and degree of reduction. Materials and methods: Nineteen non-anaemic, physically active male blood donors age 33(24–43) Biochemia Medica 2012;22(3):A54-A204
A126
were included. To determine VO2max, subjects performed a standard incremental bicycle ergometer VO2Max test. Endurance was tested using a self-paced 3 km treadmill run. Subjects were tested 2 days before and 3, 7, 14, and 28 days after donation and were drawn to analyse haematological - and iron-parameters. Results: Haemoglobin was reduced by 9.3% at day 3. After this Hb increased, and by day 28, three of 15 donors had reached pre-donation Hb level. Ferritin declined 51% from 94.8 ± 18.4 µg/L before donation to 46.8 ± 10.4 µg/L at day 14 and remained below baseline throughout the study. VO2max declined by 6.5% from 49.7 ± 2.1 mL O2 / kg/min to 45.6 ± 2.1 mL O2 /kg/min at day 3 compared with before donation. Subsequently VO2max gradually increased to 49.3 ± 2.2 mL O2 / kg/min, at day 14, thereby returning to baseline. The 3 km time trial performance declined by 5.6% from 13:55 ± 00:46 minutes before donation to 14:42 ± 01:12 minutes at day 3. At day 14 time trial performance was 13:52 ± 00:59 minutes, the same as before donation. Summary: Haemoglobin concentration was reduced by 9.3% and ferritin by 51%. Endurance was reduced by 5.6% and VO2max by 6.1% and reached pre-donation level by day 14, It seems from our study that plasma volume expansion may partly compensate for the loss in haemoglobin.
2nd European Joint Congress of EFLM and UEMS
P11 – Immunology P11-01 Comparative analysis of turbidimetry and nephelometry methods for the measurement of immunoglobulin Cachapuz I (1), Moreira S (2), Neves E (3), Lima I (3), Cerveira C (3), Alves V (1) (1) Matosinhos Local Health Unit, Clinical Pathology Service, Oporto, Portugal (2) ESTSP, Oporto, Portugal (3) Centro Hospitalar do Porto, Clinical Immunology Service, Oporto, Portugal
CLL and lGG, with p values 0.065 (CLK), 0.570 (CLL), 0.004 (IGG). Significance level was 0.05. Conclusions: Our results suggest that the performance of both methods is similar for the three parameters under study. Therefore, turbidimetry, the method most commonly used in automated large scale laboratory routine models, like CORElabs, is suitable for this propose.
P11-02 Comparison of rheumatoid factor analysis by nephelometry (BN II) and turbidimetry (ADVIA 2400)
Corresponding author: [emailprotected]
Background: The analysis of the protein content in urine samples is often required in the clinical practice. Nephelometry and turbidimetry are alternative methods, frequently used in the laboratory routine, and its choice depends on various factors including the cost, availability and characteristics of the laboratory equipments. A discussion is still ongoing, about the most suitable method, considering its sensibility and specificity, for the measurement of proteins in very low concentration levels, like monoclonal components in urine samples. The aim of this study is to compare the results of Immunoglobulin G (IGG), kappa (CLK) and lambda (CLLl) light chains concentration in urine samples, obtained by turbidimetry and nephelometry. Materials and methods: Experimental and retrospective study of 24-hour urine random samples, from 51 patients of Pedro Hispano Hospital, collected for monoclonal protein excretion screening. IGG, CLK and CLL analysis were performed by turbidimetry (Abbott Diagnostics, Architect C 8000®) and by nephelometry (Beckman Coulter IMMAGE® equipment for IGG and Siemens Dade Behring BN II equipment for CLK and CLL). Statistic analysis was performed with SPSS (vs 18) software. Results: Correlation values between results obtained by turbidimetric and nephelometric methods were 93,5%, 99,0%, 99,0% respectively for CLK,
Garcia Moreira V, Fernandez Leivas A, Bachiller Sister M, Llorente Torres A, Fernandez Rodriguez E Hospital de Cabueñes, Servicio de Analisis Clinicos, Gijon, Spain Corresponding author: [emailprotected]
Background: Detection of rheumatoid factor (RF) is a useful method in the differential diagnosis of rheumatic diseases, primarily used to help diagnose rheumatoid arthritis or Sjögren syndrome. Changes in serum RF levels may be used as indicators of disease activity, but they are also proving useful in monitoring responses to therapy. Materials and methods: Levels of FR were analyzed in 75 serum samples in parallel by our usual immunonephelometric method in a BNII analyzer (Siemens) and a new immunoturbidimetric method in Advia2400 (Siemens). Data were statistically analyzed using the MedCalc statistical package by agreement kappa statistic, Spearman correlation and regression of Passing-Bablok. To categorize the results were considered negative values RF < 14 IU/mL in Advia2400 method and < 15.9 IU/mL in the nephelometric assay, and other results were considered positive. Results: The observed agreement was 91% (expected by chance: 55%), yielding a kappa index of 0.795 (95%CI: 0.624-0.966). We calculated Spearman correlation for positive samples (N = 35) beBiochemia Medica 2012;22(3):A54-A204
A127
2nd European Joint Congress of EFLM and UEMS
tween the ratios (value/upper reference limit) of both methods: 0.810 (0.573-0.922) (P < 0.01). The Passing-Bablok regression equation was: Advia2400 = 3.56 + 1.09 x BnII (95% CI slope: 0.7941.55; 95% CI intercept:-9.26-9.47 ), so no bias was evident. Conclusion: According to the results, the strength of categorical agreement between both methods can be considered "strong” (Ladis-Koch criterion). That is, the results are qualitatively comparable. However, the results are not quantitatively interchangeable. The turbidimetric RF Advia2400 assay is a reasonable alternative to the nephelometric, as it shows a good correlation, besides being cheaper and faster.
P11-03 Evaluation of C3 and C4 immunoturbidimetric assays on ABBOTT Architect ci16200 analyzer Knezevic B, Mladina B, Čepić K, Surjan L University Hospital Split, Department of Laboratory Diagnostic, Split, Croatia
reference-change values (RCV) at two control levels were calculated. Results and conclusions: Results obtained for within run imprecision: C3 (L1 0.8%, L2 0.8%), C4 (L1 0.8%, L2 0.8%), between run imprecision: C3 (L1 1.9%, L2 1.2%), C4 (L1 2.1%, L2 3.9%) and inaccuracy: C3 (L1 3.7%, L2 4.08%), C4 (L1 2.2%, L2 6.6 %), satisfied desirable specifications for I%, B% and TE% derived from biological variation. Regression equations with 95% CI obtained from Passing-Bablok analysis were as followed: C3 y = - 0.21 (-0.24(-0.17)) + 1.7 (1.13 – 1.20)x, C4 y = 0.00 + 1.10 (1.131.20)x. Bland-Altman plot showed C3 values on average 2.2% lower (limits of agreement -9.7-5.2%) and C4 on average 9.6% higher (limits of agreement 5.1-14.1%) on Architect ci16200. Although Passing-Bablok regression showed statistically significant difference between methods, Bland-Altman plot showed differences clinically insignificant if compared to RCV values.
P11-04 Comparison of serum total IgE tests performed by four different assay systems Yoon S (1), Kim S (2)
Corresponding author: [emailprotected]
Background: Our laboratory wished to consolidate C3 and C4 testing by converting from immunonephelometry to immunoturbidimetry. We evaluated analytical performance of Abbotts C3 and C4 immnoturbidimetric assays on Abbott Architect ci16200 chemistry/immunochemistry analyzer. Materials and methods: Analytical evaluation included determination of within - run (N = 20), between - run (N = 20) imprecision, inaccuracy (N = 20) using control materials and method comparison on human samples (N = 90) comparing Architect ci16200 and Siemens BNProSpec immunonephelometer. For the sample comparison PassingBablok and Bland-Altman analyses were performed. For the evaluation of clinical significance, Biochemia Medica 2012;22(3):A54-A204
A128
(1) U2Bio, Department of Laboratory Medicine, Seoul, Korea, (South) Republic of (2) Yonsei University College of Medicine, Department of Laboratory Medicine, Seoul, Korea, (South) Republic of Corresponding author: [emailprotected]
Background: In vitro testing is commonly used to diagnose allergies and predict allergic tendency especially in pediatrics. Intermethod comparisons between four different commercial tests for serum total IgE had only been evaluated by limited studies. To determine whether IgE levels derived from different assays are precise and equivalent to those measured by ImmunoCAP. Materials and methods: Precision was determined following Clinical and Laboratory Standards Institute EP5-A2 using patients’ pooled se-
2nd European Joint Congress of EFLM and UEMS
rum. Two levels materials were analyzed in duplicates at two separate times per day for 20 days in each four systems. We performed interassay comparisons using 132 Korean patients who visited one tertiary care hospital in Seoul. For each deidentified sample, total IgE levels were measured using four different assay systems(bioMérieux VIDAS, Siemens Immulite2000, Roche Cobas e 601, Phadia ImmunoCAP). Results were analyzed to determine whether IgE measurements were comparable. Results: Four clinically used total serum IgE assays showed excellent precision performance, with coefficients of variation (CVs) below 9%. After 132 paired comparison test, Immulite2000 (y = 1.02x 1.0, R² = 0.9806) and Cobas e 601 (y = 1.14x+-0.94, R² = 0.9913) showed good correlation with ImmunoCAP assay. Although some minor outliers were noted. VIDAS (y = -4.92x + 0.91, R² = 0.9423) showed significant deviation from linearity with ImmunoCAP by Passing – Bablok analysis. Conclusions: The Immulite2000 and Cobas e601 total IgE were showed reliable performance and comparable result with ImmunoCAP assay. VIDAS showed underestimated trend in total IgE values. We should take into account the intermethod differences between those assays for clinical applications.
P11-05 Imunonephelometric quantification of monoclonal protein
produced in sheep. Since the target of this antibodies are unique junctional epitopes between the heavy chain and light chain constant regions, antibodies can separately identify the different light chain types of each Ig class (IgG kappa, IgG lambda). Aim of the study was evaluation of new test for monoclonal protein (M protein) quantification. Materials and methods: Study included 20 samples with already detected M protein. In all samples were measured total imunoglobulin A, G and M (Cobas 6000, Roche) and done capillary zone electophoresis (Capillarys2, Sebia) and immunofixation electrophoresis (Hydrasys, Sebia). After detection, M protein is quantified with The Binding Site test Hevylite on nephelometer Siemens BNII. Results and conclusions: Monoclonal protein were detected even when immunoglobulins were in reference ranges. When detected M protein is IgA class, densitometric determinated gamma globulins can not be usefull in quantification of M protein. Numerical results of total immunoglobulin concentrations and M protein are not comparable. Ratio kappa/lambda may be a good indicator of clonality. Unquestionable is importance of capillary zone electroforesis in identifying monoclonal gammapathies as simple and inexpensive routine technique in clinical laboratory. But, sometimes when the M protein (expecially IgA) migrates in the beta reagion, with either transfferin or C3, only quantification of the M protein will provide adequate follow-up. Considering the price of reagent the test probably will not find use in screening for monoclonal protein but will be usefull in monitoring the disease course.
Šegulja D (1), Matišić D (1), Batinić J (2), Nemet D (2) (1) University Hospital Centre Zagreb, Department of Laboratory Diagnostics, Zagreb, Croatia (2) University Hospital Centre Zagreb, Department of Hematology, Zagreb, Croatia Corresponding author: [emailprotected]
Introduction: Hevylite is a new test for imunonephelometric quantification of monoclonal protein. The reagent consists of polyclonal antibodies Biochemia Medica 2012;22(3):A54-A204
A129
2nd European Joint Congress of EFLM and UEMS
P11-07
P11-08
YKL-40 correlates with pro-inflammatory cytokines in rheumatoid arthritis
Epstein-Barr virus infection resembling autoimmune liver disease in 17-year-old girl – case report
Kazakova M Medical University - Plovdiv, Medical Biology, Plovdiv, Bulgaria Corresponding author: [emailprotected]
Background: The aetiology of rheumatoid arthritis remains unknown, although it is estimated that autoimmune mechanisms play a major role in the pathogenesis of the disease. YKL-40 is a candidate for a novel inflammatory marker. It is secreted by activated macrophages and neutrophils, synovial cells, arthritic chondrocytes and cancer cells. In this study we evaluated YKL-40 levels in serum and synovial fluid of patients with rheumatoid arthritis in comparison with the concentration of pro-inflammatory cytokines. Materials and methods: We examined serum and synovial YKL-40, IL-1β and TNF-α levels in 37 rheumatoid arthritis patients, aged 53.14 ± 2.73. The concentrations of these markers were measured by ELISA. Results and conclusions: The levels of YKL-40, IL1β and TNF-α in patients were remarkably higher compared to the healthy group (P < 0.01). A significant correlation between serum and synovial YKL40 levels and concentrations of IL-1β and TNF-α in patients with rheumatoid arthritis was observed (P < 0.01). These pro-inflammatory cytokines are involved in the pathogenesis of rheumatoid arthritis and are targets for the therapeutic treatment. It is shown that IL-1β and TNF-α could induce secretion of YKL-40 by chondrocytes. We determined a strong correlation between serum YKL-40 concentration and the conventional biochemical marker for assessment of disease activity - C-reactive protein (CRP) (P = 0.004; r = 0.582). Our data suggest potential involvement of YKL-40 in inflammation and disease activity of rheumatoid arthritis. Acknowledgments The study is supported by grants NO–1/2009 and NO–1/2010 from Medical University– Plovdiv. Biochemia Medica 2012;22(3):A54-A204
A130
Tesija Kuna A (1), Zaja-Franulovic O (2), Vukasovic I (1), Lesar T (2), Vrkic N (1) (1) Medical School University Hospital Sestre Milosrdnice, Clinical Institute of Chemistry, Zagreb, Croatia (2) Medical School University Hospital Sestre Milosrdnice, Department of Pediatric Gastroenterology and Hepatology, Zagreb, Croatia Corresponding author: [emailprotected]
We report a case of a 17-year-old girl admitted to Department of Pediatric Gastroenterology and Hepatology for chronic intermittent diarrhea with significant weight loss. Biochemical liver lesion confirmed with imaging techniques was found characterized with cholangitic pattern. High value of fecal calprotectin and hypoalbuminemia suggested inflammatory bowel disease with liver lesion as an extraintestinal manifestation. Autoantibody profile corresponding to autoimmune hepatitis type I was found including positive antinuclear (ANA) and smooth muscle (SMA) antibodies. Antimitochondrial antibodies (AMA) were negative on immunofluorescence test but high reactivity was observed using line immunoassay with both native AMA-M2 antigen and recombinant fusion protein AMA-M2-3E. Infective serology on admission revealed recent primary Epstein-Barr virus (EBV) infection with viral capside antigen (VCA) IgM and IgG positive, EBV early antigen (EA) IgG negative and EBV nuclear antigen (EBNA) IgG positive. Primary suspicion of ulcerative colitis was rejected regarding the normal endoscopic and histopathological findings of ileocolonic mucosa and spontaneous resolution of clinical symptoms, biochemical and ultrasonographic abnormalities. Repeat testing after 3 months revealed persistently positive ANA and SMA but absence of reactivity with AMA antigens on line immunoassay. Serologic tests documented seroconversion further supporting the diagnosis of EBV primoinfection. Repeated PCR analysis was negative for EBV DNA.
2nd European Joint Congress of EFLM and UEMS
EBV infection as trigger for autoimmune disease has been attributed to occurrence of cross-reactive antibodies, due to the mimicry of epitopes between host and EBV proteins. We presented an example of EBV primoinfection resembling autoimmune liver disease although follow up is suggested due to the persistently positive ANA and SMA.
P11-09 Concentration of IgE antibody in nasal lavage in allergic rhinitis and non-allergic rhinitis Bokulić A (1), Bukovec Megla Ž (1), Kalogjera L (2), Tomljenović D (2), Vagić D (2), Zurak K (2) (1) University Hospital Centre “Sestre milosrdnice”, Laboratory of Endocrinology, Department of Oncology and Nuclear Medicine, Zagreb, Croatia (2) University Hospital Centre “Sestre milosrdnice”, Department of Otorhinolaryngology and Head and Neck Surgery, Zagreb, Croatia Corresponding author: [emailprotected]
Background: Molecular and cellular inflammation mechanisms in nasal mucosa in allergic rhinitis (AR) and non-allergic rhinitis (NAR) have not yet been clarified. To contribute to their understanding and developing of differential diagnostics, concentration of total IgE was measured in nasal lavage in subjects with allergic rhinitis and non-allergic rhinitis. The objective of this study was to measure concentration of IgE antibody in nasal lavage and determine possible correlations. Materials and methods: Study included 60 patients of both sexes aged 18-65. Patients were divided in two groups, according to diagnosis of allergic or non-allergic rhinitis (N = 30). Nasal lavage samples were taken by modified Nacleir method. Nasal cavity was flushed with 3 mL of saline solution (0.9% NaCl). The concentration of total IgE was measured by fluoro enzyme immunoassay (FEIA) on UniCAP 100 (Phadia AB, Uppsala, Sweden). The results were evaluated by non-paramet-
ric Mann-Whitney U-test. The values P < 0.05 were considered statistically significant. Results: Results were expressed as median and interquartile range (Q1 and Q3). The concentration of total IgE was higher in patients with allergic rhinitis than in patients with non-allergic rhinitis: 40.60 kU/L (23.78-54.43) vs. 2.55 kU/L (2.05-5.80), respectively; P < 0.001. Conclusions: The results showed significantly higher concentration of IgE antibody in nasal mucosa in patients with allergic rhinitis than in patients with non-allergic rhinitis. Local IgE-mediated inflammation plays important role in allergic rhinitis and measuring the concentration of local IgE antibodies in nasal lavage may contribute to diagnostics of allergic disease.
P11-10 Immunologic laboratory diagnostic tests for patients with chronic diarrhea Mladenova T (1), Shentova R (2), Kyurkchiev D (1), Ivanova-Todorova E (1), Spassova Z (3), Altankova I (2) (1) UHAT St. Ivan Rilski, Laboratory of Clinical Immunology, Sofia, Bulgaria (2) UHATC Sofia, Pediatric Gastroenterology, Sofia, Bulgaria (3) UHAT St. Ivan Rilski, Clinic of Gastroenterology, Sofia, Bulgaria Corresponding author: [emailprotected]
Background: Patients with chronic diarrhea are difficult to diagnose and treat. Along with the conventional clinical and instrumental tools for elucidating the diagnosis there are contemporary immunologic examination for immunomediated intestinal diseases such as celiac disease (CeD), Crohn`s disease (CD) and ulcerative colitis (UC). The aim of the study was to evaluate clinical significance of antibodies against deamidated gliadin peptide (anti-DGP), antibodies against Saccharomyces cerevisiae (ASCA), fecal calprotectin (FC) and fecal lactoferrin (FL) as diagnostic tools for establishing the diagnosis of CeD, CD and UC. Biochemia Medica 2012;22(3):A54-A204
A131
2nd European Joint Congress of EFLM and UEMS
Materials and methods: We examined 137 patients – 37 with CD, 58 with CeD, 42 with UC. Sera and stool samples were tested. We also tested 25 healthy persons as control groups. The sera samples were evaluated with ELISA for anti-DGP antibodies (IgG) and ASCA (IgA), and the stool samples for FC and FL with a rapid Card test, lateral flow assay with Quantum Blue reader and ELISA for FC. Results: Anti-DGP antibodies were positive in 100% of patients with CeD and in no one of UC and CD. ASCA were positive in 14,8% of CD and 25,9% of UC patients. FC was positive in 83% of UC, 87% of CD and 37.5% of CeD patients. FL was positive in 52% of UC and 54% of CD patients. Conclusion: We found that these new immunologic markers are promising in diagnosis: anti-DGP is highly specific for CeD, ASCA are not very sensitive and discriminative for CD and UC. FL and FC are typical for active neutrophil inflammation.
Results: Patient 1: A MP Lambda was detected in serum (13.2 g/L). Bence-Jones protein was identified and measured in urine (300 mg/24 hour). Serum κ and λ FLC were measured with a concentration of 10 mg/L and 18500 mg/L, respectively. Patient 2: Serum MP was detected. Immunofixation showed two different MPs, κ FLC and IgG κ. The concentration was 2.6 g/L and 9 g/L, respectively. No Bence-Jones proteinuria was detected. The concentration of κ and λ FLC was 9060 mg/L and 7 mg/L, respectively. Both patients had a normal renal function. Conclusions: The presence of serum MP of FLC with absence or low levels of urinary BJP suggests a polymeric form of the MP with a large molecular size that prevents normal excretion by kidney. The difference between serum MP and FLC could be due to overestimation in the nephelometric measurement by the presence of polymeric forms of FLC that may have produced a more intense immunoprecipitation reaction.
P11-11 Polymeric forms of free light chains: two case reports Illana Camara F, Castillo Perez C, Cardenas M, Arroyo M
P11-12 Eosinophilic cationic protein (ECP) and total IgE concentration in children with atopic diseases
Hospital Clínico San Carlos, Laboratory Medicine, Madrid, Spain
Pancirov D (1), Ciprić M (1), Zukan I (2), Posavec M (1)
Corresponding author: [emailprotected]
(1) "Dr. Ivo Pedišić" General Hospital, Department of Biochemistry and Hematology Diagnostics, Sisak, Croatia (2) "Dr. Ivo Pedišić" General Hospital, Department of Pediatrics, Sisak, Croatia
Background: Urinary Bence-Jones protein (BJP) refers to urinary excretion of monoclonal light chains. Light chains appear in the urine when the metabolizing capacity of nephron is exceeded. Negligible urinary BJP in light chain multiple myeloma patients due to polymeric forms have been described previously. Materials and methods: Serum and 24-hour urine of two patients with multiple myeloma were studied. Capillary electrophoresis (Capillarys®) was used to detect and measure monoclonal protein (MP). Serum free light chains (FLC) were measured by immunonephelometry in an Immage 800 (Beckman Coulter®) analyzer. MP was identified by Immunofixation. Biochemia Medica 2012;22(3):A54-A204
A132
Corresponding author: [emailprotected]
Background: Eosinophilic cationic protein (ECP) is a basic protein located in the granules of eosinophil granulocytes and released during eosinophil activation. ECP serum levels are associated with the intensity of allergic inflammation. Elevated total IgE concentration is common to all atopic diseases. The aim of the study was to compare ECP and total IgE concentration in sera of patients with asthma, allergic rhinitis and atopic dermatitis, their values with values obtained from healthy children
2nd European Joint Congress of EFLM and UEMS
and to determine the relationship between these analytes in the mentioned groups of patients. Materials and methods:The study included 43 children with asthma, 20 with allergic rhinitis, 20 with atopic dermatitis and 30 healthy children as controls. ECP and total IgE serum values were determined using an automated fluorescence enzyme immunoassay (FEIA) on an UniCAP R 100 immunoanalyzer. Results: Although the ECP concentration median was highest in the group of asthmatic patients (15.3 μg/L) and lowest in the group of atopic dermatitis patients (10.4 μg/L), there was no significant difference among the groups of patients, while total IgE concentration was higher in the groups of patients with asthma and allergic rhinitis (P < 0.001). ECP and total IgE concentrations were significantly higher in patients with asthma (P < 0.001 for both) and allergic rhinitis (P = 0.018; P < 0.001) compared to controls. Weak positive correlation between these analytes was found in asthmatic patients (r = 0.478, P = 0.001). Conclusion: The results indicate that ECP values can be used as a marker of inflammation in asthmatic patients and those with allergic rhinitis. ECP and total IgE concentrations were weakly correlated only in asthmatic patients.
P12 – Kidney diseases P12-01 The performance of compensated serum creatinine in pediatric samples
pediatric population but high commercial price is limiting factor for its implementation in routine practice. We estimated proportion of compensated serum creatinine concentration below measuring range in children; following to method comparison analysis of compensate Jaffe vs. enzymatic creatinine method. Materials and methods: A total of 58 pediatric serum samples were included in the study (median age 44 months, age range 2 days-18 years). The measurement of creatinine by enzymatic method (measuring range 0-2700 μmol/L) was done on the Cobas c311 analyzer (Roche Diagnostic) and by compensated Jaffe method (measuring range 182200 μmol/L) on the Olympus AU400 analyzer (Beckman Coulter). Results: In 36 out of 58 samples (group 1, median age 66 months, age range 2 days-18 years) concentrations of compensated creatinine were in measuring range (median 34 μmol/L, range 18-195 μmol/L) but it was not a case in 22 out of 58 samples (group 2, median age 12 months, age range 15 days-6 years) with compensated creatinine concentrations below measuring range. There was a significant difference between two groups regarding age (t-test, P < 0.001). The Passing and Bablok regression analysis showed (N = 36) intercept -4.42 (95%CI 6.03 to -3.00), slope 1.02 (95%CI 1.00 to 1.06); r = 0.99; range tested 18-195 μmol/L. Conclusion: A high proportion of creatinine concentrations under measuring range was unacceptable, concerning younger children. Method comparison analysis revealed underestimation of pediatric serum creatinine by compensated Jaffe method.
Aralica M, Matica J Rijeka University Hospital Center, Clinical Institute of Laboratory Diagnostis, Rijeka, Croatia Corresponding author: [emailprotected]
Background: According to current recommendation serum creatinine measurement in adults should be performed by compensated Jaffe method. The enzymatic method is recommended for Biochemia Medica 2012;22(3):A54-A204
A133
2nd European Joint Congress of EFLM and UEMS
P12-02 The impact of different methods of creatinine measurements on MELD scoring system Perkov S (1), Flegar-Meštrić Z (1), Ožvald I (1), Kocman B (2), Čolić-Cvrlje V (3), Čakalo Lj (3)
in serum creatinine measurement may not significantly alter prognosis or have clinical consequences but may affect prioritization for liver transplantation. For this reason standardization of creatinine measurements using specific enzymatic method traceable to SRM 967 is of most importance.
(1) University Hospital Merkur, Institute of Clinical Chemistry and Laboratory Medicine, Zagreb, Croatia (2) University Hospital Merkur, Department of Abdominal Surgery and Organ Transplantation, Department of Surgery, Zagreb, Croatia (3) University Hospital Merkur, Department of Internal Medicine, Zagreb, Croatia
P12-03
Corresponding author: [emailprotected]
(1) Matosinhos Local Health Unit, Clinical Pathology Service, Oporto, Portugal, Portugal (2) Matosinhos Local Health Unit, Nephrology Service, Oporto, Portugal
Background: The Model for End-Stage Liver Disease (MELD) scoring system is used to prioritize patients for liver transplantation by their disease severity based on three laboratory parameters: serum creatinine and bilirubin concentrations and INR prothrombin ratio. The aim of this study was to investigate impact of two different creatinine methods on MELD score. Materials and methods: We assessed 40 blood samples obtained from 32 patients listed for liver transplantation. Serum creatinine concentrations was measured by the kinetic Jaffe method (Beckman Coulter OSR6178) traceable to NIST SRM 909b level 2 and enzymatic method (Beckman Coulter OSR61204) traceable to the IDMS method and SRM 967. The MELD score was calculated according to the formula currently in use by Eurotransplant. Patients are stratified in a descening order starting with the highest MELD. Results: There was a significant difference in serum creatinine among Jaffe and enzymatic methods: median 93 (range 67-744) vs. 73,5 (range 49747) μmol/L, respectively. The variation in creatinine measurements resulted in differences up to 2 points in a single patients. When the enzymatic methods was used instead of Jaffe methods, MELD scores were unchanged in 23 cases wheras in 17 cases MELD scores decreased by 1-2 point. Conclusion: Observed variability in the assessment of the MELD score due to methodological variation Biochemia Medica 2012;22(3):A54-A204
A134
“Screening” for chronic renal disease in a Portuguese population Cachapuz I (1), Branco A (2), Silva S (2), Alves V (1), Costa M (2)
Corresponding author: [emailprotected]
Background: According to the “Chronic Renal Disease (CRD) national registration” in terms of renal replacement therapy, the incidence of patients on dialysis has been increasing in Portugal, in the last decade. However, there is no precise information about prevalence of CRD at national level. We have conducted a study to assess the extent of the problem in the population of Matosinhos Local Healthcare Unit (ULSM). Materials and methods: Evaluation of glomerular filtration rate (GFR) with MDRD-4 formula, in ULSM population with scheduled medical consultation between Feb/2010 and Feb 2011. Patients from hospitalization and emergency were excluded and acute renal failure cases were avoided. Patients with at least two 60 mL/h/m2 measuring of GFR in a 3 months minimum interval. Results: Evaluation of renal function of 44869 patients (29 were excluded due to incomplete data), average: 54 years (minimum 12; maximum 99); M: 41.9% / F: 58.1%; 3949 had GFR < 60 in at least one measuring. 1547 patients met the CRD criteria, which corresponds to 3.4% prevalence. From those 74.6% are in stage 3 (prevalence = 2.57%); 20.6% in stage 4 (prevalence = 0.71%) and 4.6% in stage 5 (prevalence = 0.16%). 38.4% of the CRD population are diabetics.
2nd European Joint Congress of EFLM and UEMS
The global prevalence of CRD seems slightly inferior to the described in European studies. Diabetes mellitus is still a major risk factor for these individuals. Conclusions: The introduction of the analytical protocol for calculating GFR proved to be a good CRD screening method, like in most European countries. It helps prevent CRD and reduce the cardiovascular risk.
P12-04 Osteoporosis and kidney impairment in postmenopausal women Pavić M (1), Milevoj Kopčinović L (1), Car D (2), Nikolić T (3) (1) Medical School University Hospital Sestre Milosrdnice, Zagreb, Croatia, Clinical Unit of Medical Biochemistry in Traumatology and Orthopedics, University Department of Chemistry, Zagreb, Croatia (2) Medical School University Hospital Sestre Milosrdnice, Zagreb, Croatia, Clinical Unit of Internal Medicine, University Hospital of Traumatology, Zagreb, Croatia (3) Medical School University Hospital Sestre Milosrdnice, Zagreb, Croatia, Trauma Rehabilitation Unit, University Department of Rheumatology, Physical Medicine and Rehabilitation, Zagreb, Croatia
mal/osteopenia (N = 143; 75%) and osteoporosis (N = 47; 25%). Differences were found comparing age, weight, urine creatinine and eGFR with the CG. The prevalence of kidney impairment (GFR < 60 mL/ min/1.73 m2) according to CrCl and CG in normal/ osteopenic women was 17% and 19%, respectively; and in the osteoporosis group 34% and 36%, respectively. The prevalence was higher in the osteoporosis group (P = 0.029 for CrCl; P = 0.025 for CG). Weighted kappa between CrCl and CG was 0.500, 0.483 and 0.487 in the overall, normal/osteopenia and osteoporosis subgroup, respectively. Conclusions: Our results showed substantial prevalence of kidney impairment in postmenopausal women with osteoporosis. This must be taken into account when considering the prescription of medications with kidney clearance (like bisphosphonates). The kappa statistics classified the agreement between CrCl and CG as moderate.
P12-05 Iron content of serum ferritin: a biomarker of iron storage
Corresponding author: [emailprotected]
Mujagic R (1), Obuljen J (2), Crnokrak S (1)
Background: Both osteoporosis and kidney impairment become more prevalent with age. The aim of this study was to investigate the prevalence of compromised kidney function in women with osteopenia and osteoporosis, and to assess the agreement between GFR estimated with creatinine clearance (CrCl) and the Cockcroft-Gault equation (CG).
(1) General Hospital Pula, Clinical laboratory, Pula, Croatia (2) Children's Hospital Zagreb, Biochemical-Hematology Laboratory, Zagreb, Croatia Corresponding author: [emailprotected]
Materials and methods: We studied postmenopausal women, divided according to T-scores into two groups: normal/osteopenic and osteoporotic. They were further divided, using CrCl and CG, into groups depending on chronic kidney disease. Serum calcium, phosphorus and creatinine, 24hour-urine calcium and creatinine were determined. Estimated GFRs were calculated using the CrCl and CG. Informations about age, height, weight were collected.
Introduction: Ferritin is an iron-storage protein. The serum ferritin concentration reflects the amount of iron in the human body. Serum ferritin concentration is non-specifically elevated in the presence of infection, inflammation, liver disease and malignancies. Iron content of serum ferritin (ICF) isn’t influenced by inflammation and ICF represents an iron status biomarker regardless of inflammation. Thereafter, ICF can be confidently used to assess a functional iron deficiency in patients undergoing hemodialysis.
Results: 190 postmenopausal women (median age 67 (45-88) years) were divided into subgroups: nor-
Materials and methods: Fifty-five hemodialysed patients that had been treated with Ferrlecit and/ Biochemia Medica 2012;22(3):A54-A204
A135
2nd European Joint Congress of EFLM and UEMS
or erythropoietin past three months were included in this clinical trial. Ferritin, transferrin, serum non-ferritin iron and immaturity reticulocytes fraction index (IRF) were measured by standard methods. Total iron concentration was measured by Inductively Coupled Plasma-Mass Spectrometry (ICP-MS). ICF was calculated from total iron, nonferritin iron and ferritin concentration. Results: There were significant positive association between logarithm-transformed ICF with IRF (rP = 0.377; P = 0.005) and transferrin (rP = 0.555; P < 0.001). ICF median (14.1 µmol/mg) was below reference range. Conclusion: Transferrin concentration firstly depends on nutritional status, and ferritin concentration firstly depends on inflammation rather than iron status. Concussively, ferritin and/or transferrin don’t represent confidential biochemical predictors of iron deficiency in hemodialysed patients. Regardless of inflammation, ICF can confidently predict functional iron availability for erythropoiesis. ICF represents clinically relevant biomarker which reflects the true iron needs and can be safely used to optimize iron supplementation for correcting iron deficiency in patients undergoing hemodialysis.
P12-06 Nitric oxide and homocysteine in patients with chronic kidney disease Mikulić I (1), Petrik J (2), Čepelak I (2), Tomić M (3), Baković V (3), Mikulić V (1) (1) Mostar University Hospital, Department of Laboratory Diagnosis, Široki Brijeg, Bosnia and Herzegovina (2) Faculty of Pharmacy and Biochemistry, Department of Medical Biochemistry and Hematology, Zagreb, Croatia (3) Mostar University Hospital, Department of Nephrology and Hemodialysis, Clinic for Internal Diseases, Mostar, Bosnia and Herzegovina Corresponding author: [emailprotected]
Background: Nitric oxide (NO) and homocycteine have an important role in the complex pathogenesis of chronic kidney disease (CKD). Biochemia Medica 2012;22(3):A54-A204
A136
Material and methods: The levels of NO (spectrophotometric assay, Griess reaction) and homocysteine (chemiluminescence microparticle immunoassay) were determined in plasma/serum and/or urine of patients with CKD (N = 101) and of the control subjects (N = 51). The total number of patients with CKD was divided into subgroups according to: a) the primary cause of impaired renal function, b) the National Kidney Foundation guidelines, and c) the intensity of proteinuria. Results: The results show that serum concentrations of NO (median 9.20 μmol/L), and homocysteine in plasma (median 16.95 μmol/L) in patients with CKD was significantly higher (P = 0.009, P < 0.001) compared with the control group (median 7.27 μmol/L and 13.04 μmol/L, respectively). The concentration of homocysteine in plasma showed a relatively good diagnostic sensitivity (60.0 to 89.5%) and diagnostic specificity (63.4 to 90.2%) in distinguishing between groups of patients with CKD and a group of control subjects. Conclusions: Results of logistic regression showed that the increase of both NO level and homocysteine level for 2.72 µmol/L raises the possibility for development of CKD (2 times for NO: OR = 2.021, or 6.5 times for homocysteine: OR = 6.512). Moreover, the increase of NO level in serum and increase of homocysteine level in plasma is a risk factor for progression to higher stages of CKD (P = 0.002 and P < 0.05, respectively).
2nd European Joint Congress of EFLM and UEMS
P12-07 Estimated glomerular filtration rate, hypertension and blood mercury in a hospital working population Diaz Diaz A, Morales C, Trasobares E, Castillo Perez C, Fuentes M, González-Estecha M Hospital Clínico San Carlos, Laboratory Medicine, Madrid, Spain Corresponding author: [emailprotected]
Background: Blood mercury has been associated with nephrotoxicity and cardiovascular disease among the general population. The aim of this study is to evaluate the association between blood mercury concentrations and estimated glomerular filtration rate (eGFR) and hypertension in a hospital working population. Material and methods: We recruited 395 employees (64 men and 331 women) who were given the EMA® exposure questionnaire. Blood mercury concentration (µg/L) was measured by atomic absorption spectrometry and thermal decomposition amalgamation. Serum selenium concentration (µg/L) was measured by electrothermal atomic absorption spectrometry. eGFR was assessed using the abbreviated MDRD formula (Levey et al. 2000). Results: The median of blood mercury was 8.00 μg/L (IQR: 5.20-11.60) and the mean of eGFR was 74.44 mL/min/l.73 m² (SD: 10.29). A statistically significant correlation was found between blood mercury and the eGFR (r = -0.127, P = 0.014). This significant correlation was observed in women (r = -0.109; P = 0.05) but not in men. In order to explore possible hidden kidney disease we used a cutoff of 60 mL/min/l.73 m² for eGFR and a cutoff of 5.8 μg/L for mercury and no significant differences were observed. A positive correlation between eGFR and selenium/mercury ratio was found (r = 0.11; P = 0.034). This significant correlation was seen in men (r = 0.255; P = 0.049) but not in women. No differences were observed between hypertension and blood mercury or with the selenium/ mercury ratio.
Conclusions: The association between eGFR and mercury is different when considering the selenium status. It is necessary to consider the interactions between these elements in order to evaluate mercury toxicity.
P12-08 Cadmium, estimated glomerular filtration rate and hypertension in a hospital working population Diaz Diaz A, González-Estecha M, Castillo Perez C, Morales C, Fuentes M, Arroyo M Hospital Clínico San Carlos, Laboratory Medicine, Madrid, Spain Corresponding author: [emailprotected]
Background: Low cadmium levels (0.43 μg/L) have been associated with nephrotoxicity and cardiosvascular disease among the general population. The aim of this study is to evaluate the association between blood cadmium concentrations with estimated glomerular filtration rate (eGFR) and hypertension in a hospital working population. Material and methods: We recruited 395 employees (64 men and 331 women) who were given the PESA® exposure questionnaire. Blood cadmium was measured by electrothermal atomic absorption spectrometry. eGFR was assessed using the abbreviated MDRD formula (Levey et al. 2000): eGFR [mL/min/l.73 m²] = 186 × (Serum creatinine [mg/dL])-1.154 × (Age [y]) 0.203 (× 0.742 if female). Results: The median of blood cadmium was 0.29 μg/L (IQR:0.18-0.50) and the mean of eGFR was 74.44 mL/min/l.73 m² (SD:10.29). A statistically significant correlation was found between blood cadmium and the eGFR (r = -0.176, P = 0.001). This significant correlation was also observed in men (r = -0.301; P = 0.019) and women (r = -0.147; P = 0.009). In order to explore possible hidden kidney disease we used an eGFR cutoff of 60 mL/min/l.73 m² and a cadmium cutoff of 0.43 μg/L and no significant differences were found. In connection Biochemia Medica 2012;22(3):A54-A204
A137
2nd European Joint Congress of EFLM and UEMS
with hypertension, we found no significant differences between blood cadmium concentrations and hypertension were found. Conclusions: The association found between eGFR and cadmium levels in men as well as in women supports the role of cadmium in kidney disease. However, using a cutoff of 60 mL/min/l.73 m² we cannot confirm this correlation, probably due to the low number of events under the cutoff used.
P12-09 uNGAL in deceased donors as a marker of early kidney transplant injury Kalachyk A (1), Oganova E (2), Naderi U (3), Sadovski D (1), Dolgolikova A (1) (1) National Organs and Tissues Transplant Center, Minsk, Belarus, Transplant Department, Minsk, Belarus (2) National Organs and Tissues Transplant Center, Minsk, Belarus, Clinical Laboratory, Minsk, Belarus (3) National Organs and Tissues Transplant Center, Minsk, Belarus, Department of coordination, Minsk, Belarus
comparative analysis showed that delayed or SGF occured in the 1st group in 11.5% (3/26) patients, and in the 2nd group-21.7% (5/23) patients respectively. RGF was significantly better in the 1st recipients group: creatinine 117.4 ± 25.5 mmol/L, eGFR using MDRD 78.4 ± 29.5 mL/min, the 2nd recipients groups: creatinine 141.4 ± 72.4 mmol/L and eGFR-68.9 ± 20.1 mL/min (P < 0.05) 1 month after the transplantation. Conclusion: The study showed that uNGAL level in deceased donor with brain death can be used as a biomarker for prediction of RGF in early postoperative period.
P12-10 Low-grade inflammation and iron metabolism in chronic kidney disease (CKD) patients Tandara L, Salamunić I, Gugo K, Bilopavlovic N, Pauković-Sekulić B, Tandara M University Hospital Centre Split, Department of Medical Laboratory Diagnosis, Split, Croatia
Corresponding author: [emailprotected] Corresponding author: [emailprotected]
Background: The standard approach to the selection of deceased donors by assessment of urine output and serum creatinine levels do not allow to predict delayed graft function (DGF) or slow GF. Materials and methods: We analyzed the association between the concentration of uNGAL and renal graft function (RGF) in early postoperative period in deceased donors with brain death. Urine was collected in 25 deceased donors and analyzed using Architect. Results: DGF was diagnosed in 10% and SGF in 6% kidney recipients. All patients were devided into 2 groups according to uNGAL concentrations. 1st group - 26 recipients that obtained renal graft from deceased donors with low NGAL concentration (≤ 18 ng/mL), the 2nd - with high NGAL(≥ 18 ng/mL). There were no significant differences in mean donor age, sex ratio (male/female), time of cold storage and cause of death in these groups. A Biochemia Medica 2012;22(3):A54-A204
A138
Background: Patients with CKD are exposed to persistent low-grade inflammation. Increased level of proinflammatory cytokines cause cellular iron retention and repress iron efflux from sites of main iron flow into the blood reducing thus iron availability and contributing to development of anemia in CKD patients. This study aimed to assess relationship between indicators of inflammation and routine biochemical markers of iron metabolism as well as hematological indicators of iron availability in group of HD patients with low-grade inflammation (defined as CRP < 15 mg/L, without clinical manifestation of inflammation). Materials and methods: The study was conducted in 45 HD patients and 21 healthy subjects. Biochemical markers (serum iron, transferrin, transferrin saturation, ferritin), haematological indexes (%Hypo and CHr) were determined by routine lab-
2nd European Joint Congress of EFLM and UEMS
oratory methods. As inflammatory markers, CRP and hsIL-6 were determined. Results: Levels of serum CRP [2.9 (0.9-10.9) vs. 0.8 (0.4-3.2) mg/L] and IL-6 [5.03 (1.69-10.5 vs. 1.20 (0.14-10.55) ng/L] were significantly higher in HD patients compared with control group (P < 0.05). We found a statistically significant (P < 0.05) lower level of serum iron, transferrin, transferrin saturation and higher level of ferritin and %Hypo but there were no significant correlations between CRP/IL-6 and biochemical markers of iron status or hematological indexes in HD group. Conclusions: Parameters of iron metabolism are changed but inflammation indicators do not correlate with parameters of iron metabolism in our studied group. Since levels of serum inflammatory markers are subjected to a substantial variability over time repeated versus single measurements in a future studies could give a more valuable information about examined relationship.
P12-11 Cystatin C in early diagnosis of contrast-induced acute kidney injury Vavilova T, Klyusova S, Gaykovaya L North-West State Medical University named after Mechnikov, Laboratory Department, Saint-Petersburg, Russia (Russian Fed.) Corresponding author: [emailprotected]
Objective: Contrast-induced acute kidney injury (AKI) may take place in some patients after percutaneous coronary interventions and diagnostic procedures. Early diagnosis allows beginning the treatment in time and preventing severe renal insufficiency. Recent data showed cystatin C (CysC) as useful marker in AKI, but less information is about the contrast-induced one. Materials and methods: The level of serum creatinine (Cr) and CysC was measured before and after 18-20 h of contrast administration in 30 patients, admitted for coronary angiography and/or balloon angioplasty in university clinic. All patients
had risk factors but not confirmed kidney injury; 28 men and 2 women, 63.0 ± 9.9 ears old. CysC was measured by immunoturbidimetry method (KONELAB-20 analyser, Alfresa kit, Japan). Glomerular filtration rate (GFR) was calculated by MDRD and the equation Levey for cystatin C. Results: The average increase in cystatin C level in 18-20 hours was 21.0%, and in serum creatinine 5.5% (P < 0.05). The cystatin C sensitivity was found to be 94%, and specificity - 69% for ACI. Decreasing of the GFRCysC and GFRMDRD also showed significant differences: from 92.4 ± 36.8 to 73.1 ± 26.6 mL/min/1,73 m2 (20.9%); and from 81.3 ± 19.1 to 77.2 ± 20.7 mL/min/1,73 m2 (5.0%), respectively (P < 0.05). Conclusion: Cystatin C proved to be an early marker in interventional cardiology and may be used for the diagnosis of contrast-induced acute kidney injury.
P12-12 Protein:Creatinine ratio in spot urines to predict proteinuria in chronic kidney disease Shrestha R (1), Gyawali P (2), Neopane P (3), Jha B (2) (1) Nepal Medical College and Teaching Hospital, Clinical Biochemistry, Kathmandu, Nepal (2) Institute of Medicine, TU Teaching Hospital, Biochemistry, Kathmandu, Nepal (3) Medicare National Hospital, Laboratory Medicine, Kathmandu, Nepal Corresponding author: [emailprotected]
Background: The measurement of protein in 24hr urine collection has been regarded as the gold standard. Spot urine protein:creatinine ratio (PCR) have been widely used as alternative to 24h urine protein (UTP). Objective: The aim of this study was to examine the ability of PCR to predict urinary 24h protein loss in Nepalese patients with CKD. Materials and methods: This Study comprises 933 CKD patients (178 had stage 1 CKD, 230, 222, 158, and 145 patients with stage 2, 3, 4, and 5 reBiochemia Medica 2012;22(3):A54-A204
A139
2nd European Joint Congress of EFLM and UEMS
spectively). Patients were instructed to collect 24h urine and spot urine, both were subjected for determination of UTP and spot PCR. Results: 24.3%, 25%, 46.8%, 75.7% and 90.1% of stage 1, 2, 3, 4 and 5 CKD respectively had proteinuria as determined by UTP (> 150 mg/day). There was good correlation between UTP and PCR, with correlation coefficients(r) of 0.923. Receiver operator characteristic (ROC) curve analysis showed PCR to be a good predictor of proteinuria, with area of 0.973 (95% CI; 0.961-0.985, P < 0.001). At PCR cutoff of 20 mg/mmol sensitivity of 93% and specificity of 92% can be achieved. The optimal cutoff varies among stage of CKD. The optimal cutoff of PCR is 13.2 mg/mmol in stage 1, 15.1 20 mmol in stage 2, 20.5 mg/mmol in stage 3, 26.1 mg/mmol in stage 4 and 33 mg/mmol in stage 5. Conclusions: Spot PCR can be a good alternative to 24 hour UTP. By careful choice of cutoffs, PCR can be used in patients with CKD to identify significant proteinuria.
ted to the Pediatric Clinic. 62 of them had acute pyelonephritis, while 28, who represented controls, had cystitis or fever of other etiology. Routine laboratory analyses (CBC, CRP and urine analysis), urine culture, kidney ultrasound and static scintigraphy were also done. Urine NGAL was measured using CMIA method (ARHITECT i1000, ABBOT). We have compared single NGAL concentrations with mg NGAL/g creatinine in same patient. Statistical calculation was performed using Mann-Whitney rank sum test and ROC analyses. Results: At a cut-off value of 29.1 ng/ml NGAL, sensitivity and specificity for detecting acute pyelonephritis was 0.964 (95% CI, 0.901 to 0.992), while at cut-off value of 120.1 mg NGAL/g creatinine sensitivity and specificity was 0.988 (95% CI, 0.938 to 1.000). Mann-Whitney rank sum test also showed significant difference in median values between the two groups for both measurements (P < 0.001). Conclusion: Although the values of NGAL / g creatinine were a bit superior, there is no statistical significant difference between measurements.
P12-13 Sensitivity and specificity of NGAL in acute pyelonephritis Mandić S (1), Arambašić J (2), Horvat V (1), Mandić D (3), Šerić V (1) (1) University Medical Center Osijek, Department of Clinical Laboratory Diagnostics, Osijek, Croatia (2) University Medical Center Osijek, Pediatric Clinic, Osijek, Croatia (3) Institute of Public Health for Osijek-Baranya Country, Health Ecology Department, Osijek, Croatia
P12-14 Assessment of the new CKD-EPI formula to estimate CKD in hospitalized patients Fernandez Rodriguez E (1), Garcia Alonso S (1), Fernandez Garcia E (2) (1) Cabuenes Hospital, Biochemistry, Gijon, Spain (2) Central Hospital, Intensive Care Unit, Oviedo, Spain Corresponding author: [emailprotected]
Corresponding author: [emailprotected]
Background: Neutrophil gelatinase-associated lipocalin (NGAL) is a small protein expressed in renal tubules where it is considerably induced in ishemic or nephrotoxic injury. A single urine NGAL measurement is proposed test for distinguish acute kidney injury. However, single urine can sometimes be concentrated or diluted, which can influence results of measurements.
Background:A recent report by the CKD-EPI group describes a new equation to estimate the GFR. The CKD-EPI equation improved the accuracy and precision of results of the current first-choicec MDRD IDMS formula, specially for GFR > 60 mL/ min/1.73m2. A high percentage (28.3%) of hospitalized patients in Spain have deteriorated renal function stages 3-5 as measured by MDRD 4 formula.
Materials and methods: We have analyzed 90 children under 5 years of age, which were admit-
Materials and methods:The goal of our study was to compare the estimated GFR by using the
Biochemia Medica 2012;22(3):A54-A204
A140
2nd European Joint Congress of EFLM and UEMS
new equation CKD-EPI with MDRD 4 in a wide cohort of hospitalized patients (14,658 adults) and to analyze the impact of the new CKD-EPI formula on the satging of patients with CKD. Results: The concordance correlation coefficient beetween both formulas was 0.9949 (95% CI: 0.9947 to 0.9951). The distribution of KDOQI stages were: CKD-EPI (1 + 2, 69.9%; 3a, 14.9%; 3b, 9.4%; 4, 4.3%; 5, 1.5%),MDRD (1 + 2, 72.7%; 3a, 14.9%; 3b, 7.7%; 4, 3.4%; 5, 1.2%). Weighted Kappa statistics was 0.861 (very good agreement). Overall, CKD-EPI detected an additional 2.8% of patients with GFR < 60 mL/min/1.73m2. Conclusions: CKD-EPI equation reclasified an additional 2.8% of patients to stages of worse GFR
P13 - Lung, liver and gastrointestinal diseases P13-01 Serum copper concentrations and cardiomyopathy in cystic fibrosis patients
addition, we estimated the Cu/Ceruloplasmine ratio. The left ventricular ejection fraction (LVEF) was determined by a Philips IE33 Echocardiogram. We defined systolic dysfunction as an LVEF less than 55% (Simpson’s method). Results: The mean copper concentration was 131.8 µg/dL (SD: 37.7). The mean serum ceruloplasmine was 34.00 mg/dL (SD: 9.1). The mean Cu/Ceruloplasmine ratio was 3.9 (SD: 0.4). No correlation was found between total copper and LVEF or between the Cu/ceruloplasmine ratio and LVEF. However, upon considering patients with an LVEF under the cutoff of 55 % we found a lower serum copper concentration (117.8 µg/dL SD:18.4 vs. 132.6 µg/dL SD: 38.3), although this difference was not statistically significant. Conclusion: In spite of the malabsorption associated with CF, we did not observe copper deficiency in this population. Since we found a decrease in copper concentrations in patients with lower LVEF, more studies should be performed with a greater sample size in order to clarify the role that copper may play in the cardiomyopathy of CF patients.
Castillo Perez C (1), González-Estecha M (1), Morales C (1), Ruiz L (2), Díaz Díaz A (1), Segovia J (2)
P13-02
(1) Hospital Clínico San Carlos, Laboratory Medicine, Madrid, Spain (2) Hospital Puerta de Hierro Majadahonda, Cardiology, Madrid, Spain
Castillo Perez C (1), González-Estecha M (1), Díaz Díaz A (1), Ruiz L (2), Maíz L (3), Segovia J (2)
Corresponding author: [emailprotected]
Background: Copper deficiency has been reported in cardiomyopathy and may occur in patients with intestinal malabsorption, as occurs in cystic fibrosis (CF). The aim of this multicenter study is to evaluate copper in CF patients, who have a high prevalence of cardiomyopathy. Materials and methods: We studied 123 adult CF patients (63 male and 60 female) with a mean age of 31 (SD: 8.90). Serum copper concentrations were measured using flame atomic absorption spectrometry. The concentration of serum ceruloplasmine was measured by immunonephelometry. In
Lead and cadmium in cystic fibrosis
(1) Hospital Clínico San Carlos, Laboratory Medicine, Madrid, Spain (2) Hospital Puerta de Hierro Majadahonda, Cardiology, Madrid, Spain (3) Hospital Ramon y Cajal, Neumology, Madrid, Spain Corresponding author: [emailprotected]
Background: Exposure to lead and cadmium is a public health problem due to the broad exposure to these toxic substances among the general population, and in recent years they have been associated with an increased cardiovascular risk. The objective of this multicenter study is to determine blood lead and cadmium concentrations in a population of unselected patients diagnosed with Biochemia Medica 2012;22(3):A54-A204
A141
2nd European Joint Congress of EFLM and UEMS
cystic fibrosis (CF), who have a higher prevalence of cardiomyopathy than expected. Material and methods: We studied 123 adult CF patients (63 male and 60 female) with a mean age of 31 (SD: 8.90). The blood lead (μg/dL) and cadmium (μg/L) concentrations were measured by electrothermal atomic absorption spectrometry with Zeeman background correction in a Perkin-Elmer spectrometer. The left ventricular ejection fraction (LVEF) was determined by a Philips IE33 echocardiogram. We defined systolic dysfunction as an LVEF less than 55% (Simpson’s method). Results: The median of lead was 0.80 μg/dL (IQR: 0.48-1.13). Blood lead percentiles (5, 25, 50, 75, 95) were: 0.07, 0.48, 0.80, 1.13, 1.91 µg/dL respectively. Eighty per cent of the patients had blood cadmium concentrations below the detection limit (0. 07 μg/L). Blood cadmium percentiles (75, 90, 95) were: 0.07, 0.3, 0.7 μg/L. No significant differences were found between lead and cadmium levels and LVEF. Conclusion: The concentrations of blood lead in this population are low and similar to the reference values for this age group in Spain. Most of these patients showed blood cadmium levels below the detection limit probably because of their low level of smoking.
13-03 Interaction of blood mercury with essential trace elements in a cystic fibrosis population Morales C (1), González-Estecha M (1), Castillo Perez C (1), Ruiz L (2), Prados C (3), Segovia J (2) (1) Hospital Clínico San Carlos, Laboratory Medicine, Madrid, Spain (2) Hospital Puerta de Hierro Majadahonda, Cardiology, Madrid, Spain (3) Hospital Universitario La Paz, Neumology, Madrid, Spain Corresponding author: [emailprotected]
Background: Exposure to mercury is a public health issue. Cystic fibrosis is characterized by an obstructive pulmonary pattern and a pancreatic Biochemia Medica 2012;22(3):A54-A204
A142
exocrine deficiency, frequently associated with malabsorption. We studied the relationship between mercury, copper, zinc and selenium in this multicenter population. Materials and methods: We studied 123 adult CF patients (63 male and 60 female) with a mean age of 31 (SD: 8.90). Serum copper (µg/dL) and zinc (µg/dL) were measured using flame atomic absorption spectrometry. Serum selenium (µg/L) was measured using electrothermal atomic absorption spectrometry. Blood mercury (µg/L) was measured by atomic absorption spectrometry and thermal decomposition amalgamation. Results: The median of blood mercury was 5.7 µg/L (IQR: 2.9-9.6). Forty eight per cent of the patients had blood mercury levels higher than the levels established by the EPA (5.8 µg/L). The mean of serum copper was 131.8 µg/dL (SD: 37.7). The mean of serum zinc was 86.9 µg/dL (SD: 13.3) and for selenium was 71.9 µg/L (SD: 14.8). A negative correlation was found between mercury and zinc (r = -0.125) which was not statistically significant; no correlation was observed between mercury and copper. A positive correlation was found between mercury and selenium (r = 0.308, P < 0.001). This correlation was observed in both patients with blood mercury levels under 5.8 µg/L (r = 0.304, P = 0.012) and in those with levels above 5.8 µg/L (r = 0.333, P = 0.007). Conclusion: We found high mercury levels in patients with CF. Further studies are desirable to investigate the interactions with essential trace elements and different compounds of the diet which could prevent mercury toxicity.
2nd European Joint Congress of EFLM and UEMS
P13-04 Selenium and mercury and the left ventricular ejection fraction in adult cystic fibrosis patients González-Estecha M (1), Ruiz L (2), Castillo Perez C (1), Morales C (1), Díaz Díaz A (1), Segovia J (2) (1) Hospital Clínico San Carlos, Laboratory Medicine, Madrid, Spain (2) Hospital Puerta de Hierro Majadahonda, Cardiology, Madrid, Spain Corresponding author: [emailprotected]
Background: Cystic fibrosis (CF) is frequently associated with malabsorption. Certain studies have described a rare form of cardiomyopathy (CMP), similar to the one seen in Keshan’s disease. The aim of this multicenter study is to measure serum selenium and blood mercury and their relation with cardiomyopathy in CF patients. Materials and methods: We studied 123 adult CF patients (63 male and 60 female) with a mean age of 31 (SD: 8.90). Blood mercury concentration (µg/L) was measured by atomic absorption spectrometry and thermal decomposition amalgamation. Serum selenium concentration (µg/L) was measured by electrothermal atomic absorption spectrometry. The Left Ventricular Ejection Fraction (LVEF) was determined by echocardiography. We defined systolic dysfunction as an LVEF of less than 55% (Simpson’s method). Results: The patients with serum selenium concentrations below 60 µg/L had a lower mean LVEF (58.86% SD: 12.10) than the ones with serum selenium concentration above 60 µg/L (65.21% SD: 6.23) and this difference was statistically significant (P = 0.001). The difference between these means (B = 6.36; P < 0.001 CI 95%: 2.76-9.95) can be explained in 9.4% by selenium (R2 = 0.094; P < 0.001) and 9.8% can be explained by the selenium/ mercury ratio (R2 = 0.098; P < 0.002). However, once adjusted for mercury this last difference can be explained only by selenium (B = 6.45; P = 0.001 CI 95%: 2.58-10.33). No significant differences were found between mean LVEF and blood mercury
levels (cutoff of 5.8 µg/L) and the selenium/mercury ratio (cutoff of 10.34). Conclusion: Studying the association between mercury, selenium and LVEF, we observed that only low selenium concentrations are related to alterations in LVEF.
P13-05 Monitoring faecal occult blood test positivity in the NHS Bowel Cancer Screening Programme Halloran S, Burtonwood C, Butler P, Young M NHS Bowel Cancer Screening Programme, Southern Hub, Guildford, United Kingdom Corresponding author: [emailprotected]
Background: The guaiac-based faecal occult blood test kit used by the NHS Bowel Cancer Screening Programme (BCSP) relies on subjective visual assessment to determine positivity. BCSP participants apply two samples from three separate bowel motions to each of six test kit windows lined with filter paper impregnated with guaiac. Test kits are returned to the Hub for analysis where development of an unstable blue-green colour in response to a peroxide developer indicates the presence of haemoglobin. Individuals who test positive are referred for further investigation, usually colonoscopy. The subjective nature of the test kit reading and possible inaccuracies have consequences for the national programme, participants and colonoscopy services. Materials and methods: Test kit readers are tested for colour blindness and visual acuity when they start work in the Hub. The percentage of positive test spots is recorded weekly for readers completing > 100 kits. The acceptable spot positivity range (1-4%) is based on an approximation of ± 2 standard deviations from the rolling six-month mean percentage positivity for all Hub staff. Results: Since monitoring began in 2010 there has been a reduction in reader imprecision and outliers. New staff attend training sessions to learn about Biochemia Medica 2012;22(3):A54-A204
A143
2nd European Joint Congress of EFLM and UEMS
the concept of reader positivity and the interventions that may be put in place if their positivity falls outside the acceptable range and supervised refresher training for all staff is conducted annually. Conclusion: Monitoring of test kit readers’ performance is essential whilst adoption of an automated immunochemical test analysis is the long-term solution.
colonoscopies performed. About 40% of the screened population that undergoes colonoscopy has significant neoplasia (cancer, high- or intermediate-risk adenomas). The prevalence of significant neoplasia is greater in men and increases with age. The proportion of significant neoplasia detected in screening episode 2 is lower than in episode 1. Conclusion: The screening data are encouraging and indicate that the BCSP in England is likely to achieve its goal of reducing bowel cancer mortality.
P13-06 The NHS Bowel Cancer Screening Programme, Southern Hub – screening activity and outcomes Halloran S, Seaman H, Young M NHS Bowel Cancer Screening Programme, Southern Hub, Guildford, United Kingdom Corresponding author: [emailprotected]
Background: The NHS Bowel Cancer Screening Programme (BCSP) in England invites individuals aged 60-74 years to be screened every two years. The BCSP Southern Hub serves a population of about 14.4 million people and manages the screening activity in the south of England. Invitees are sent a guaiac-based faecal occult blood (gFOB) test kit, asked to provide a faecal sample and to return the test kit to the Hub for analysis. Participants with a positive (‘abnormal’) test are referred to a Specialist Screening Practitioner (SSP) for further investigation, usually colonoscopy. Materials and methods: All screening activity, including uptake, gFOB test results, SSP referrals and colonoscopy outcomes are stored on the Bowel Cancer Screening System. Data for the period 2006-2011 were extracted and analysed. Results: The uptake of screening invitations (the proportion of invitees that is adequately screened) is approximately 56% overall (higher for women than men [61% vs. 55%]). The proportion of positive test kits is higher for men than women (2.6% vs. 1.6%) and positivity has increased over time, with a consequent increase in the number of Biochemia Medica 2012;22(3):A54-A204
A144
P13-07 Comparative study of the risk of developing NAFLD in individuals without liver disease Almeida A, Pires C, Paulo R, Leitao C, Bellem F, Abreu R Escola Superior Tecnologia Saude Lisboa, Laboratory Sciences and Technologies and Comunity Intervention, Lisbon, Portugal Corresponding author: [emailprotected]
Introduction: Nonalcoholic fatty liver disease (NAFLD) has been recognized as the most common liver disease in Western countries, since its prevalence is high (20-30%) in developed countries. The objective of our work is a comparative study of biochemical parameters (glucose, cholesterol, HDL-cholesterol, LDL-cholesterol, triglycerides, AST and ALT) between individuals with NAFLD and others without NAFLD. Materials and methods: Control group includes 47 subjects without NAFLD (25 females and 13 males) and other group with 11 subjects (2 females and 9 males) diagnosed with NAFLD by liver biopsy or ultrasound. After blood collection, the biochemical parameters were assessed on the equipment TARGA3000®. Results: Significant differences were found between both groups on: triglycerides, AST and ALT. Aminotransferases are the variables that showed a more marked difference, AST and ALT values were elevated in 100% and 72,7%, respectively, of the subjects with NAFLD. One subject from control
2nd European Joint Congress of EFLM and UEMS
P13-08
Conclusions: The citrulline levels were significantly lower in patients compared to the control group. Low citrulline levels were found in patients shortly after stem cell transplantation and in patients with GvHD. Observations in larger groups of patients are necessary. Supported by specific fund SVV 262 806 and by the project Ministry of Health, Czech Republic for conceptual development of research organization 00669806 - Faculty Hospital in Pilsen, Czech Republic
Citrulline – marker of enterocytes mass and function in patients after stem cells transplantation
P13-09
group had higher values of aminotransferases then the average of subjects with NAFLD. Conclusion: These results enlighten the need for surveying and monitoring apparently healthy population in order to be effective in primary prevention of NAFLD and other metabolic disorders.
Sedlackova T (1), Rajdl D (1), Ladislav T (1), Racek J (1), Vokurka S (2), Svoboda T (2) (1) Charles University in Prague, Faculty of Medicine in Pilsen and Faculty Hospital in Pilsen, Department of Clinical Biochemistry and Haematology, Pilsen, Czech Republic (2) Faculty Hospital in Pilsen, Department of HaematoOncology, Pilsen, Czech Republic Corresponding author: [emailprotected]
Background: Citrulline is an amino acid produced by enterocytes. Plasma citrulline concentration is considered as a marker of enterocytes mass and function. The determination of citrulline could be helpful in patients with intestinal failure, intestinal diseases (celiac and Crohn disease, etc.) or intestinal damage caused by toxicity of chemotherapy or graft-versus-host disease (GvHD). Materials and methods: We measured plasma citrulline levels in 12 patients (20 patient´s samples) with diarrhea after allogeneic stem cell transplantation and in 20 healthy controls. Results: The median value of citrulline levels was significantly lower in the transplanted patients group compared to healthy controls: 10.2 (0.6– 63.8) vs. 33.3 (19.1–45.9) μmol/L, P < 0.001. The median values of citrulline levels in patients with post-transplant toxic intestinal damage (mucositis) (N = 8, day 1–22 post-transplant) vs. GvHD (N = 7, day 43-142) vs. “others” (usually dysmicrobia, N = 5, day 120-570) were: 9.6 (0.6–18.6) vs. 3.4 (1.9–9.9) vs. 19.5 (15.3–63.8) μmol/L.
Prevalence of AMHA and ANA in patients with suspicious primary biliary cirrhosis Padjen I (1), Kozmar A (2), Mayer M (1), Sentić M (1), Malenica B (2) (1) University Hospital Centre Zagreb, Zagreb, Croatia, Department of Internal Medicine, Zagreb, Croatia (2) University Hospital Centre Zagreb, Zagreb, Croatia, Department of Laboratory Diagnostics, Division of Immunology, Zagreb, Croatia Corresponding author: [emailprotected]
Primary biliary cirrhosis (PBC) is characterized by the presence of disease-specific autoantibodies that are primarily directed against mitochondrial antigens (AMA). However, a subgroup of patients’ sera is also positive for antibodies to nuclear autoantigens (ANA). PBC-specific antinuclear antibodies are of diagnostic and clinical relevance since they can be used as a „positivie tool“in the diagnosis of AMAnegative PBC while at the same time identifying of patients with more advanced liver disease. Because both AMA and ANA testing are a critical part of diagnosis in PBC, it is important to detect them very carefully. Indirect immunofluorescences (IIF) on frozen sections of rat kidney and stomach, human epithelial HEp-2 cells and immunoblot to different target antigens have been used for this purpose. The aim of our study was to determine prevalence of PBC-specific ANA and their target antigens in 210 patients with suspicious PBC. AMA were positive in 91.9% (193/210) and PBC-specific ANA in 8.1% (17/210) of sera. Both AMA and ANA were found in 35.2% (74/210) of patients sera. AMA positive sera Biochemia Medica 2012;22(3):A54-A204
A145
2nd European Joint Congress of EFLM and UEMS
recognized specific target autoantigens M2 and ANA specific autoantigens: Sp100 (57.1%; 52/91), PML (52.7%; 48/91) and gp210 (41.7; 38/91). Futhemore, they recognized the Ro-52 autoantigen (33.3%; 70/210). Sera that were ANA positive in the IIF recognized only nuclear specific autoantigens Sp100, PML and gp210. Our results show the compatibility of IIF and immunoblot in detecting AMA and ANA, and that IIF on HEp-2 cells and frozen sections of rat kidney and stomach detects PBC-specific ANA.
P13-10 Comparison of fecal calprotectin and CRP in pediatric inflammatory bowel disease Linarić I (1), Obuljen J (1), Margetić S (2), Mišak Z (3), Hojsak I (3), Žižić V (1) (1) Children's Hospital Zagreb, Department of Laboratory Diagnostics, Zagreb, Croatia (2) Sestre milosrdnice University Hospital Center, Department of Laboratory Haematology and Coagulation, Universitiy Department of Chemistry, Zagreb, Croatia (3) Children's Hospital Zagreb, Department of Gastroenterology and Nutrition, Zagreb, Croatia Corresponding author: [emailprotected]
Background: The aim of the study was to evaluate the diagnostic accuracy of C-reactive protein (CRP) and fecal calprotectin (FC) as markers of inflammatory bowel disease (IBD) in pediatric patients. Materials and methods: The study included 66 pediatrics patients; 41 patients with IBD (confirmed by colonoscopy as the gold standard) and 25 patients with excluded IBD (non-IBD). The serum concentrations of CRP were measured by an immunoturbidimetric high sensitive latex CRP assay (Beckman Coulter, AU 400 analyzer). The concentrations of FC were measured with commercially available enzyme-linked immunosorbent assay (Calprest, Eurospital). Results: The medians (95% confidence interval (95%CI); interquartile range (IQR)) of both markers were significantly higher (P < 0.001) in IBD pediatric patients: CRP 8.7 mg/L (4.4-16.0; 2.7-20.6) and Biochemia Medica 2012;22(3):A54-A204
A146
FC 368.0 μg/g (234.0-457.0; 215.8-559.0) compared with non-IBD patients (CRP 0.3 mg/L; (0.2-0.6; 0.20.7) and FC 15.6 μg/g (15.6-17.0; 15.6-20.8). However, the receiver operating characteristic (ROC) analysis showed significantly higher diagnostic accuracy (P = 0.039) of FC (area under curve (AUC) of 0.977; 95%CI = 0.905-0.998, sensitivity (Se) of 90.2%; 95%CI = 76.9-97.3, specificity (Sp) of 100%; 95%CI = 86.3-100.0, likelihood ratios LR- 0.09 and LR+ 22.6 at optimal cut-off value of 56 μg/g) compared with those of CRP (AUC = 0.903; 95%CI = 0.805-0.962, Se = 75.6% (95%CI = 59.7-87.6), Sp = 96.0% (95%CI = 79.6-99.9), LR- 0.25 and LR+ 18.9 at optimal cut off value of 2.6 mg/L). Conclusion: Although both markers, CRP and fecal calprotectin, can be used for estimating mucosal inflammation in pediatric IBD patients, FC showed a higher diagnostic accuracy in discriminating between IBD and non-IBD pediatric patients with better sensitivity, specificity and likelihood ratios.
P13-11 Lipid profile in patients with chronic obstructive pulmonary disease Milevoj Kopčinović L (1), Pancirov D (2), Simić L (3), Čepelak I (3), Žanić Grubišić T (3), Rumora L (3) (1) Medical School University Hospital Sestre Milosrdnice, University Department of Chemistry, Clinical Unit of Medical Biochemistry in traumatology and orthopedics, Zagreb, Croatia (2) Dr. Ivo Pedišić General Hospital, Department of Biochemistry and Hematology Diagnosis, Sisak, Croatia (3) Faculty of Pharmacy and Biochemistry, University of Zagreb, Department of Medical Biochemistry and Hematology, Zagreb, Croatia Corresponding author: [emailprotected]
Background: Chronic obstructive pulmonary disease (COPD) is a complex systemic disease associated with many comorbidities. Cardiovascular diseases (CVD) are the leading cause of death among patients with COPD. Our aim was to assess the lipid profile and the relationship between lipid and lung function parameters in COPD patients.
2nd European Joint Congress of EFLM and UEMS
Materials and methods: The study included 38 healthy subjects (15 smokers, 10 ex-smokers, 13 non-smokers) and 103 COPD patients (31 smokers, 25 ex-smokers, 47 non-smokers). COPD patients were also subdivided according to disease severity (GOLD stages II-IV). FEV1 predicted and FEV1/FVC were determined by spirometry. Total cholesterol, triglycerides, HDL cholesterol, ApoA and ApoB were measured in sera of all participants, while LDL cholesterol and LDL/HDL and ApoB/ApoA ratios were calculated. Results: Total cholesterol, triglycerides, LDL cholesterol and ApoA were lower, and ApoB/ApoA ratio was higher in patients with COPD (P = 0.003, P < 0.001, P = 0.043, P < 0.001, P = 0.005, respectively). No differences were found in HDL cholesterol and ApoB concentrations and LDL/HDL ratio when comparing patients with healthy subjects. However, HDL was weakly negatively correlated with FEV1/ FVC in the patient group (r = -0.27, P = 0.006). In addition, neither disease severity nor smoking status influenced lipid parameters in COPD patients. Conclusions: Although COPD is associated with an increased risk of CVD, our results do not confirm a pro-atherogenic lipid pattern in these patients. However, further research including a larger number of participants is needed to clarify this dilemma.
P13-12 MMP-9 and TIMP-1 concentrations in plasma of patients with chronic obstructive pulmonary disease Somborac Bačura A (1), Rumora L (1), Šribar D (1), Popović-Grle S (2), Čepelak I (1), Žanić Grubišić T (1) (1) University of Zagreb, Faculty of Pharmacy and Biochemistry, Department of Medical Biochemistry and Hematology, Zagreb, Croatia (2) University Hospital Centre Zagreb, University of Zagreb, School of Medicine, Zagreb, Croatia
metalloproteinases (TIMPs) has been implicated in the pathogenesis of chronic obstructive pulmonary disease (COPD). The aim of this study was to evaluate MMP-9 and TIMP-1 concentrations in COPD patients in relation to the severity of disease. Materials and methods: The study included patients with stable COPD (N = 59) and healthy volunteers (N = 21). COPD patients were divided into subgroups (GOLD stages II to IV) according to the spirometry results. Plasma MMP-9 and TIMP-1 concentrations were determined using a commercially available ELISA kits. Classic inflammatory markers were also measured (differential leukocyte counts and CRP). Results: MMP-9 concentration in COPD patients (204.13 (115.70-351.24) ng/mL) was significantly increased comparing to healthy controls (70.25 (52.48-104.96) ng/mL) with P < 0.001. There were no significant differences in TIMP-1 concentration. MMP-9/TIMP-1 ratio differed significantly between COPD patients (1.659 (0.965-2.687)) and healthy controls (0.627 (0.424-0.890)) with P < 0.001. Similar pattern was found already in GOLD II stage of disease. Very good diagnostic accuracy for MMP-9 was determined (AUC = 0.884; sensitivity of 66.1% and specificity of 100.0%; P < 0.001). The multivariate logistic regression model showed that the use of MMP-9 in combination with neutrophils, lymphocytes and CRP improved significantly (P = 0.023) diagnostic strength (AUC = 0.975). Conclusions: Increased concentration of MPP-9 and higher MMP-9/TIMP-1 ratio found in COPD patients as early as in GOLD II stage highlight the significance of protease/antiprotease imbalance for the development of COPD and potential use of these parameters as biomarkers for early diagnosis of COPD.
Corresponding author: [emailprotected]
Background: An imbalance of matrix metalloproteinases (MMPs) and tissue inhibitors of matrix Biochemia Medica 2012;22(3):A54-A204
A147
2nd European Joint Congress of EFLM and UEMS
P13-13 The relationship between BAL IL-8 and DLCO in patients with COPD Fijačko M (1), Fijačko V (2), Dobrošević B (1), Pavela J (1), Sikora I (1), Šerić V (1) (1) Clinical Hospital Center Osijek, Department of Clinical Laboratory Diagnostic, Osijek, Croatia (2) Clinical Hospital Center Osijek, Department of Pulmonary Disease, Osijek, Croatia Corresponding author: [emailprotected]
Background: Most patients with chronic obstructive pulmonary disease (COPD) develop emphysema with alveolar destruction and small airway inflamation, most caused by smoking. The inflamation is characterised by increased neutrophils, CD8+ T lymphocyte, macrophages and associated cytokines, chemokines and proteases. Interleukin 8 (IL-8) is one of the best characterised members of the chemokine family and one of the most important neutrophil chemoattractants. Lung function measurements included FEV1, FEV1/FVC % and diffusing capacity (DLCO). Decrease DLCO results showed that gases do not diffuse normally across lung membranes, this indicate that certain lung disease are present: COPD or interstitial lung disease. The aim was to investigate relationship between DLCO and BAL IL-8. Material and methods: 51 patients (82% man) with COPD and 16 controls (43% man) were studied. The concentrations of IL-8 was measured by ELISA method (eBioscience). DLCO was performed with single breath method on Master screen (Jaeger). Results of DLCO is usually reported as the percent of predicted amount of carbon monoxide inhaled that should be absorbed. Results: Statistically significant differences were not found between BAL levels IL-8 in COPD patients and control group (504 ± 666 pg/mL vs. 264 ± 256 pg/mL). Statistically significant differences were found between DLCO in COPD patients and control group (66.4 ± 22.8 % vs. 4.4 ± 23.0 at P level < 0.05). In addition, BAL IL-8 in COPD patients Biochemia Medica 2012;22(3):A54-A204
A148
showed a strong negative correlation to DLCO (r= - 0.40, P < 0.05.). Conclusions: These results showed that BAL IL-8 was signifficantly associated with DLCO in patients with COPD.
P14 - Microbiology - Infection P14-01 Screening for the urinary tract infections using Sysmex UF 1000i flow cytometer Konderak J (1), Elmardi A (2), Farkas B (2), Pinter E (1) (1) Synlab Hungary Ltd., Medical Biochemical Department, Budapest, Hungary (2) Synlab Hungary Ltd., Microbiological Department, Budapest, Hungary Corresponding author: [emailprotected]
Background: Bacterial cultures for the urinary tract infections (UTI) are the most common microbiological tests. Huge amounts of negative cultures demand for effective screening method reducing cost and time. The aim of this study was to evaluate the efficacy of UF 1000i flow cytometer for preselecting of negative results. Material and methods: Urine specimens (N = 1226) suspected UTI were simultaneously cultured and analyzed by UF 1000i for bacterial (BC) and leukocyte (WBC) counts. Population: male:female = 1:2; ages: 0-15 years: 8%; 16-65 years: 59%; > 65 years: 33%; outpatient:inpatient = 4:1. For culture samples were inoculated using a 10 μL loop on selective agar plates. After standard incubation results were evaluated. A sample was considered negative for UTI if growth was < 103 CFU/mL (colony forming unit). Positive samples were attributed to one of the levels of CFU/mL (> 103, > 104, > 105). Results: Using culture results as gold standard, we performed ROC analysis to determine area under curve (AUC), cutoff values (CO), sensitivities (SE), specificities (SP), negative predictive values (NPV) in pointing to BC and WBC measured by UF1000i.
2nd European Joint Congress of EFLM and UEMS
Results of ROC statistics at CFU/ml of > 103, > 104, > 105 AUC: 0.88, 0.91 and 0.93, CO BC and/or WBC/ µL: > 9, > 22, > 100, SE%: 95, 96, 96; SP%: 31, 45, 54; NPV%: 97, 98, 98, respectively. Conclusions: By screening out method, UF 1000i saved 23, 32 and 42% of the total urine cultures at > 103, > 104 and > 105 CFU/mL respectively.
P14-02 Comparison between a syphilis screen of two immunoassays for the diagnosis of syphilis Saue E, Viia V, Mägi M East-Tallinn Central Hospital Diagnostic Clinic, Central Laboratory, Tallinn, Estonia Corresponding author: [emailprotected]
Background: Syphilis can be asymptomatic, serologic screening is recommended for persons at high risk, pregnant women, blood donors and routine survey. Treponema pallidum, the bacterium that causes syphilis, cannot be cultured. Serologic testing is treponemal method most often used to diagnose syphilis in patients with suspected disease. To evaluate the performance of the Immulite 2000 Syphilis Screen assay to the Cobas 6000 Treponema pallidum latex agglutination assay-TPLA. Parallel studies were carried out in order to switch to another method of analysis of the syphilis screening. Materials and methods: Out of total 43 human serum specimens, belonging to three different categories (routine laboratory screening for syphilis, N = 16; syphilis patients, N = 15; potential falsepositive results, N = 12), parallel studies were carried out in 16 unselected and 27 previously maintained positive serums. During the analysed period, parallel studies were performed by using the chemiluminescent immunoassay (Immulite 2000) and latex agglutination assay (Cobas 6000) method. All samples were tested with immunoblot Treponema IgG and IgM kits for confirmation.
Results: 6 results differed from the total number of 43 parallel studies carried out. 6 results were found to be false-positive since the confirmatory tests affirmed negative results. Syphilis screening can be switched over to the Cobas 6000 analyzer. Conclusions: In comparison with the Syphilis Screen assay, the TPLA assay is more specific than the Immulite kit and has the advantage of reducing the number of confirmatory tests (mostly pregnant sera).
P14-03 Procalcitonin as early diagnostic marker for infection in febril neutropenia cancer patients Alonso T (1), Cuadrado-Cenzual M (2), De Gracia Y (2), Ortega De Heredia D (2), Perez-Segura P (1) (1) Hospital Clinico San Carlos, Oncologia, Madrid, Spain (2) Hospital Clinico San Carlos, Analisis Clinicos, Madrid, Spain Corresponding author: [emailprotected]
Introduction: Febrile neutropenia (FN) in cancer patients is a complication related to antineoplasic therapy with serious impact on their morbidity and survival. The main cause is the infectious process. The aim of this study was to evaluate Procalcitonin (PCT) like early diagnostic marker in patient with FN associated with chemotherapy. Materials and methods: A prospective double blind study was developed. We included 78 patients with FN and with treatment to them of the fever according to clinical practice protocol. Of all of them, 61 patients completed all the study with serial studies of PCT serum levels. Results: Bateriemia was detected in 36 patients and PCT showed higher significantly levels (P < 0.001) in this patients in comparision with non bacteriemia patients. Relation between Procalcitonine values and treatment non–response was significant (p 0.999). The point of cut-off PCT level, with better sensitivity (74.6 %) and specificity (80%) was 0.21µg/L. The multivariant analysis Biochemia Medica 2012;22(3):A54-A204
A149
2nd European Joint Congress of EFLM and UEMS
showed that the value of PCT over 0.5 µg/L in patients with FN and bacteriemia it was an independent variable like marking diagnosis of bacteriemia in patients with febrile neutropenia (Odss Ratio 3.5 with an interval of 95% confidence 1.67.8) and P < 0.001. Conclusion: PCT values in cancer patients with FN and infections; was higher and descend after restoring antibiotic treatment agreeing with clinical improvement. These data suggest it determination of PCT in cancer patients with FN; could be an useful early diagnostic marker for detection of bacteriemia
aspirate cultures; 26% were positive for antigenuria. Mean SOFA: 9 (SD 3.9);APACHE II: 20 (SD 8.5). Length of stay: 11.1 days. Mortality:14.7%. On admission day, PCT mean value: 23.7 ng/mL and CRP: 20.9 mg/dL. On day 1 serum PCT revealed a significant correlation with APACHE II index (P < 0.05) and was positive for blood cultures (P < 0.05).But, CRP value did not reveal these correlations. There was no relation either between PCT and SOFA index or with mortality. Conclusions: This study revealed that even though PCT is related to APACHE II severity index, it is not related to ICU mortality. We found a significant correlation between PCT value and positive blood cultures, as mentioned in previous publications.
P14-04 The role of procalcitonin in sepsis patients with pneumonia Cachapuz I (1), Amaro A (2), Granja C (2), Alves V (1) (1) Matosinhos Local Health Care Unit, Clinical Pathology Service, Oporto, Portugal (2) Matosinhos Local Health Care Unit, Intensive Care Medicine Service, Oporto, Portugal
P14-05 Extended spectrum Beta-lactamases and AmpC Beta lactamases producing uropathogenes among children Deo P Monash University, Department of Biochemistry and Molecular Biology, Melbourne, Australia
Corresponding author: [emailprotected] Corresponding author: [emailprotected]
Background: The value of procalcitonin (PCT) in acutely ill sepsis patients has been studied and the use of this biomarker revealed its clinical relevance in ICU setting. The study purpose was to evaluate the role of PCT on septic patients management with community-acquired pneumonia (CAP) and invasive ventilator-associated pneumonia (IVAP). Materials and methods: Prospective study. Adults admitted in ICU (April - December 2010) with CAP and/or IVAP diagnosis. PCT and C-reactive protein (CRP) were dosed daily. All patients underwent microbiological testing. Severity indexes were assessed. SPSS(v16) was used for statistical analysis. Results: Total of 43 patients: 37 with sepsis related to CAP; 6 with sepsis related to IVAP. 74%male, average age:57 years old. 70% of the cases had a septic shock and 21% had severe sepsis. Blood cultures were positive in 11.6%; 25.6% had positive tracheal Biochemia Medica 2012;22(3):A54-A204
A150
Objectives: UTI are common in childhood. Gram negative bacilli are the common isolates from the UTI. ESBL and AmpC beta-lactamase are the most significant enzymes involved in conferring resistance to Beta-lactam antibiotics in Gram negative bacteria. This study was aimed to find out the prevalence of multidrug resistant (MDR), ESBL and AmpC beta-lactamases producing isolates among children with UTI in Nepal. Materials and methods: A prospective study was carried out from July, 2006 to March, 2008 at IOM,Kathmandu, Nepal). 820 urine specimens were obtained from clinically suspected UTI children (age < 12 years, female to male ratio 2.1:1). Most of samples were midstream urine, 25 supra pubic aspiration and 4 from catheter. Culture, organism identification and antibiotic susceptibility
2nd European Joint Congress of EFLM and UEMS
test were done by following the protocol of American Society for Microbiology (ASM). Results: Among 820 urine samples, 23.51% (201/820) had significant bacterial growth with 184 (91.54%) non-repeat gram-negative isolates in which most were E. coli (58.15%) followed by Klebsiella species (15.2%).The prevalence of MDR, ESBL and AmpC were 115 (62.5%), 43 (23.36%) and 15 (8.15%) respectively. Maximum incidence of ESBL producer was E. coli (39.5%) followed by Klebsiella (16.2%) and Pseudomonas species (13.9%). Highly AmpC producing species were Klebsiella (40%) and Pseudomonas (26.6%). ESBL producers and non producers MDR isolates were highly resistant to amoxycillin-clavunic acid, aztrenome, cefepime and ceftazidime-clavunic acid. Imepenum and pipracilin were most effective drug among ESBL producers and non producers. Infection was more common in age group 6 ± 2.3 years with female to male ratio 2.04:1. Conclusion: Result shows high percentage (62.5%) of MDR pathogens in childhood UTI.
P14-06 Mass spectrometry in identification of antibiotic resistance Pilatova K (1), Frostova T (1), Dolejska M (2), Budinska E (3), Sterba J (4), Zdrazilova Dubska L (1) (1) Masaryk Memorial Cancer Institute, Department of Laboratory Medicine, Brno, Czech Republic (2) University of Veterinary and Pharmaceutical Sciences, Department of Biology and Wildlife Diseases, Brno, Czech Republic (3) Masaryk University, Institute of Biostatistics and Analyses, Brno, Czech Republic (4) Clinic of Pediatric Oncology, Children's Medical Center, Brno, Czech Republic
major problems. Currently, mass spectrometry (MS) represents new approach for bacterial identification. The goal of this study is assessment of correlation between MS profile and bacterial antibiotic fenotype. Materials and methods: We analyzed 56 isolates of Enterobacteriacea obtained from children treated for malignant disease. Bacteria were cultivated on agar plates; antibiotic susceptibility was tested using disc diffusion method, ESBL strains by double-disc synergy test. Sample preparation for SELDI-TOF MS: one bacterial colony was resuspended in 100 μL of distilled water and frozen at -70 °C. After thawing, suspension was applied on protein gold chip and after drying, covered twice by sinapinic acid matrix. Measurement was carried out on SELDI-TOF MS (Ciphergen) in the range of m/z = 3000 - 20000. Results: We were able to clearly distinguish between individual bacterial species on the basis of cluster analysis of acquired protein spectra. We also found peaks which differ significantly in individual strains and correlates with antibiotic fenotype in Klebsiella pneumoniae. Conclusions: Our pilot data show that protein profiling of bacteria using mass spectrometric methods allows discovery of new antibiotic resistance biomarkers and might represent new approach for identification of resistant bacteria strains as alternative method for current cultivation and molecular biology methods. This study was supported by European Regional Development Fund (RECAMO; CZ.1.05/2.1.00/03.0101) and by the Czech Science Foundation (grant P502/10/ P083).
Corresponding author: [emailprotected]
Background: The assessment of antibiotic resistance is essential for appropriate treatment initiation of infection disease. Increasing frequency of multiresistant bacterial strains and development of new antibiotic resistance mechanisms represent Biochemia Medica 2012;22(3):A54-A204
A151
2nd European Joint Congress of EFLM and UEMS
P14-07
P14-08
Prevalence of syphilis among blood donors in years 2009-2011 and our experiences with Abbott and Siemens reagents
HBV pre-existing immunity of healthcare workers in occupational exposures in University Hospital Dubrava
Sopić T
Serdar T (1), Markovic D (2), Marijancevic D (1), Petrovecki M (3), Romic Z (1)
University Clinical Centre of Maribor, Centre of Transfusion Medicine, Maribor, Slovenia Corresponding author: [emailprotected]
Background: Syphilis is sexually transmitted disease, caused by spirochaete organism Treponema pallidum. Although rare, it can be transmitted with blood transfusion and also is a good indicator of high risk sexual behaviours. Testing blood donors for syphilis is mandatory in most countries of the world. Materials and methods: Specimens of blood donors were tested with Abbott reagents on Architect with CMIA (Chemiluminiscent Immunoassay) method and with Siemens reagents on BEP 2000 with EIA (Enzyme Immunoassay) method. Initially reactive samples were tested in duplicate. Repeatedly reactive samples were sent to confirmatory testing in reference laboratory. Results: In years 2009-2011 we have tested 94,328 blood donations, 42,543 with Abbott reagents and 51,785 with Siemens reagents. There were 97 (0,23 %) initially reactive and then 93 (0.22%) repeatedly reactive with Abbbott reagents and 62 (0.12%) initially reactive and then 43 (0.08) repeatedly reactive with Siemens reagents. Confirmatory testing was positive for 17 (0.02 %) samples. Conclusion: All confirmed positive results were reactive with Abbott and Siemens reagents, so both are suitable for screening for syphilis. We noticed more more false reactive results with Abbott reagents, then with Siemens reagents. The prevalence of syphilis among blood donors is staying the same in the last three years. Biochemia Medica 2012;22(3):A54-A204
A152
(1) University Hospital Dubrava, Clinical Department for Laboratory Diagnostic, Zagreb, Croatia (2) University Hospital Dubrava, Department for Clinical Microbiology and Hospital Infections, Zagreb, Croatia (3) Clinical Department for Laboratory Diagnostics, University Hospital Dubrava, Zagreb, Department of Medical Informatics, Rijeka University School of Medicine, Rijeka, Croatia Corresponding author: [emailprotected]
Background: Occupational exposure in healthcare workers represents every contact with a material that carries the risk of acquiring an infection during the working activities. Viral infections are the main blood transmitted infections and the most frequent are hepatitis B virus (HBV), hepatitis C virus (HCV) and human immunodeficiency virus (HIV). Materials and methods: Since 2002, every reported occupational exposure of healthcare worker was registrated by the Committee for hospital infections of University Hospital Dubrava. Database among others contains qualifications of the exposed worker and his pre-existing immunity to HBV (aHBs-titer). Results: In 10 year period of occupational exposures monitoring (from 2002 to 2011) 451 cases were reported. The majority of occupational exposures were reported by nurses or medical technicians (55.4%), followed by medical doctor resident (12.6%), medical doctor specialist (8.0%), cleaner (6.9%), laboratory technician (3.3%) and others (13.8%). In 59.4% of the exposed healthcare workers aHBs-titer status was assessed as satisfactory (aHBs > 100 IU/L), in 19.5% was not satisfactory (aHBs < 100 IU/L) and in 21.1% was not measured. Conclusion: The 10-year follow-up of occupational exposures in healthcare workers in University Hospital Dubrava has shown an increase in the
2nd European Joint Congress of EFLM and UEMS
number of reported events. Implementation of preventive measures, such as universal precaution measures and HBV vaccination results a significant reduction in the incidence of HBV infection among healthcare workers. In many countries, the number of healthcare workers that underwent HBV vaccination usually does not exceed 65%, indicating that HBV vaccination is not applied in a sufficient number of healthcare workers.
P14-09 Trends in antibiotics prescribed for bacterial pneumonia with higher CRP in preschool children Bajraktarevic A (1), Kaljic Junuzovic A (1), Miokovic M (1), Penava S (1), Mulabegovic J (2), Delic M (3) (1) Public Health Institution of Canton Sarajevo, Pediatrics department, Sarajevo, Bosnia and Herzegovina (2) Pharmacy Faculty, Department for Biochemistry and Clinical Pharmacologz, Sarajevo, Bosnia and Herzegovina (3) Krankenhaus Hietzing Stadt Wien Mit Neurologishem Zentrum Rosenhögel, Insitute Für Labormedizin Mit Serologie Und Infektiondiagnostik Mit Ambulanz , Vienna, Austria Corresponding author: [emailprotected]
Objective: To estimate the incidence rates of preschool children outpatient bacterial pneumonia, examine time trends in antibiotics prescribed for bacterial pneumonia with higher CRP, and determine factors associated with broad-spectrum antibiotic prescribing for pneumonia in this population.
children were cephalosporins, macrolides, penicillins and aminoglycoside antibiotics. Cephalosporins were most commonly prescribed, ranging from 33.3% to 44.5% of all antibiotics prescribed for pneumonia in ages from 6 months to 6 years. Macrolides were the second most commonly prescribed antibiotic, ranging from 22.5% to 35.3% of all antibiotics prescribed for this diseases. There was no statistical difference in serum CRP values among the four groups with pneumonia after antibiotics therapy. Conclusions: A strong association has been found between the level of circulating C-reactive protein (CRP) and the severity of pneumonia and success of antibiotics therapy in control laboratory data after three or four days in outpatients conditions .
P15 - Molecular diagnosis 1 P15-01 The association of postprandial triglycerides with hsCRP, TAS, ICAM-1 and APOA5 and HL gene variants Kackov S (1), Simundic AM (2), Nikolac N (2), Celap I (2), Dukic L (2), Bilusic M (1) (1) Policlinic Bonifarm, Medical biochemistry laboratory, Zagreb, Croatia (2) Sestre milosrdnice University Hospital Center, University Department of Chemistry, Zagreb, Croatia Corresponding author: [emailprotected]
Materials and methods: The material consists of 902 small preschool Bosnians children aged 6 months to 6 years or half year more who took part in this retrospective study in pediatrics settings in six municipalities from nine municipalities of Canton Sarajevo. Within 72 or 96 hours after establition of diagnosis and beginning of antibiotics therapy blood samples CRP were taken for first and control analysis.
Background: Several authors have reported the association of postprandial hypertriglyceridemia with oxidative stress, systemic inflammation and endothelial dysfunction. Our aim was to investigate the effect of postprandial hypertriglyceridemia on oxidative stress and endothelial dysfunction. We also assessed the association of APOA5 -1131T/C and -250G/A hepatic lipase polymorphisms with different response to the highcalorie meal in the group of healthy middle aged male individuals.
Results: The four most commonly prescribed antibiotic classes for bacterial pneumonia in preschool
Materials and methods: We recruited 102 healthy male volunteers (52-68 years). All participants conBiochemia Medica 2012;22(3):A54-A204
A153
2nd European Joint Congress of EFLM and UEMS
sumed a high-calorie meal (800 calories, 50 g fat, 28 g protein, 60 g carbohydrates). Blood samples were drawn at 8 a.m., after an overnight fast and 3 hours after the meal. Glucose, total cholesterol, triglycerides, HDL-cholesterol, LDL-cholesterol, bilirubin, uric acid, hsCRP, TAS and ICAM-1 were measured at fasting state and postprandially. APOA5 -1131T/C and -250G/A hepatic lipase promoter genetic polymorphisms were determined for all participants. Results: Postprandial triglycerides were significantly increased (1.4 (1.1 - 2.1) vs. 2.4 (1.9 - 3.3) mmol/L, P < 0.001). Average triglyceride increase was 1.0 ± 0.7 mmol/L (65%). Although concentrations of triglycerides, HDL-cholesterol, LDL-cholesterol, TAS and ICAM-1 differed significantly between fasting state and postprandial measurements (P < 0.001), differences were within the limits of analytical imprecision and are not considered as clinically relevant. Other parameters did not change 3 hours after the meal. Triglycerides response did not differ respective to the APOA5 and HL polymorphisms. Conclusion: Postprandial hypertriglyceridemia is not associated with increased concentrations of hsCRP, TAS and ICAM-1. Furthermore, APOA5 and HL polymorphisms are not associated with different response of triglycerides.
P15-02 Association of three polymorphisms of scavenger receptor class BI gene in with coronary stenosis Bouslama A Sahloul University Hospital, Biochemistry, Sousse, Tunisia Corresponding author: [emailprotected]
Background: The potential role of scavenger receptor class BI in the regulation of lipoproteins metabolism and atherosclerosis has attracted considerable interest. We tested the relationship of Biochemia Medica 2012;22(3):A54-A204
A154
three SCARB1 polymorphisms with significant coronary stenosis (SCS) and lipid profile in a coronary Tunisian population. Materials and methods: Three SCARB1 polymorphisms (exon8 (C/T), exon1 (G/A), intron5 (C/T)) were studied in 316 tunisians undergoing coronary angiography. SCS was defined as a luminal narrowing of ≥ 50% in at least one major coronary artery. Lipid profile was measured. Genotyping was performed using PCR-RFLP. Statistical analysis was performed by SPSS. Results and conclusion: TT genotype of exon8 was associated with higher concentrations of HDLC and ApoAI in the group without SCS. The T allele of exon 8 was associated with 41% lower risk of SCS. This protective effect seemed to be particularly significant in women, non diabetics and nonsmokers. The T allele of intron 5 was associated with an increased risk of SCS, particularly in smokers. AA genotype of exon1 was associated with an increased risk of SCS in diabetics and in patients with metabolic syndrome. The (CAT) haplotype was associated with an increased risk of SCS compared to the wild haplotype and had a 4-fold greater risk of SCS than patients with haplotype (TGC) which seems to be the most protective against SCS. The T allele of exon 8 in SCARB1 seemed to increase the HDL-C and ApoAI concentrations and reduce the risk of SCS. The intron 5, exon 1 polymorphisms and (CAT) haplotype seemed to have an atherogenic effect.
2nd European Joint Congress of EFLM and UEMS
P15-03 Six lipoprotein lipase gene polymorphisms, lipids and coronary stenosis in a Tunisian population
HindIII and D9N polymorphisms and CTGTAG haplotype seem to be considered as marker of coronary stenosis. In another hand, HindIII and haplotypes were related to coronary stenosis severity.
Rebhi L (1), Kchok K (1), Omezzine A (1), Rejeb J (1), Boughzala E (2), Bouslama A (1)
P15-04
(1) Sahloul University Hospital, Biochemistry, Sousse, Tunisia (2) Sahloul University Hospital, Cardiology, Sousse, Tunisia
Toll-like receptors tlr-2 and tlr-4 gene polymorphisms in patients with cerebral atherosclerosis
Corresponding author: [emailprotected]
Background: Lipoprotein lipase (LPL) is the ratelimiting enzyme in the hydrolysis of triglyceriderich lipoprotein particles. LPL polymorphisms’ effects on lipids and coronary artery disease are controversial among studies and populations. Our aim was to study the association between six polymorphisms and significant coronary stenosis (SCS), disease severity and lipid parameters in Tunisian patients. Materials and methods: LPL PvuII, 93 T/G, 188 G/E, HindIII, N291S and D9N polymorphisms were analyzed in 316 patients who underwent coronary angiography. SCS was defined as the presence of stenosis ≥ 50% in at least one major coronary artery. The stenosis severity was determined by using Gensini score (GS). Results and conclusions: A significant association of SCS with TT and TG genotypes of the HindIII polymorphism was showed: OR = 2.84, 95% CI, 1.19-7.40, P = 0.017; OR = 1.77, 95% CI, 1.99-2.82, P = 0.033), respectively. The TT genotype was significantly associated with increased triglyceride level and ApoB/ApoA-I ratio and with decreased HDL-C. Haplotype analysis showed that OR of SCS associated with the CTGTAG haplotype was 2.12 (95% CI 1.05-4.25,P = 0.032) and with CGGGAA was 0.71 (95% CI 0.26-1.95, P = 0.022) compared to the CTGTAA. Significant difference in GS was observed among HindIII genotypes and haplotypes. A significant association between the mutated genotype of HindIII polymorphism with decreased HDL-C level and increased ApoB/ApoA-I ratio and triglyceride level was showed. Our results suggest that
Galovic R (1), Flegar-Mestric Z (2), Matokanovic M (3), Brajsa K (4), Vidjak V (5), Barisic K (3) (1) University Hospital Centre Zagreb, Clinical Dept. of Laboratory Diagnostics, Zagreb, Croatia (2) University Hospital Merkur, Institute of Clinical Chemistry and Laboratory Medicine, ZAGREB, Croatia (3) University of Zagreb Faculty of Pharmacy and Biochemistry, Dept. of Medical Biochemistry and Hematology, Zagreb, Croatia (4) Galapagos Research Center Ltd., Research Center, Zagreb, Croatia (5) University Hospital Merkur, Institute for Radiology, Zagreb, Croatia Corresponding author: [emailprotected]
Background: The innate immunity proteins TLR-2 and TLR-4 are possible connection between immune response and inflammation involved in the pathogenesis of atherosclerosis. Previous studies suggest that tlr-2 and tlr-4 gene polymorphisms could contribute to the differences in the disease development. Materials and methods: A group of patients with cerebral atherosclerosis, evaluated by digital subtraction angiography, with > 50% stenosis of cerebral artery (N = 47) was compared to the control group (N = 27) concerning single nucleotide polymorphisms G/A753 in the tlr-2 gene and A/G299 in the tlr-4 gene, determined by the real-time PCR. Serum proinflammatory cytokines IL-6 and TNF-α, were determined by enzyme immunoassays. Results: Examined polymorphism of tlr-2 gene was present in 3.7% and 5.3% subjects of the control and of the cerebral atherosclerosis group, respectively, while polymorphism of tlr-4 in 9.3% and 8.5% subjects, respectively. There was no difference in Biochemia Medica 2012;22(3):A54-A204
A155
2nd European Joint Congress of EFLM and UEMS
proportions of the polymorphisms between the groups. The concentrations of IL-6 and TNF-α were higher in the cerebral atherosclerosis than in the control group (1.54 pg/mL, (1.04-3.50 pg/mL) vs. 1.21 pg/mL, (0.45-2.32 pg/mL), P = 0.048 and (0 pg/ mL (0-1.8 pg/mL) vs. 0 pg/mL, (0-0 pg/mL), P = 0.039, respectively), presented as median (1-3 quartile).
0.221, mean age 48.41 ± 10.92 years, mean BMI 36.6 ± 4.8 kg/m2). Genotyping was performed using PCR-RFLP. Lipids parameters were measured. BMI and HOMA-IR were calculated. MetS was defined according to IDF-2005, obesity was defined according to WHO-1995. The study was approved by the Medical Hospital Ethic Committee.
Conclusion: Higher concentrations of IL-6 and TNF-α in the cerebral atherosclerosis group are indicators of inherent inflammation. Single nucleotide polymorphisms G/A573 of the tlr-2 and A/ G299 of the tlr-4 gene had no influence on the concentrations of circulating IL-6 and TNF-α. The polymorphisms were not significant for the diagnosis of cerebral atherosclerosis with more than 50% stenosis of cerebral arteries.
Results and conclusion: 420GG were associated with higher waist circumference and BMI. 44G/A polymorphism was associated with increased total cholesterol and LDL-C levels. The others genotypes showed no association with all MetS parameters. Concerning association between SNPs and MetS risk, only mutated genotypes at 44G/A increase the risk of MetS after adjustment to confounders parameters (OR = 1.93, P = 0.023). About obesity risk, only 420C/G seems to contribute in obesity. Adjusted ORs of obesity associated to mutated genotypes were 2.17, 95%CI [1.28-3.68]; P = 0.004.
P15-05 Four resistin polymorphisms, metabolic syndrome parameters and obesity risk in Tunisian volunteers Boumaiza I, Omezzine A, Rejeb J, Rebhi L, Ben Rejeb N, Bouslama A Sahloul University Hospital, Biochemistry, Sousse, Tunisia Corresponding author: [emailprotected]
Background: Resistin is a protein hormone produced by adipocytes. Some studies found increased circulating resistin levels and its mRNA expression in adipose tissue in patients with obesity. While other studies failed to confirm this finding. Genes encoding adipokines are important functional candidates for development of obesity. Many resistin gene polymorphisms were described and their implication in obesity and metabolic syndrome (MetS) was controversial. Our aim was to study the relationship between four resistin polymorphisms (420C/G, 44G/A, 62G/A and 394C/G) and MetS parameters and the risk of obesity in Tunisian volunteers. Materials and methods: We have recruited 169 non obese BMI < 30 kg/m2 (sex-ratio = 0.594, mean age 43.25 ± 13.12 years; mean BMI 24.73 ± 3.50 kg/ m2) and 160 obese BMI ≥ 30 kg/m2 (sex-ratio = Biochemia Medica 2012;22(3):A54-A204
A156
P15-06 Simultaneous detection of mutations within genes associated with familial hypercholesterolemia Latten M (1), Murray H (1), Graham C (2), Martin R (2), Crockard M (1), Fitzgerald S (1) (1) Randox Laboratories Limited, Molecular Diagnostics, Crumlin, United Kingdom (2) Northern Ireland Regional Genetics Centre, Belfast Health and Social Care Trust/ City Hospital, Belfast, United Kingdom Corresponding author: [emailprotected]
Background: Familial Hypercholesterolemia (FH) is a genetic disorder characterised by high levels of low density lipoprotein in the cardiovascular system and early onset of cardiovascular disease. Currently in the UK, 1 in 500 people suffer from FH, with 85% of this population un-diagnosed. The genes apo-lipoprotein B, low density lipoprotein receptor, and proprotein convertase subtilisin/kexin type 9 are known to be associated with FH. This study reports the development of an assay enabling the simultaneous analysis of 20 common mutations within these genes.
2nd European Joint Congress of EFLM and UEMS
Materials and methods: The assay is based on a combination of two multiplex PCRs and biochip array hybridisation. Innovative PCR priming technology permits high discrimination between multiple wild-type and mutant DNA regions, which in combination with spatially organised biochip array technology increases the multiplexing capacity of the assay. Dedicated software processes results automatically, with analysis completed within 3 hours, from template DNA. Results: Assay specificity was confirmed using DNA from 100 FH positive patient samples. Subsequently, a further 100 blinded samples were assessed with a correlation of 98% with patient samples that had previously been sequenced. The cohort included patient samples containing the 20 common mutations. Conclusions: Data indicates applicability of this assay for the rapid simultaneous analysis of 20 common mutations within three genes associated with FH. Treatment from adolescence age with lipid modifying drug therapy, combined with lifestyle changes, can restore normal life expectancy. Therefore, this assay can be used as an analytical tool to facilitate FH diagnosis.
P15-07 Apolipoprotein A5 genetic polymorphisms and fasting serum lipidogram in elderly subjects with MetS Feher Turkovic L (1), Pasalic D (2), Pavlovic M (3) (1) University of Applied Health Studies-Zagreb, Department of Chemistry, Biochemistry and Clinical Chemistry, Zagreb, Croatia (2) University of Zagreb, School of Medicine, Department of Medical Chemistry, Biochemistry, and Clinical Chemistry, Zagreb, Croatia (3) Institute for Medical Research and Occupational Health, Department of Occupational Health and Environmental Medicine, Zagreb, Croatia
lipidemia, hyperglycemia, and hypertension. It represents a risk factor for many disorders. Apolipoprotein A5 which is located on VLDL and HDL regulates the concentration of triglycerides. Therefore, genetic isoforms might affects triglyceride concentration and contribute in pathogenesis of MetS. Materials and methods:The cross-sectional study included 155 men and 187 women older than 70 years. Fasting serum concentration of biochemical parameters were determined by standardized methods. International Diabetes Federation criteria was used for determination of MetS. Two Apo A5 genetics polymorphisms (c.1259T>C-SNP1 andS19W) were genotyped using PCR-RFLP method with detection of fragments with chips on Agilent 2100 bioanalyzer. Results: SNP1 genotype frequencies for T/T, T/C and C/C genotype were 183, 41 and 1 in MetS(+), and 96, 15 and 11 in MetS(-), respectively, (P = 0.478). S19W genotype frequencies for SS, SW and WW were 184, 41 and 2 in MetS(+) and 92, 22 and O in MetS(-), (P = 0.586). Mean total-cholesterol concentration was significantly higher in SNP1-C allele carriers then in non-carriers for women in MetS(+) group (6.23 ± 1.36 and 5.58 ±1.14, respectively, P = 0.020). LDL-cholesterol was also significantly higher for same group and same allele (4.05 ± 1.04 and 3.53 ± 0.97, P = 0.026). In subjects with MetS(+), higher triglycerides concentration were observed in W-carriers than in non-carriers of S19W (median were 1.79 and 1.97, respectively, P = 0.050). Total-cholesterol were higher in men Wcarriers vs. non-carriers (5.55 ± 1.17 and 4.97 ± 1.11 respectively, P = 0.048). Conclusions: Apo A5 genetic polymorphisms SNP1 and S19W are associated with dislypidemia in elderly subjects with MetS.
Corresponding author: [emailprotected]
Background: Metabolic syndrome (MetS) is a cluster of metabolic abnormalities as are obesity, dysBiochemia Medica 2012;22(3):A54-A204
A157
2nd European Joint Congress of EFLM and UEMS
P15-08 Adiponectin gene variants, and gene-environment interactions as predictors of early central obesity Karmelić I (1), Lovrić J (1), Sertić J (2), Božina T (1), Božina N (2), Jelaković B (3) (1) School of Medicine, Department of Chemistry, Biochemistry and Clinical Chemistry, Zagreb, Croatia (2) University Hospital Centre Zagreb, Department of Laboratory Diagnostics, Zagreb, Croatia (3) University Hospital Centre Zagreb, Department of Internal Medicine, Division of Nephrology and Arterial Hypertension, Zagreb, Croatia Corresponding author: [emailprotected]
Background: Adiponectin is an adipose tissue-derived adipokine linked to central obesity and ADIPOQ variants are promising markers for understanding the genetic base of obesity. We aimed to evaluate the relation of adiponectin concentrations and ADIPOQ gene variants to abdominal obesity and hypertension in young subjects. In addition, influence of the gene-environment (diet) interaction was estimated. Materials and methods: The study included 149 subjects. Clinical examination and anthropometric measurements were done. Adiponectin levels were estimated by ELISA assay. ADIPOQ -11377C>G and -11391G>A were genotyped by real-time PCR. Results: Waist circumference, systolic and diastolic blood pressure showed inverse correlations with adiponectin concentrations. ADIPOQ -11377GG and -11391GA significantly increased the risk for the development of central obesity (OR 5.57 and OR 3.37, respectively). The test of overall association showed significant correlation of central obesity with -11377C>G and -11391G>A haplotypes (P < 0.001). We found a significant association of -11391G/A variants with triglycerides and BMI, with A allele more frequent in subjects with BMI ≥ 25 kg/m2 (P = 0.021), while GG genotype predispose for lower concentrations of triglycerides (P = 0.005). A significant correlation was found for -11377GG variant with the concentration of total Biochemia Medica 2012;22(3):A54-A204
A158
cholesterol (G/G vs. other P = 0.043) and hypertension (P = 0.035), where -11377G allele carriers have significantly higher risk for elevated blood pressure (OR 2.73). When a diet was introduced as a covariable, correlation was significant between -11391G>A and HDL-C only (P = 0.015). Conclusion: Analysis of adiponectin concentration and ADIPOQ -11391G>A and -11377C>G promoter gene variants could be clinically meaningful for estimation of obesity and obesity-related syndrome risk in young adult population.
P15-09 Gender-specific effects of PPARG, APOE, ACE, LPL, IL-6 and AT1R gene variants on metabolic syndrome Božina T (1), Sertić J (2), Lovrić J (1), Jelaković B (3), Merkler M (4), Reiner Ž (4) (1) School of Medicine, University of Zagreb, Department of Medical Chemistry, Biochemistry and Clinical Chemistry, Zagreb, Croatia (2) University Hospital Centre Zagreb, School of Medicine, University of Zagreb, Department of Laboratory Diagnostics, Department of Medical Chemistry, Biochemistry and Clinical Chemistry, Zagreb, Croatia (3) University Hospital Centre Zagreb, Department of Internal Medicine, Division of Nephrology and Arterial Hypertension, Zagreb, Croatia (4) University Hospital Centre Zagreb, Division of Metabolic Diseases, Zagreb, Croatia Corresponding author: [emailprotected]
Background: Metabolic syndrome (MS) is a cluster of modifiable risk factors including hypertension, abdominal obesity, dyslipidemia and insulin resistance, associated with nonmodifiable risk factors, such as age, sex and genetic background. We investigated the possible role of gene polymorphisms of PPARG (Pro12Ala), ApoE (ε2, ε3, ε4), LPL (P+/-), IL-6 (-174G>C), ACE (I/D) and AT1R (1166A>C) in MS. Materials and methods: 516 individuals were investigated including 263 patients with MS and 253 subjects without MS criteria. Genotyping was performed using PCR based methods.
2nd European Joint Congress of EFLM and UEMS
Results: In female group associations were found for: LPL and ACE with MS (P = 0.04); PPARG and LPL with blood pressure, (P = 0.04); LPL with cholesterol and LDL (P = 0.01 and P = 0.05, respectively). Significant gene interactions observed between: APOE and PPARG, ACE and APOE were associated with BMI (P = 0.01 and P = 0.05, respectively); LPL and PPARG were associated with triglycerides (P = 0.03). For males we found associations of: LPL variants with MS (P = 0.02), BMI (P = 0.002) and waist circumference (P = 0.008); PPARG and APOE with BMI (P = 0.05); IL-6 with CRP (P = 0.02). Significant gene interactions observed between: PPARG and AT1R were associated with blood pressure (P = 0.05); PPARG and APOE with triglycerides (P = 0.02); PPARG and APOE, PPARG and IL6 (P = 0.03), ACE and APOE (P < 0.001) with cholesterol; PPARG and LPL (P = 0.003), PPARG and IL6 (P = 0.06) with HDL; PPARG and IL6 (P = 0.01), ACE and APOE (P = 0.04); PPARG and APOE, LPL and ACE (P = 0.01), AT1R and ACE (P = 0.06) with CRP. Conclusion: Gene variants of PPARG, APOE, LPL, ACE, AT1R and IL-6 could be susceptibility factors of obesity, lipid status, and glucose intolerance.
P15-10 Contribution of 5-HTTLPR and BDNF gene variants to obesity risk Božina T (1), Sertić J (2), Lovrić J (1), Jelaković B (3), Jovanović N (4), Božina N (2) (1) School of Medicine, University of Zagreb, Department of Medical Chemistry, Biochemistry and Clinical Chemistry, Zagreb, Croatia (2) University Hospital Centre Zagreb, Department of Laboratory Diagnostics, Zagreb, Croatia (3) University Hospital Centre Zagreb, Department of Internal Medicine, Division of Nephrology and Arterial Hypertension, Zagreb, Croatia (4) University Hospital Centre Zagreb, Department of Psychiatry, Zagreb, Croatia
control of energy balance and is involved in several biological processes including mood, appetite, sleep, libido, memory, and body weight regulation. Brain-derived neurotrophic factor (BDNF) is also currently recognized as an important participant in the regulation of food intake. The aim of this study was to evaluate whether the 5-HTTLPR S/L and BDNF Val66Met gene variants are associated with obesity in a sample of adults of Croatian origin. Materials and methods: 462 individuals were investigated including 301 obese (BMI ≥ 30 kg/m2) and 161 lean (BMI < 25 kg/m2) (mean age ± SD was 49 ± 8 years). Genotyping of triallelic structure of 5-HTTLPR (LA, LG, S) and of BDNF Val66Met polymorphisms was performed using the RealTimebased allele specific PCR methods. Results: In the whole group we found no associations between 5-HTTLPR S/L and BDNF Val66Met polymorphisms and obesity. When we compared male and female samples, we observed statistically significant differences in the distribution of 5-HTTLPR genotypes: 5-HTTLPR LALA genotype was more frequent in the group of lean women comparing to the group of lean men (41% and 28% respectively, P = 0.022). The 5-HTTLPR S and BDNF Met allele carriers have higher risk to develop obesity (OR 2.07) than non carriers (P = 0.038). Discussion: Our findings indicate that 5-HTTLPR polymorphism may be linked with obesity in adult female population, reinforcing the role of the serotonin transporter as a risk factor for the obesity phenotype. SERTPR and BDNF gene interactions could additionally predispose to obesity risk.
Corresponding author: [emailprotected]
Background: 5-Hydroxytryptamine (5-HT, serotonin) plays an important role in the central nervous Biochemia Medica 2012;22(3):A54-A204
A159
2nd European Joint Congress of EFLM and UEMS
P15-11 ABCG2 gene variant and fluvastatin adverse drug reactions in renal transplant recipients Mirošević Skvrce N (1), Božina N (2), Pejnović L (2), Barišić I (3), Zibar L (4), Macolić-Šarinić V (1) (1) Agency for Medicinal Products and Medical Devices, Zagreb, Croatia (2) University Hospital Centre Zagreb, University of Zagreb School of Medicine, Department of Laboratory Diagnostics, Zagreb, Croatia (3) University Hospital Centre Zagreb, University of Zagreb School of Medicine, Department of Nephrology, Arterial Hypertension and Dialysis, Zagreb, Croatia (4) Clinical Hospital Center Osijek, University of Osijek School of Medicine, Department of Internal Medicine, Osijek, Croatia Corresponding author: [emailprotected]
Background: Polymorphisms in genes encoding drug transporters could be valuable pharmacogenetic markers. The ABCG2 efflux transporter is expressed in multiple tissues and plays an important role in the disposition of different drugs including statins. The functional 421C>A polymorphism in the ABCG2 that reduces transporter activity has been found to be associated with increased systemic exposures to certain statins, including fluvastatin. Although genetic variability in the ABCG2 distinctively affects the pharmacokinetics, there are no published data that would indicate that variability can result in fluvastatin induced adverse drug reactions (ADRs). The aim of this case-control study is to show the contribution of pharmacogenetic predisposition (ABCG2 gene variant) to the development of fluvastatin ADRs (myotoxicity, hepatotoxicity, other side effects) in renal transplant recipients. Patients and methods: 108 renal transplant recipients were included in the study, 54 patients with ADRs to fluvastatin therapy, and 54 controls without ADRs (matched according to fluvastatin dose, age, gender, concomitant therapy, and other conditions). Genotyping of the ABCG2 421C>A polymorphism was performed using the TaqMan allele-specific PCR assay (AppliedBiosystems). Biochemia Medica 2012;22(3):A54-A204
A160
Results: According to the statistical analysis, ABCG2 421CA genotype carriers have significantly higher incidence of ADRs to fluvastatin therapy comparing to ABCG2 421CC genotype carriers (OR 3.77, 95%CI 1.26-11.28, P = 0.024). Conclusion: Our data are the first to indicate there is an association between adverse drug reactions to fluvastatin therapy in renal transplant recipients with ABCG2 421C>A polymorphism.
P15-12 Association of soluble and -2518 A>G CCL2 polymorphism with inflammation markers and IR Guzman Ornelas M (1), Valdez Alvarado E (1), Aguilar Aldrete M (2), Sierra Amor R (3), Castro Albarran J (4), Navarro Hernandez R (5) (1) Universidad de Guadalajara, Doctorado en Ciencias Biomedicas, Centro Universitario de Ciencias de la Salud., Guadalajara, Mexico (2) Universidad de Guadalajara, Salud Publica, Centro Universitario de Ciencias de la Salud., Guadalajara, Mexico (3) Iniciativa Privada, Laboratorios LAB-QUIM, Veracruz, Mexico (4) Secretaria de Salud Jalisco, Nutricion, H.M.I.E.L.M., Guadalajara, Mexico (5) Universidad de Guadalajara, Biologia Molecular y Genomica, Centro Universitario de Ciencias de la Salud., Guadalajara, Mexico Corresponding author: [emailprotected]
Introduction: Insulin resistance (IR) is a disease with genetic susceptibility characterized by an abnormal inflammatory response. CCL2 is secreted by adipocytes, plays a central role in macrophage accumulation in white adipose tissue (WAT), promoting the inflammatory process, remain suggested to be involved in the progression of obesity to IR. The CCL2 gene is overexpressed in WAT and decreases insulin-stimulated glucose uptake into adipocytes. The -2518A>G polymorphism in the regulatory region of CCL2 may regulate gene expression. The aim was to investigate the relationship of CCL2 -2518A>G polymorphism and sCCL2 with inflammation markers and IR.
2nd European Joint Congress of EFLM and UEMS
Materials and methods: In a cross-sectional study we included 309 individuals Mexican-mestizo, classified by HOMA-IR index. Body composition, anthropometrics and inflammation markers were measured by routine methods, and -2518A>G variants by PCR-RFLP and sCCL2 by ELISA methods. Results: In this study group we found differences in: 1) sCCL2 levels (191 ± 14.9, 280 ± 21.6 ng/mL, P = 0.001); and 2) the genotype frequencies (AA: 33%, 30%; GA: 53%, 41% and GG: 14%, 29%, P = 0.007) between healthy and IR individuals, respectively. The G allele carriers showed lower measures (101 ± 9.7 cm) of hip circumference than the A allele carriers (104 ± 11.4 cm). While in IR individuals, the GG genotype carriers showed higher levels of: CRP, WBC and triceps skin fold thickness than the GA+AA genotype carriers. The sCCL2 levels showed correlations with CRP, glucose, sInsulin, HOMA-IR, weight, BMI and hip-circumference (r = 0.190 to 0.350, P < 0.05). Conclusions: We suggest that CCL2 allele -2518G is associated with inflammatory course and distribution of body fat in IR Mexican-mestizo.
P15-13 Association of PAI-1 5G allele with inflammation markers and body fat in Mexican with obesity Diaz-Rubio G, Navarro Hernandez R, Chavarria-Avila E, Aguilar-Uscanga B, Reyes-Blanco M, Ruiz-Quezada S
tor-1 (PAI-1) is the main inhibitor of fibrinolysis, representing approximately 60%. The sources of PAI-1 are endothelium cells, platelets, adipocytes and stromal cells of adipose tissue. The gene variants of PAI-1 are associated with cardiovascular diseases. Our aim was to investigate the relationship of PAI-1 4G/5G polymorphism with inflammation markers and body fat distribution in obesity. Materials and methods: In a cross-sectional study we included 179 individuals Mexican-mestizo, classified by BMI index. Body composition, anthropometrics and inflammation markers were measured by routine methods, 4G/5G polymorphism by PCR-RFLP, and insulin by ELISA methods. Results: In this study group we found differences in hsCRP levels (1.87 ± 0.36, 4.73 ± 1.64 mg/L, P = 0.031); and body-fat-index (2.17 ± 0.07, 2.54 ± 0.18, P = 0.032) between 5G/5G and 4G/4G genotype carriers. The hsCRP levels showed correlations with distribution and body fat mass (r = 0.259 to 0.557, P < 0.05). While, the genotype frequencies were (Lean: 16%, 41%, 43%; overweight: 12%, 48%, 40%; obese: 16%, 58%, 26%) for 4G/4G, 4G/5G and 5G/5G genotype carriers, respectively. In individuals classified as obesity, we found the following differences: total body fat (43% vs. 34%; P = 0.015), waist-hip-ratio (0.8710 ± 0.18 vs. 0.9328 ± 0.18; P = 0.024) and platelet-count (237.200 ± 10.595 vs. 288.500 ± 15.743; P = 0.010) in 5G/5G vs. 4G/5G + 4G/4G genotype carriers. Conclusions: We suggest that 4G/5G PAI-1 polymorphism may be associated with inflammatory process and body fat distribution in Mexican-mestizo individuals with obesity.
Universidad de Guadalajara, Departamento de Farmacobiologia, Centro Universitario de Ciencias Exactas e Ingenierias, Guadalajara, Mexico Corresponding author: [emailprotected]
Introduction: Comorbidity in obesity are diseases with genetic susceptibility and characterized by an inflammatory response. An association between obesity, inflammation and reduced fibrinolysis activity contributes to a higher risk of cardiovascular events. The plasminogen activator inhibiBiochemia Medica 2012;22(3):A54-A204
A161
2nd European Joint Congress of EFLM and UEMS
P16 - Molecular diagnosis 2 P16-01 Genotyping of 13 α-thalassaemia point mutations using primer extension reaction and dipstick assay
methods. The proposed method is simple, rapid, cost-effective, does not require specialized instrumentation or purification of the PCR products and could be a particularly useful tool in laboratories with limited resources.
Petropoulou M
P16-02
National and Kapodistrian University of Athens, Department of Chemistry, Athens, Greece
Descriptive study of genotype frequencies of Ala9Val polymorphism of SOD-Mn in chagasic patients
Corresponding author: [emailprotected]
Background: Alpha-thalassaemia is inherited as an autosomal recessive disorder characterized by a clinical phenotype varying from almost asymptomatic to a lethal hemolytic anemia. Reduced or absent a-globin synthesis, mainly caused by deletions of one or both a-globin genes and by point mutations, leads to a-thalassemia (a-thal). Numerous techniques have been developed for the identification of the underlying genetic defect in affected individuals. Materials and methods: The method consists of: (i) PCR amplification of a single fragment (1087 bp) of the a1 and a2 globin gene flanking all 13 mutations; (ii) three 10-plex primer extension reactions of the unpurified amplification product using allele-specific primers, carrying at their 5’end a unique recognition sequence. The reaction takes place in the presence of biotin-dUTPs and a DNA polymerase that lacks 3’→5’ exonuclease activity and (iii) dry-reagent multi-allele dipstick assay for visual detection of the primer extension reaction products within minutes. The detection of the products is achieved by naked eye using anti-biotin conjugated gold nanoparticles. Results and conclusions: Parameters that affect the performance of the PCR amplification, PEXT reaction and the multiallele biosensor were investigated in order to optimize the specificity and detectability of genotyping assay. The method was evaluated by analyzing 83 samples of known genotypes and the results were found to be fully concordant with those obtained by the reference Biochemia Medica 2012;22(3):A54-A204
A162
D'arrigo M (1), Gerrard G (1), Lioi S (1), Zumoffen C (1), Beloscar J (2) (1) Facultad de Ciencias Bioquimicas Y Farmaceuticas Universidad Nacional de Rosario, Bioquimica Clinica, Rosario, Argentina (2) Facultad de Ciencias Medicas Universidad Nacional de Rosario, Cardiologia, Rosario, Anguilla Corresponding author: [emailprotected]
Introduction: The existence of individuals infected with Chagas disease (CD) living in endemic areas without apparent heart damage shows that a proportion of them are able to contrarest the infection by Tripanosoma cruzi. Genetic factors of each patient ere actively involved in the evolution of clinical manifestations of CD. In this work we decided to do a descriptive study of genotype frequencies (GF) of Ala9Val polymorphism of SODMn and determine the enzyme activities of superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT) in chagasic patients (CP) with cardiomyopathy (CwC) and without cardiomyopathy (CnC) compared with healthy controls (HC). Materials and methods: The molecular characterization was performed by PCR-RFLP. Enzyme activities were determined by spectrophotometric methods. The hypothesis test under normal theory proportions and Kruskal Wallis test were carried out. Results: The GF of SOD-Mn (IC 95%) were: HC: Ala/ Ala: 0.54, Ala/Val: 0.33, Val/Val: 0.13; CwC: Ala/Ala: 0.35, Ala/Val: 0.30, Val/Val: 0.35; CnC: Ala/Ala: 0.36, Ala/Val: 0.46, Val/Val: 0.18. The enzyme activities
2nd European Joint Congress of EFLM and UEMS
were: CAT (K/gHb): HC: 185 ± 28, CwC: 316 ± 68, CnC: 332 ± 41; GPx (U/gHb): HC: 61 ± 1, CwC: 98 ± 17, CnC: 102 ± 20; SOD (USOD/gHb): HC: 895 ± 314, CwC: 3270 ± 833, CnC: 2590 ± 188. The study of SOD-Mn GF and the activities of CAT, SOD and GPx showed significant differences (P 1/year) attack frequency compared to the rest of patients. Conclusions: Our findings indicate that missense mutations of the C1INH gene are associated with favourable manifestation of the disease. Information on the mutation type can be used in clinical practice for predicting the severity of HAE.
P16-05 Serotonin transporter polymorphism (5-HTTLPR) in Croatian population Culej J (1), Štefanović M (2), Karlović D (3) (1) Sestre Milosrdnice University Hospital, Department of transfusiology and haemostasis - Clinic for tumors, Zagreb, Croatia (2) Sestre Milosrdnice University Hospital, Clinical Institute of Chemistry, Zagreb, Croatia (3) Sestre Milosrdnice University Hospital, Department of Psychiatry, Zagreb, Croatia Corresponding author: [emailprotected]
Background: Serotonin transporter is responsible for serotonin reuptake in the brain as well as other tissues. The most studied polymorphism is consisted of 44 base pairs deletion/insertion in the promoter region (5-HTTLPR) that classifies alleles into „short“ (S) or „long“ (L) variant. Short allele is associated with reduced transcriptional acBiochemia Medica 2012;22(3):A54-A204
A164
tivity. Some previous family based studies support thesis of genetic basis of psychiatric disorders. According to previous research, this polymorphism is related to depression and other psychiatric disorders, as well as with response to selective serotonin reuptake inhibitors treatment. Genetic heterogeneity of population might cause misleading association between cases and controls in case-control studies. The aim of this study was to assess frequency of S and L alleles in Croatian population. Materials and methods: 307 healthy individuals of both sexes, (age 18 to 50) were included in this study. Their physical and psychical condition was assessed by physical examination, and MINI psychiatric interview for mental disorders exclusion. Genotyping was performed by simple PCR reaction followed by the gel electrophoresis which is used for separation of 375 bp (allele S) from 419bp (allele L). Results: Genotype frequencies were in accordance with Hardy-Weinberg equilibrium (P = 0.114). L allele frequency is 59% and for S allele 41%. Frequency distribution for studied genotype is: LL – 37%; LS – 44%; SS – 19%. Conclusion: Based on Hardy – Weinberg equilibrium, investigated population is homogenous and reported genotype frequencies are in accordance to some other studies performed on European population.
2nd European Joint Congress of EFLM and UEMS
P16-06 Q192R and L55M pon1 gene polymorphisms associated with peripheral arterial disease Kardum Paro M (1), Perkov S (1), Flegar- Meštrić Z (1), Vidjak V (2), Novačić K (2), Barišić K (3) (1) Merkur University Hospital, Institute of Clinical Chemistry and Laboratory Medicine, Zagreb, Croatia (2) Merkur University Hospital, Institute for Radiology, Zagreb, Croatia (3) University of Zagreb, Faculty of Pharmacy and Medical Biochemistry, Zagreb, Croatia Corresponding author: [emailprotected]
Background: Development of peripheral arterial disease (PAD) could be due to human serum paraoxonase (pon1) gene polymorphisms or lower catalytic concentrations of serum paraoxonase-1 (PON1). The aim of study was to examine the association of pon1 gene polymorphisms (Q192R and L55M) with the catalytic concentrations of serum PON1 in patients with PAD. Materials and methods: Healthy subjects (118) and patients with angiographically confirmed diagnosis of PAD (110) were investigated. The catalytic concentration of serum PON1 was determined by spectrophotometric method using paraoxon as the supstrate in the presence of NaCl. pon1 gene polymorphisms were determined by PCR-RFLP method accredited according to ISO15189. The assay performance was assessed by interlaboratory specimens exchange. Allele and genotype frequencies were compared by the χ2 or Fisher exact test. Results: For pon1 gene polymorphism Q192R allele and genotype frequency were significantly different betweeen patients with PAD and healthy subjects (both < 0.001), while for pon1 gene polymorphism L55M allele (P = 0.649) and genotype (P = 0.327) frequencies did not differ significantly. The catalytic concentrations of serum PON1 were significantly lower in patients with PAD (P = 0.001). In both groups studied the lowest catalytic concentration were found in QQ and MM genotypes of pon1 gene. Conclusions: Lower catalytic concentrations of serum PON1 and Q192R pon1 gene polymorphism
could play a role in the development of PAD. Differences in the Q and R allelle frequencies between healthy subjects and patients with PAD may be one of the causes for the reduced catalytic concentrations of serum PON1.
P16-07 Platelet receptor for von Willebrand factor: gene polymorphism and cerebrovascular disease Laskovets A (1), Zaytseva N (1), Goldobin V (2), Klocheva E (2), Vavilova T (1), Sirotkina O (3) (1) North-West State Medical University named after Mechnikov, Clinical Laboratory Diagnostics, St-Petersburg, Russia (Russian Fed.) (2) North-West State Medical University named after Mechnikov, Neurology, St-Petersburg, Russia (Russian Fed.) (3) B.P. Konstantinov Petersburg Nuclear Physics Institute, Human Molecular Genetics, St-Petersburg, Russia (Russian Fed.) Corresponding author: [emailprotected]
Objective: Platelet activation and aggregation are pivotal in the pathogenesis of cerebrovascular disease. The aim of our study was to determinate the von Willebrand (vWF) factor platelet receptor gene polymorphism in patients with different pathogenetic variants of stroke. Materials and methods: We examined the T(-5)C and Thr145Met GP Iba in 47 healthy controls and 123 patients with stroke due to macroangiopathy (1 group), microangiopathy (2 group) and subjects with pathological tortuosity of brachiocephalic arteries, but without ischemic stroke (3 group) by PCR-RFLP. Results: The analysis of T(-5)C GP Iba showed the following genotypes distribution: 75.6% and 24.4%; 65.9% and 34.1%; 82.9% and 17.1%; 78.7% and 21.3% for TT and TC in 1, 2, 3 and control groups, respectively. There was no CC genotype in investigated subjects. The distribution of T(-5)C GP Iba genotype between patients and controls was not significantly different. However, the significant difference was observed in the Thr145Met GP Iba Biochemia Medica 2012;22(3):A54-A204
A165
2nd European Joint Congress of EFLM and UEMS
genotypes’ frequencies – 73.2%, 22.0%, 4.8% and 89.4%, 10.6%, 0% for ThrThr, ThrMet and MetMet in 1 group and controls, respectively (P = 0.045). The Thr145Met GP Iba genotypes’ frequencies were not significantly different in 2 and 3 groups – 87.8%, 9.8%, 2.4% and 90.2%, 9.8%, 0%. Thereby, the presence of the 145Met GP Iba allele in the homo-or heterozygous state was a risk factor for stroke due to macroangiopathy (OR = 3.08 [95% CI 1.02-9.33]). Conclusion: The prevalence of the Thr145Met GP Iba in patients with macroangiopathy suggests an association of carriage of this allele and the development of ischemic stroke.
coma Phyllodes). Microsatellites were studied in these tumors too. Results: The whole study of BRCA1 and BRCA2 genes was negative for mutations and deletions/ insertions. Sequences of TP53 in germ-line showed a deletion of 9 nucleotides in exon 5 (c.436del9), but it maintains the open reading frame. The deletion is located in binding DNA domain. However, not deletereus effect has been predicted in silico analysis. The study of the TP53 protein by immunohistochemistry (IHC) in the Cystosarcoma Phyllodes showed high expression of the protein. Microsatellites and the sequencing of TP53 in tumors showed the loss of the normal allele (LOH).
Li-Fraumeni Syndrome: description of a new pathogenic TP53 mutation
Conclusions: The positive IHC, the segregation of the variant with the disease and the LOH of wild type allele in this family indicates that new variant c.436del9; p.Trp146_Asp148del would be pathogenic.
Illana F (1), Llovet P (2), de la Hoya M (2), Olivera H (3), Perez-Segura P (3), Caldes T (2)
P16-09
P16-08
(1) Hospital Clinico San Carlos, IdISSC, Laboratory Medicine Department, Madrid, Spain (2) Hospital Clinico San Carlos, IdISSC, Molecular Oncology Laboratory, Madrid, Spain (3) Hospital Clinico San Carlos, Department of Medical Oncology, Madrid, Spain Corresponding author: [emailprotected]
Background: Li-Fraumeni syndrome is associated to germ-line mutations of one TP53 allele. The most common cancers associated with LFS are breast cancer (30.6%), soft tissue sarcomas (17.8%), brain tumors (14%) and osteosarcome (13.4%). In this work, we found a novel germline TP53 mutation in a high risk breast/ovarian cancer family that's not reported in the P53_IARC database. Material and methods: We studied BRCA1 and BRCA2 genes in blood of a high risk breast/ovarian family by Denaturing Gradient Gel Electrophoresis and Multiplex Ligation-dependent Probe Amplification. Also, exons 5 to 9 and splice junctions of TP53 were sequenced in blood and tumors of two relative’s patients (30 and 32 years old) who suffered breast cancer (one of them was a CystosarBiochemia Medica 2012;22(3):A54-A204
A166
ATP7B gene mutations in Wilson disease patients from Croatia Ljubić H (1), Božina T (2), Merkler A (1), Relja M (3), Kalauz M (4), Sertić J (1) (1) University Hospital Centre Zagreb, Department of Laboratory Diagnostics, Zagreb, Croatia (2) School of Medicine University of Zagreb, Department of Medical Chemistry, Biochemistry and Clinical Chemistry, Zagreb, Croatia (3) University Hospital Centre Zagreb, Department of Neurology, Zagreb, Croatia (4) University Hospital Centre Zagreb, Department of Internal Medicine, Zagreb, Croatia Corresponding author: [emailprotected]
Background: Wilson disease (WD) is a rare autosomal recessive disorder of copper metabolism associated with progressive liver disease and degenerative changes in the basal ganglia. More than 400 mutations of the ATP7B gene have been proven to cause the absence or dysfunction of copper transporting P-type ATPase. If WD is diagnosed early enough, effective treatments are available that prevent or reverse many manifestations of this disorder.
2nd European Joint Congress of EFLM and UEMS
Materials and methods: 84 unrelated patients, with hepatic, neurological and/or psychiatric symptoms. Biochemical analyses considering copper levels in serum, urine and liver were indicative of WD diagnosis. Genomic DNA was used to amplify 21 exons of the ATP7B gene. Sequencing analysis was performed by PCR and capillary electrophoresis with BigDye Terminator v3.1 kit on Applied Biosystems Genetic analyzer 3130xl. Preliminary screening for most frequent His1069Gln mutation was performed on Roche LightCycler real-time PCR instrument. Results: Out of total number of WD patients, molecular-genetic analysis confirmed clinical diagnosis in 41 patient (48.8%). 19 (22.6%) patients were found to be heterozygous for certain WD mutation, while in 24 patients (28.6%) no mutations were found. The most frequent mutation in Croatian population is His1069Gln in exon 14 of ATP7B gene, which accounts for 63.3% of total number of WD mutations. Other frequent mutations are mostly located in exons 5, 13, 15 and 21 of ATP7B gene. Conclusions: Sequencing analysis is the most adequate method for diagnostics of Wilson disease because of its genetic heterogeneity and also in cases with atypical clinical presentation or equivocal copper studies.
P16-10 Molecular genetic analysis of primary immunodeficiency diseases in Croatian patients
in individuals born with malfunctioned immune system. The aim was to confirm clinical diagnosis at molecular genetic level in patients with PID and to define carrier status by analyzing DNA samples of individuals with PID in family history. Materials and methods: 15 samples of genomic DNA was analyzed: 7 samples of patients with suspicion on one of the PID and 8 samples of their family members. For identification of mutations in the coding region of analyzed genes, we used the sequencing method on Applied Biosystems 3130xl Genetic analyzer and BigDye® Terminator v3.1 Cycle Sequencing Kit. Results: In 2 patients with cyclic and severe congenital neutropenia, we found 2 mutations occurred de novo in mother's egg cells. In a patient with suspicion on Omenn syndrom, mutation wasn't found. In 2 patients with X-linked agammaglobulinemia, mutations in the BTK gene were found. In the first patient, mutation occurred de novo in mother's egg cells and, in the other, mutation was inherited from the mother. The third patient with XLA suspicion did not have mutation in the BTK gene. In a patient with suspicion on chronic granulomatous disease, mutation wasn't found, so there is a possibility for further analysis of other genes associated with this disease. Conclusions: Molecular genetic analysis is the final diagnostic step in confirmation of the PID diagnosis as it provides comprehension of disease mechanisms at the molecular level and correlation between phenotype and genotype of PID.
Merkler A (1), Richter D (2), Kelečić J (2), Rako I (1), Marodi L (3), Sertić J (1) (1) University Hospital Center Zagreb, Department of Laboratory Diagnostics, Zagreb, Croatia (2) University Hospital Center Zagreb, Department of Pediatrics, Zagreb, Croatia (3) University of Debrecen, Department of Infectious and Pediatric Immunology, Debrecen, Hungary Corresponding author: [emailprotected]
Background: Primary immunodeficiency diseases (PID) are a group of inherited conditions that occur Biochemia Medica 2012;22(3):A54-A204
A167
2nd European Joint Congress of EFLM and UEMS
P16-11
P16-12
HLA-DQ genotyping of celiac disease in Mersin Province of Turkey
Sequencing analysis of CFTR gene in Croatian patients with cystic fibrosis
Polat G (1), Yesiltepe Y (1), Altıntas E (2), Ates F (2)
Zekušić M (1), Ljubić H (1), Juričić L (1), Tješić-Drinković D (2), Rako I (1), Sertić J (1)
(1) Mersin University, Faculty of Medicine, Department of Medical Biochemistry, Mersin, Turkey (2) Mersin University, Faculty of Medicine, Department of Gastroenterology, Mersin, Turkey Corresponding author: [emailprotected]
Background: Celiac disease is a common autoimmune disease caused by a chronic inflammatory reaction to the dietary protein gluten in the intestine. It is characterized by gluten-induced symptoms and signs, specific antibodies, specific HLA class II types and enteropathy. The objective of this study is to determine the genetic profile of celiac disease in Mersin Province of Turkey. Materials and methods: HLA-DQ2, -DQ8 and -DR4 molecular typing was performed in 220 patients. The molecular typing was analyzed using polymerase chain reaction sequence-specific primers and/or reverse polymerase chain reaction sequence-specific oligonucleotides techniques. Results: We have found HLA-DQ2, -DQ8 and -DR4 types or their combinations in 133 patients. 27.7% of all patients (61/220) were HLA-DQ2, 3.1% (7/220) were -DQ8 and 8.6% (19/220) were -DR4. The combinations were 10.4%, 5.9%, 3.6% and 0.9% for – DQ8 + DR4, DQ2 + DQ8 + DR4, DQ2 + DQ8 and DQ2 + DR4, respectively. 45.8 % of patients showing polymorphisms (61/133) were HLA-DQ2, 5.2% (7/133) were -DQ8 and 14.2% (19/133) were -DR4. The combinations were 17.3%, 9.77%, 6.0% and 1.5% for DQ8 + DR4, DQ2 + DQ8 + DR4, DQ2 + DQ8 and DQ2 + DR4, respectively. Conclusion: These results support the evidence that HLA-DQ status influences the development of celiac disease. Further, determination of patient’s HLA-DQ genotype allows establishing clinically relevant genetic risk profiles. Biochemia Medica 2012;22(3):A54-A204
A168
(1) University Hospital Centre Zagreb, Department of Laboratory Diagnostics, Zagreb, Croatia (2) University Hospital Centre Zagreb, Department of Pediatrics, Zagreb, Croatia Corresponding author: [emailprotected]
Background: Cystic fibrosis (CF) is the most common autosomal recessive hereditary disease in Caucasians with an average incidence of 1:2500 newborns. CFTR gene codes for CFTR protein (cystic fibrosis transmembrane conductance regulator) which regulates the transport of chloride ions through cell membranes. Materials and methods: Genomic DNA of 65 CF patients was screened for 32 mutations by Cystic Fibrosis Genotyping Assay CFv3 (Abbott). Twelve patients who were found to be heterozygous for one CF mutation were further analysed by sequencing method for specific exons (7, 10, 11, 12 and 19) on AB Genetic Analyzer 3130XL, using BigDye Terminator Cycle Sequencing Kit v3.1 (Applied Biosystems). Results: Since year 2001, out of 65 patients detected to have CF, 42 were homozygous with the deltaF508 mutation and 11 were compound heterozygous. By using commercial kit we revealed 14 different mutations in patients with CF. Through sequencing exons 7, 10, 11, 12 and 19 in 12 heterozygous patients so far we have found five polymorphisms: M470V (c.1408A>G), 1898+152T/A (c.1766+152T>A), 3601-65C/A (c.3469-65C>A), 1525-61A/G (c.139361A>G) and R75Q (c.224G>A). These DNA polymorphisms will certainly contribute to understanding the sequential variants and clinical correlation. Other CFTR exons still remain to be analyzed. Conclusions: This paper investigates variants in CFTR gene other than the panel covered by commercial kit. By collecting information about similar
2nd European Joint Congress of EFLM and UEMS
cases and studying them, we are likely to notice a link between new mutations, polymorphisms and clinical features.
P17 - Oncology - Tumor marker 1 P17-01 New strategies to improve the quality and efficiency of health assistance to chronic cancer patients Torrubia B, Guessous A, De Gracia Y, Cuadrado-Cenzual M Hospital Clinico, Analisis Clinicos, Madrid, Spain
Conclusion: The optimized and preferred care circuits implementation, in both models have proven to be safe, effective, improving the accessibility of patients to diagnostic testing.
P17-02 Usefulness of inflammatory markers in assessing the severity of colorectal cancer Đerek L (1), Servis D (2), Unic A (1), Juricek J (1), Romic Z (1) (1) University Hospital Dubrava, Clinical Department for Laboratory Diagnostics, Zagreb, Croatia (2) University Hospital Dubrava, Department for Abdominal Surgery, Zagreb, Croatia
Corresponding author: [emailprotected] Corresponding author: [emailprotected]
Background: Integral care to the cancer patient, has a relevant social impact while it requires a multidisciplinary vision of the different areas of medicine to the patient care. Patients in oncological units are the most common class of potentially difficult draws because they are subject to more frequent laboratory testing. It is necessary to evaluate how care is applied to cancer patients and to develop strategies for improvement oncological care process. The aim of the present work was to evaluate of the health care process of the cancer patient and to improve strategies aimed at incorporating the patients' prospects to the care provision measures. Materials and methods: Development of two care models: 1º: High Resolution Model (MCAR) that include analytical process and the administration of the therapy at the hospital in the same morning; and 2º: Accessibility Analytics Patient Model (MAAP) improve the accessibility to the patients at the analytical tests, making possible the access to different flebotomy services points. Results: The percentage of cancer patients who are in treatment has undergone a biannual 8.76% increase. MCAR are used by 58% oncology patients. In relation to MAAP model there was an increase in the use of this model up to 42% in the two years of its implementation.
Background: Severe abdominal surgeries, including colorectal cancer (CRC) surgery lead to systemic inflammatory response, which is followed by the increase of inflammatory markers. Intensity of inflammatory response follows the severity of surgical injury and influences postoperative recovery. Considering that the duration of the surgery, as well as the time that the colon has been open during the surgery (open colon time, OCT) define the severity of CRC surgery, and consequently influence the postoperative recovery, we wanted to investigate if the inflammatory markers correlate with those two factors and thus provide useful information. Materials and methods: The study included 20 patients who underwent CRC surgery. Duration of the surgery, and OCT were measured for the duration of the procedure. C-reactive protein (CRP), interleukine-6 (IL-6), ferritin and soluble triggering receptor expressed on myeloid cells-1 (sTREM-1) were measured 24 hours after the surgery. CRP and ferritin were measured using immunoturbidimetric method (Beckman-Coulter, Tokyo, Japan), IL-6 and sTREM-1 using ELISA method (Quantikine, R&D Systems, Minneapolis, USA; IQ products BV, Groningen, The Netherlands). Biochemia Medica 2012;22(3):A54-A204
A169
2nd European Joint Congress of EFLM and UEMS
Results: Statistical analysis showed no correlation between tested markers and the duration of the surgery. We found strong correlation for IL-6 (r = 0.8147, P < 0.001) with OCT. No correlation between CRP, ferritin and sTREM-1 and the OCT was found. Conclusions: Our results showed that CRP, ferritin and sTREM-1 do not point to the injury and recovery after CRC surgery. We found strong positive correlation of IL-6 with the OCT during the surgery, so the rise of IL-6 could provide useful information in following CRC surgery severity and postoperative recovery.
P17-03 The phosphorescence as a predictive basis of early diagnosis of oncopathology Gramatiuk S Kharkiv National Medical University and Medical and diagnostic center 'Ekomed', Clinical biochemistry, Kharkiv, Ukraine Corresponding author: [emailprotected]
Serum proteins phosphorescence state was studied and evaluated in 59 patients with stomach adenocarcinoma, aged from 35 to 68 years. Diagnosis was confirmed by clinical and histomorphological methods. Among patients with gastroadenocarcinoma phosphorescence was studied in 33 men and 26 women. I, II, III and IV stages of disease were detected in 8, 7, 9 and 9 men and 6, 7, 7, and 6 women, accordingly. The study of luminol-dependent serum films phosphorescence in patients with adenocarcinoma of the stomach revealed increasing in men at 94.5%, 41.9%; 44.5%, 73.95%, 286.8% and 217.9% at such activation spectral lines as 297, 313; 334, 365, 404 and 434 nm. In women, the intensity of phosphorescence in similar lines of activation with monochromatic light of 297, 313, 334, 365, 404 and 434 nm, rised up to 93.2%, 33.7%, 29.6%, 67.4%, 286.7% and 216.7%. The mostly significant indexes of phosphorescence were detected at activation with monochromatic Biochemia Medica 2012;22(3):A54-A204
A170
light of 297 nm, 404 nm and 434 nm. Serum phosphorescence intensity in patients at activation with monochromatic light of 297 nm wavelength rised up by 1.94 and 1.93 times accordingly in men and women in comparison with a group of conditionally healthy people. At activation with wavelength of 404 nm serum phosphorescence in patients increased by 3.7 and 3.6 times, at 434 nm by 3.2 and 3.1 times in comparison with a group of conditionally healthy people.
P17-04 Study of cytokines, as potential biomarkers in the diagnosis of oral squamous cell cancer Hamad A Al-Balqa Applied University, Medical Allied of Science, Amman, Jordan Corresponding author: [emailprotected]
Introduction: Oral cancer is one of the prevalent cancers of the body and is one of the 10 most common causes of death. Oral squamous cell carcinoma (OSCC) accounts for over 90% of these tumors. The aim of this study was designed to detect biochemical markers in serum and saliva of oral squamous cell carcinoma patients and to evaluate their validity in monitoring and diagnosis. Materials and methods: The level of certain proinflammatory cytokines in the serum and saliva of (30) patients with OSCC and (20) healthy individuals as control group was measured. Levels of proinflammatory cytokines Interleukin 1" (IL-1"), Interleukin (IL-6), Interleukin (IL-8) and Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF) was detected by enzyme linked immunosorbent assay (ELISA). Results: Serum IL-6 and IL-8 level was detected at higher concentrations in patients with OSCC than the control group (P < 0.001). No significant differences in serum IL-1 alpha and GM-CSF of patients with OSCC as compared with control group.The levels of IL-1 alpha, IL-6, IL-8 and GM-CSF in saliva
2nd European Joint Congress of EFLM and UEMS
showed significant increase in patients with OSCC when compared with control group. Conclusion: Salivary IL-1 alpha and GM-CSF was useful in the diagnosis of OSCC patients. Serum IL-6 was useful in the diagnosis of OSCC patients than salivary IL-6. Serum and salivary IL-8 were very useful in the diagnosis of OSCC patients and separating between OSCC patients and control group. From the results of the presents study, it can be concluded that cytokines are important in proinflammatory and proangiogenic responses and are detectable in serum and saliva of patients with OSCC. These cytokines increase the pathogenicity of OSCC and prove useful as biomarkers for diagnosis.
P17-05 Germline polymorphisms in LRIG1 gene predict clinical outcome in metastatic colorectal cancer Paez D (1), Pare L (2), Sebio A (2), Del Rio E (2), Barnadas A (1), Baiget M (2) (1) Hospital de la Santa Creu i Sant Pau, Medical Oncology, Barcelona, Spain (2) Hospital de la Santa Creu i Sant Pau, Genetics, Barcelona, Spain Corresponding author: [emailprotected]
Background: Leucine-rich and immunoglobulinlike domains (LRIG) 1, 2, and 3 are integral membrane proteins. LRIG1 negatively regulates EGF signaling and increasing evidence indicates that LRIG1 is a tumor suppressor in certain cancer types. Lrig1 is expressed at low levels in several cancer types but is overexpressed in some colorectal tumors. We postulate that polymorphisms in the LRIG1 gene could influence the EGFR signalling pathway and be related with the clinical outcome in metastatic colorectal cancer (mCRC). Materials and methods: We studied 126 Spanish mCRC treated with a first-line oxaliplatin/5-fluorouracil regimen. Polymorphisms in the LRIG gene
were selected using public literature resources and databases (NCBI, PubMed, db SNP, Ensembl and GeneCards Version 3). Two non-synonymous (c3158A>C and c1843A>G) and two synonymous (c2221T>C and c1317C>T) common and putatively functional polymorphisms were selected. These germline polymorphisms were analyzed using a Fluidigm equipment in DNA samples extracted from peripheral blood. Overall survival (OS) was evaluated according to each genotype. Results and conclusions: There were significant associations between LRIG1 c1317C>T and c3158A>C polymorphisms and clinical outcome. The analysis of the c1317C>T polymorphism revealed that OS was lower for patients harboring a T/T genotype than for patients with the C/C or C/T genotypes (P = 0.01). In the case of the c3158A>C polymorphism, patients harboring a C/C genotype had a lower OS than patients with a A/A or A/C genotypes (P = 0.047). We identified germline variants in LRIG1 gene predicting clinical outcome in patients with mCRC receiving first-line oxaliplatin/5-fluorouracil chemotherapy.
P17-06 Proantocyanidins in cytoprotection of antitumor drugs-treated cells Katanic J (1), Stankov K (1), Srdjenovic B (2), Pavlovic N (2), Bogdanovic V (3), Bogdanovic G (3) (1) Medical Faculty Novi Sad, Institute of Biochemistry, Novi Sad, Serbia (2) Medical Faculty Novi Sad, Institute of Pharmacology, Novi Sad, Serbia (3) Oncology Institute of Vojvodina, Experimental Laboratory, Sremska Kamenica, Serbia Corresponding author: [emailprotected]
Introduction: Antitumor drugs Doxorubicin (DOX) and Mitomycin C (MMC) are broadly used antitumor drugs, but their clinical use is significantly limited due to their systemic toxicity. The main cause of their toxic effects is the oxidative stress as a consequence of ROS (reactive oxygen species) generBiochemia Medica 2012;22(3):A54-A204
A171
2nd European Joint Congress of EFLM and UEMS
ation during the DOX and MMC metabolism. The main aim of our study was to investigate the efficacy of different antioxidative agents in oxidative stress prevention and protection from DOX and MMC effects, in normal (CHO – Chinese hamster ovary) and malignant cells (K562 – human erythroleukemia cells). Materials and methods: The antioxidative agents used in our study were the glutathione precursor N-acetylcystein (NAC) and proantocyanidins (PAC), natural antioxidative compounds derived from the plant polyphenols. According to the aim of this study, the parameters of oxidant status, activity of antioxidant enzymes glutathione-S-transferase and glutathione reductase were tested in human erythroleukemia (K562) and Chinese hamster ovary (CHO) cells lines. Both cell lines were pretreated with potentially protective agents (NAC and PAC) 30 minutes before DOX and MMC, as prooxidant agents. Cell supernatants were prepared after 24 hours and GST and GR were determined, as parameters of oxidant status. Results and conclusions: Results of our study suggest that proantocyanidins and N-acetylcysteine exert the antioxidative activity and therefore also the potentially protective activity to the effects of DOX and MMC. Comparative analysis of activities of antioxidant enzymes GST and GR in healthy (CHO) and malignant (K562) cells lines shows that none of those antioxidant agents expresses selective activity to non-malignant, CHO cells.
P17-07 YKL-40, IL-1β and TNF-α in malignant glioma Kazakova M Medical University - Plovdiv, Medical Biology, Plovdiv, Bulgaria Corresponding author: [emailprotected]
Background: Glioblastoma multiforme (GBM) is the most aggressive brain tumour. YKL-40 is a glycoprotein that has been associated with glioma Biochemia Medica 2012;22(3):A54-A204
A172
grade and a poorer clinical outcome. The aim of the investigation was to analyze tissue and serum YKL-40 levels in glial tumours in comparison with pro-inflammatory cytokines. Materials and methods: Serum YKL-40, IL-1β and TNF-α levels were measured in 7 patients with GBM, 14 patients with lower-grade glioma (grades II and III) and 40 healthy controls, by ELISA method. An immunohistochemical analysis of YKL-expression was performed. Results and conclusions: Feeble reactivity was determined in single glial cells in the normal brain. In astrocytoma the glycoprotein was present in numerous malignant cells. The most intensive reactivity was detected in GBM. Our study revealed a significant difference in YKL-40 serum concentrations between healthy subjects and lower-grade glioma, as well as between astrocytoma and GBM. The level of the glycoprotein in tumors was twofold higher than in controls (P < 0.01). We found an association between serum YKL-40 values and tissue YKL-40 expression. Enhanced IL-1β concentrations in glioma patients were detected. The secretion of TNF-α showed a similar pattern, but the increase towards the controls was lower compared to IL-1β. In conclusion, YKL-40 correlates positively with pathological tumor grades and might serve as a novel biomarker in malignant glioma. We revealed IL-1β and TNF-α – dependent (Th1) immune response in these malignant tumors. Acknowledgments: The study is supported by grants NO–1/2009 and NO–1/2010 from Medical University – Plovdiv.
2nd European Joint Congress of EFLM and UEMS
P17-08
P17-09
Tumour markers in fluid analysis in the differential diagnosis of cystic lesions of the pancreas
Multiplex biochip arrays allow simultaneous assessment of serum markers for colon cancer screening
Albuquerque M (1), Rego de Sousa M (2)
Bunger S (1), Fitzgerald S (2), Toner V (3), Buning J (4), von Eggeling F (5), Habermann J (1)
(1) Laboratório de Patologia Clínica Hospital CUF Infante Santo / Centro de Medicina Laboratorial Dr. Germano de Sousa, Clinical Pathology / Immunology, Lisbon, Portugal (2) Centro de Medicina Laboratorial Dr. Germano de Sousa, Clinical Pathology / Immunology, Lisbon, Portugal Corresponding author: [emailprotected]
Pancreatic cystic lesions include the most common pseudocysts and a variety of cystic tumours with different biologic and pathologic characteristics. With the increasing technological tools at our disposal, cystic neoplasm became more visible. Preoperative differential diagnosis of cystic lesions of the pancreas may be difficult because decision making through reliable clinical and, or imageological criteria is uncertain. In the last decade, the cystic fluid analysis using a panel of tumours markers and in some studies, serum pancreatic enzymes like amylase and lipase, combined with endoscopic ultrasonography fine needle aspiration, became an important tool in the pancreatic lesions workup and even in follow up. The authors propose to make the evaluation of the assay of tumour markers, CEA, CA 19.9, CA 125 in net drainage of cystic lesions and their application in the differential diagnosis of cystic pancreatic lesions. Drainage fluids were obtained in 195 patients with pancreatic cysts for a period of 6 years (2006 to 2012) with variable histological diagnoses: pseudocysts, mucinous cystic carcinomas, ductal carcinomas and cystadenomas. The aims of this study are to establish the cut off levels for our laboratory in the light of the study population as well as verify the correlation between the determinations of these biological markers and severity of histological lesions.
(1) University of Lubeck, Department of Surgery, Lubeck, Germany (2) Randox Laboratories Limited, Research and Development, Crumlin, United Kingdom (3) Randox Laboratories GmbH, Research and Development, Wulfrath, Germany (4) University Hospital of Schleswig-Holstein, Internal Medicine I, Lubeck, Germany (5) Jena University Hospital, Institute of Human Genetics, Jena, Germany Corresponding author: [emailprotected]
Background: Colorectal cancer is the third most commonly diagnosed cancer in males and the second in females. Despite the implementation of screening programs such as colonoscopy, approximately 50% of patients are diagnosed at advanced tumor stages resulting in poor prognosis. Innovative, patient-friendly screening tools could aid in the early detection and allow curative treatment interventions. Materials and methods: A nine target multiplex protein biochip was generated based on a thorough literature review. Diagnostic performance was evaluated using a training- and validation-set of a highly standardized, liquid nitrogen preserved serum collection of 317 samples comprising controls, adenomas, and colon cancers. Results: Serum levels of CEA, IL-8, VEGF, S100A11, C3adesArg, CD26, MCSF and CRP showed significant differences between colon cancer cases and controls (P < 0.05). The largest areas under the receiver operating characteristics curve were observed for CEA(0.69), IL-8(0.68), and CRP(0.64). At threshold levels yielding specificities of 90%, the sensitivities for CEA, IL-8 and CRP were 26%, 22%, and 17%, respectively. Testing all possible combinations of these markers at the predefined threshold levels, CEA + IL-8 reached a sensitivity of 37% Biochemia Medica 2012;22(3):A54-A204
A173
2nd European Joint Congress of EFLM and UEMS
at 83% specificity and CEA + CRP obtained a sensitivity of 35% at 81% specificity for detecting colon carcinomas. Conclusions: Multiplex biochip array technology offers an innovative and patient-friendly approach to colorectal cancer screening. The diagnostic value of identifying further serum biomarkers and the potential advantage of combining biochip analysis with fecal occult blood has the potential to improve the performance of colorectal cancer screening and warrants further investigation.
P17-10 Identification of novel cancer biomarkers using the Randox-QuantiPlasm69 monoclonal antibody chip Antal-Szalmas P (1), Lazar J (2), McGregor S (3), Muller M (4), Kurucz I (2), Kappelmayer J (1) (1) Deptartment of Laboratory Medicine, University of Debrecen MHSC, Debrecen, Hungary (2) Biosystems International Ltd., Debrecen, Hungary (3) Randox Laboratories Ltd., Crumlin, United Kingdom (4) Adware Research Ltd., Balatonfüred, Hungary Corresponding author: [emailprotected]
Background: Recently a novel monoclonal antibody based protein chip – QuantiPlasm69 (QP69) – has been introduced by Randox Laboratories. This system uses 69 monoclonal antibodies (mAbs) – developed by BioSystems International – that are immobilized on 9x9 mm ceramic chips. The QP69 assay can recognize concentration changes of several human plasma proteins simultaneously and in this way can identify novel plasma markers in a wide variety of diseases. Materials and methods: Plasma samples and clinical data of 150-150 patients with prostate and lung cancer and 300 healthy controls were collected. Individual and pooled samples of the patients and controls were evaluated by the QP69 system. The plasma pools were created from the individual samples based on clinical, histopathological and laboratory data. Other biochemical parameters and Biochemia Medica 2012;22(3):A54-A204
A174
the classical tumormarkers were also measured. To find the most predictive parameters principal component, binary logistic regression and ROC analysis was performed beside the classical statistics. Results: A set of mAbs (three antibodies) present on the QP69 chip were able to discriminate between healthy controls and lung cancer patients, and a different set of mAbs (three antibodies) were able to distinguish samples of healthy controls from those of prostate cancer patients. These differences were independent of age and smoking habits. Combination of these antibodies in themselves or with classical tumormarkers could further improve their efficacy. Conclusions: The QP69 kit can be an effective tool in biomarkers’ search and discovery. This work was supported by the National Office for Research and Technology of Hungary (TECH-09-A1-2009-0113; mAB-CHIC).
P18 - Oncology - Tumor marker 2 P18-01 Evaluation of tumor marker HE4 assay on the Elecsys 2010 analyzer Ćorić J Clinical Center of Sarajevo University, Department of Clinical Chemisty, Sarajevo, Bosnia and Herzegovina Corresponding author: [emailprotected]
Background: Whey-acidic protein human epididymis protein4 (HE4), a new promising biomarker for epithelial ovarian cancer (EOC). The measured HE4 value of patients sample can depending on the testing procedure use. Methods: We evaluated a HE4 method on Elecsys 2010 analyzer. The method for quantitative determination of HE4 is direct, competitive electrochemiluminescence immunoassay. For quality control we use Elecsys PreciControl HE4 1 and 2. HE 4 was measure on sera obtained from 96 women (40 healthy and 56 with epithelial ovarian cancer).
2nd European Joint Congress of EFLM and UEMS
Results: The Roche HE 4 assays showed a good linearity (r = 0.99) and precision (intrassay and total CV < 5%). The median HE4 serum concentrations was significantly higher among EOC patients than healthy females (P < 0.05). As a single marker, HE4 had a sensitivity of 78.4 % with a specificity of 95 %. Conclusions: The presented results of the analytical evaluation methods for the determination of HE 4 on the Elecsys 2010 analyzer showed an acceptable accuracy and precision.
P18-02 CYFRA 21-1 new tumor marker in Abbott iArchitect family Županić D, Crnokrak S, Mujagić R, Honović L Pula General Hospital, Department of Laboratory Diagnostics, Pula, Croatia Corresponding author: [emailprotected]
Introduction: CYFRA 21-1 is a tumor marker that measures fragments of Cytokeratins 19 using two monoclonal antibodies KS 19-1 and BM 19-21, but its main role is to monitor the course and success of the therapy of NSCLC. Objective: Our goal was to investigate the acceptability of Abbott reagents for use in daily routine on immunochemical analyzer Architect i2000SR to ensure traceability of our results. Validation of immunoassays was performed using CLSI / NCCLS procedure EP 15-A2. Materials and methods: The following parameters were assessed: Precision (random error) in which we determined repeatability, interprecision and overall laboratory precision by determination of a small group (N = 30) and systematic error (deviation from expected values). Results: • Control Level 1: 5 ng / mL (3.5 to 6.5) (Repeatability x = 5.23, Sr = 0.22, KV% = 4.2%) • (Interprecision x = 5.23, Sb = 0.123, KV% = 2.4%)
• (Overall laboratory precision SL = 0.218, KV% = 4.2%) • (Systematic error is 0.23 (4.6%)) • Control Level 2: 35 ng / mL (24.5 to 45.5) (Repeatability x = 34.92 ng / ml, Sr = 0.44, KV% = 1.3%) • (Interprecision x = 34.92, Sb = 0.524, KV% = 1.5%) • (Overall laboratory precision SL = 0.635, KV% = 1.8%) • (Bias -0.08 (-0.23%)) Conclusion: Based on the results of this study, we can conclude that the tested reagent CYFRA 21-1 Abbott is acceptable and we recommend its use on automated immunochemical analyzer iArchitect Abbott.
P18-03 Analytical performance of tumor markers on UniCel DxI 600 using Sigma metrics Unic A, Derek L, Marijancevic D, Serdar T, Romic Z University Hospital Dubrava, Clinical Department for laboratory diagnostics, Zagreb, Croatia Corresponding author: [emailprotected]
Background: The Sigma concept of a tolerance limit provides guidance for defining intended medical use in the form of an allowable total error. Performance is characterized on a sigma scale. In terms of Sigma, if a method has a value less than three is considered to be unreliable and should not be used in routine laboratory practice. The aim of this study was to assess the performance of automated immunodiagnostic chemiluminescence system UniCel DxI 600 (Beckman-Coulter, Tokyo, Japan) for qualitative detection of tumor markers. Materials and methods: For CA19-9, CA15-3, CA125, and PSA method validation Lypochek Tumor Marker (Bio-Rad Laboratories, Marnes-la-Coquette, France; Control lot 54530- Level 1 and 3) control samples were obtained. All immunoassays were preformed on the UniCel DxI 600 analyzer Biochemia Medica 2012;22(3):A54-A204
A175
2nd European Joint Congress of EFLM and UEMS
according to the manufacturer instructions. Control samples were tested in triplicate every day during the 5 days according CLSI/NCLLS protocol EP15-A2. Bias was calculated using method comparison. Sigma was calculated using Ricos quality requirements. Results: Calculated sigma metrics for CA15-3 were 2.5 and 1.4; for CA125 6.5 and 5.9; for CA19-9 6.4 and 9.2; and for PSA-1.0 and 2.2. Conclusion: Analytical performance for CA125 and CA19-9 is world class. On the other hand, CA15-3 and PSA analytical performance is poor or even unacceptable. Using graphic tool it becomes apparent that bias is the main problem with CA153 and PSA performance. With lower bias CA15-3 assay performance might even reach Six Sigma zone. Therefore, it is necessary to use the same method for individual patient. In that case, even these two methods became of excellent quality.
P18-04 Prognostic value of Cyfra 21-1, CEA and NSE in patients with NSCLC Musliu A Faculty of Natural Science, Department of Biology, Tirana, Albania Corresponding author: [emailprotected]
Background: The aims of this study were to evaluate prognostic effects, sensitivity and specificity of Cyfra 21-1 in detecting non-small cell lung cancer. Subjects and methods: The study included 118 randomly selected patients with NSCLC compared with control group of 30 patients with nonmalignant pulmonary disease. Histology tumor diagnosis was based on biopsy specimens obtained at bronchoscopy, lymphode biopsy or thoracotomy. Tumor markers (Cyfra 21-1,CEA and NSE) were assayed in Immunological Abbot’s Axsym System analyzers. Chemotherapy included cisplatin and carbocisplatin were aplicated in doses (80-100 mg/ Biochemia Medica 2012;22(3):A54-A204
A176
m2 day 1.3 wk cycle and 200-300 mg/m2 day 1.3 wk cycle for six cycles). Results: The level of tumor markers was determined in both population in different stage of age before and after chemotherapy. Median level of Cyfra 21-1 at diagnosis was 36.2 ng/mL with range 2.8-215.0 and decreased significantly after second cycles of chemotherapy 24.3 ng/mL (range 3.4145.0; P < 0.01). Same results we obtained in the NSE concentration (23.4 μg/mL before and 14.2 μg/mL after therapy, but CEA shows not significantly changes. Cyfra 21-1 were elevated in 22.3%, NSE in 10.8% and CEA in 16.5% of patients respectively. Conclusion: Cyfra 21-1 reflects the extent of the disease and has an independent prognostic role along with performance status and disease stage in NSCLC.Combining Cyfra 21-1, NSE and CEA correlated with prognosis in a significant and independent manner.
P18-05 Computational screening for non-small cell lung carcinoma biomarker candidates Dundovic S (1), Debeljak Z (2), Seric V (2) (1) General Hospital "Nasice", Department of Medical Biochemistry, Nasice, Croatia (2) Clinical Hospital Center "Osijek", Department of Clinical Laboratory Diagnostics, Osijek, Croatia Corresponding author: [emailprotected]
Introduction: Comparison of gene expressions obtained on control and patient samples based on computational methods allows selection of relevant genes and their protein products which may serve as biomarker candidates. For the purpose of non-small cell lung carcinoma biomarker screening a multivariate classification method known as k-nearest neighbors has been selected in this study. Materials and methods: The study was conducted on a publicly available set of gene expressions
2nd European Joint Congress of EFLM and UEMS
(Showe MK et al. Cancer Res 2009;69:9202-10) which consists of 291 gene expression profiles (137 non-small cell lung carcinoma patient samples and 91 control samples), each containing 15 227 gene expressions. k-nearest neighbors method was applied to all pairs of gene expressions. t-test with Benjamini-Hochberg correction for multiple testing has been used as a univariate filter. Results: Application of k-nearest neighbors resulted in the test set accuracy of 81.6-86.8% which corresponds to 1000 top ranking gene pairs. Less than a half of highly ranked genes were statistically significant according to the corrected t-test. The majority of the top ranking genes are responsible for the processing of gene information and for the regulation of signaling pathways. Conclusion: k-nearest neighbors method provides insight into the relevance of single genes and gene interactions for differentiation of patient and control samples. Low correspondence between univariate results and the results of k-nearest neighbors underlines importance of the latter. This might influence the quality of candidates for non-small cell lung carcinoma biomarkers and corresponding diagnostic approaches which laboratory evaluation is underway.
P18-06 Prognostic and predictive significance of ERβ1 in primary breast cancer Jelisavac Ćosić S (1), Jakić-Razumović J (2), Sirotković-Skerlev M (3)
positivity (ERa and PgR). Estrogen receptor β (ERβ) is a second estrogen receptor and its role in endocrine treatment is not fully elucidated. The aim of this research was to determine prognostic and predictive value of isoform EBβ1 in breast cancer. Materials and methods: In the study were included 150 consecutive primary breast cancer cases operated at University hospital Zagreb (year 2002-2003). Overall and disease free survival were recorded until January 1st, 2011. Immunohistochemistry was used for EBβ1 determination (antiERβ1 clone PPG5/10, Dako, Denmark). Results: ERβ1 and ERα expressions were not correlated (P = 0.178, r = 0.123). There was no association of ERβ1 with age, menopausal status, lymph node status, tumor size, histological grade, histological type, nuclear grade, Nottingham prognostic index, proliferation index Ki67 expression, steroid receptor status and HER-2/neu status. In univariate Cox-regression analysis, ERβ1 was associated with overall survival (P = 0.026; HR = 0.46; 95%CI 0.24-0.92) but there was no association with disease free survival (P = 0.054; HR = 0.51; 95%CI 0.251.04). Kaplan-Meyer survival curve analysis confirmed prognostic significance of ERβ1 for overall survival. In the subgroup of patients that received endocrine treatment (N = 93), there was no statistical difference in survival between patients with high and those with low ERβ1 [overall survival (P = 0.27; HR = 1.73; 95%CI 0.65-4.7), disease free survival (P = 0.08; HR = 2.25; 95%CI 0.91-4.94)]. Conclusions: Cancer tissue expression of ERβ1 is a prognostic, but not a predictive marker in primary breast cancer.
(1) University Hospital Centre Zagreb, Department of Oncology and Radiotherapy, Zagreb, Croatia (2) University Hospital Centre Zagreb, Zagreb University Medical School, Department of Pathology and Cytology, Zagreb, Croatia (3) Zagreb University Medical School, Department of Pathophysiology, Zagreb, Croatia Corresponding author: [emailprotected]
Background: Adjuvant endocrine therapy is effective treatment in breast cancer. Patients receive endocrine treatment according to steroid receptor Biochemia Medica 2012;22(3):A54-A204
A177
2nd European Joint Congress of EFLM and UEMS
P18-07
P18-08
Comparative study of two chemiluminescent methods for determining free PSA
Soluble urokinase plasminogen activator receptor (suPAR) in breast cancer
Fernandez Leivas A, Garcia Moreira V, Llorente Torres A, Beridze N, Garcia Alonso S, Fernandez Rodriguez E
Tarapacz J, Rychlik U, Wójcik E, Stasik Z, Kulpa J
Hospital de Cabueñes, Servicio de Analisis Clinicos, Gijon, Spain
Center of Oncology – Maria Sklodowska-Curie Memorial Institute, Cracow Division, Poland, Clinical Biochemistry, Kraków, Poland
Corresponding author: [emailprotected]
Corresponding author: [emailprotected]
Background: The % free PSA (fPSA) is, on average, lower in prostate cancer than the benign prostatic hyperplasia and has been commonly used as an aid in the diagnosis of prostate cancer when PSA is between 4-10 ng/mL.
Introduction: The urokinase plasminogen activator system plays an important role in many processes involved with cancer invasion and metastasis. The elevated levels of uPAR as well as uPA and PAI-1 are associated with poor prognosis in cancer patients. Recently, similar suggestions are brought forward to soluble form of this receptor – suPAR. The aim of study was the evaluation of relationship between suPAR and selected biochemical and clinical parameters in breast cancer patients.
Materials and methods: In our study, fPSA were analyzed in 50 serum samples of patients with total PSA between 4-10 ng/mL. The results obtained using the methods ADVIA Centaur® fPSA test (Siemens) and fPSA Immulite2000 (Siemens), were compared in order to ensure transferability of results between both methods. Total PSA was measured by ADVIA Centaur® method. Analysis of data was performed using the MedCalc®, using PassingBablok nonparametric regression test, Pearson´s correlation and agreement between methods was evaluated using the Kappa statistics, according to the following groups: positive > 15% and negative < 15% fPSA. Results: The following values of fPSA were obtained (range, median) 0.39-2.72, 1.191 (95% CI: 1.037 to 1.346) and 0.27-2.46, 1.135 (95% CI: 0.985 to 1.285) for Immulite2000 and ADVIA Centaur® respectively, yielding a correlation coefficient of 0.9519 between both methods (P < 0.001). The Passing-Bablok regression equation obtained had a Slope = 0.9583 (95% CI 0.8729 to 1.0596) and Intercept = 0.0196 (95% CI: -0.0837 to 0.1084). The concordance of % freePSA is good, yielding a Kappa index of 0.797 (95%CI: 0.628-0.966). Conclusions: Both methods of fPSA show a good correlation (0.9 1/250) screen positive, and low risk group (< 1/250) screen negative. Aim: Assessment of correlation between the screening high risk pregnancies and the amniocentesis in our center. Material and methods: Study included 472 pregnant women tested between 02/2011-04/2012, 2242 years, gestation 11+0 to 13+6 weeks. The testing of the maternal biomarkers has been performed on Roche Elecsys2010. Measurement of the NT has been performed by Toshiba-XarioXG ultrasound. The risk has been calculated on FMF’s software, where the woman age, CRL, parity, smoking, BMI and ethnic origin has been evaluated too. Amniocentesis has been performed between 17w 1d-20w 6d weeks of gestation on ultrasound guided free hand technique using the 22G needle.
2nd European Joint Congress of EFLM and UEMS
Results: Among 472 tested, with the high risk on Trisomy21 (> 1/250) resulted 26 (5.5%) which then have been recommended for amniocentesis. 23 of 26 woman have undergone the amniocentesis whilst 3 refused the procedure. Among 23 performed amniocentesis 2 of 23 (7.69%) or (4.2‰) cases of the total number tested, resulted with Trisomy21 and have been referred for pregnancy termination. Conclusions: Antenatal screening is a non invasive test, able to be realized in the early gestation, technically easy to be performed and cost effective.
P19-03 Standard and new laboratory markers and their usefulness in prediction of preeclampsia Štefanović M (1), Delić R (2), Krivec Š (3) (1) Sestre milosrdnice University Hospital, Clinical Institute of Chemistry, Zagreb, Croatia (2) General Hospital Celje, Department of Obstetrics and Gynecology, Celje, Slovenia (3) General Hospital Celje, Department of Laboratory Medicine, Celje, Slovenia Corresponding author: [emailprotected]
Background: Syndrome preeclampsia occurs in 5 to 8% of pregnancies and is a leading cause of maternal and neonatal morbidity and mortality. There are no clinically available tests that perform well in predicting development of preeclampsia at the moment. The aim of this study was to investigate the predictive potential of standard laboratory parameters and to evaluate new angiogenic markers at third trimester of pregnancy in prediction of preeclampsia. Patients and methods: First, retrospective designed study included 113 patients with preeclampsia and a control group of 95 uncomplicated pregnancies. It evaluates erythrocytes, leukocytes, thrombocytes, hemoglobin, hematocrit, AST, ALT, GGT, alkaline phosphatase, total bilirubin, urea, creatinine, uric acid, body mass index, parity, age, and blood type in prediction of preeclampsia
based on multivariate logistic regression model. In the second, prospective study data from 34 patients with preeclampsia and 35 patients with uncomplicated pregnancies were evaluated. New angiogenic markers, PlGF, s-Flt-1 were evaluated together with standard laboratory parameters, the same way as in the first study. Results: When parameters such as uric acid and urea were included into logistic regression model, we correctly classified 79.6% patients. Additional parameters (thrombocytes, hematocrit, aspartate aminotransferase and leukocytes) raised correct classification to 83.8% patients and employing PlGF, sFlt-1 with other parameters, percentage of correctly classified patients reached 94,3%. Conclusion: Standard laboratory parameters, when used as laboratory test panel have significant prognostic value in the prediction of preeclampsia, but new angiogenic markers showed to be superior and can sucesfully predict occurence of preeclampsia at lower gestational age.
P19-04 Comparison of the „SsdwLab 5.0.9“ and „Fetal Medicine Foundation“ Down syndrom screening softwares Radeljak A (1), Podobnik Brlecic P (2), Ožvald I (1), Gebauer Vukovic B (2), Podobnik M (2), Stojanovic N (1) (1) University Hospital Merkur, Institute of Clinical Chemistry and Laboratory Medicine, Zagreb, Croatia (2) Clinic „Podobnik", Clinic „Podobnik", Zagreb, Croatia Corresponding author: [emailprotected]
Background: First trimester screening by a combination of maternal age, fetal nuchal translucency and maternal serum free-β-hCG and PAPP-A can indentify about 85-90% of fetuses with trisomy 21 and other mayor aneuploidies. The aim of this study was to compare risk calculations perfomed using two different softwares. Materials and methods: PAPP-A and free-β-hCG concentrations were measured by electrochemiluBiochemia Medica 2012;22(3):A54-A204
A181
2nd European Joint Congress of EFLM and UEMS
minescent immunoassay (ECL) on Roche Elecsys analyzer. The obtained results were within the target values of internal and external quality assurance programme (UKNEQAS scheme for First Trimester Downs Syndrome Screening). Nuchal translucency was measured by sonographers with FMF Certificate of Competence. Risk calculations were performed using „SsdwLab 5.0.9“(Roche, certified by FMF) and „Fetal Medicine Foundation“ softwares. Results: Patients (N = 247) were devided into high, intermediate and low risk grupe. The obtained distributions per group were: high risk 18 (7.29%), intermediate risk 36 (14.57%) and low risk 193 (78.14%) patients using SsdwLab 5.0.9 software vs. high risk 12 (4.86%), intermediate risk 26 (10.53%) and low risk 209 (84.61%) patients using FMF software. 18 patients were classified as high risk by SsdwLab 5.0.9 vs. 10 (55.6%) as high risk and 8 (44.4%) as intermediate risk using FMF software. Only one fetus from the high risk group (by both softwares) was trisomy 21 positive, which was confirmed by amniocentesis. Conclusions: Comparing risk calculations performed using „SsdwLab 5.0.9“ and „Fetal Medicine Foundation“ softwares, we concluded that SsdwLab 5.0.9 calculates higher risks for the same input parameters. Further research will be aimed at discovering the causes of observed differences.
P19-05 What do the triple test results mean? Popovic D (1), Vujovic S (2), Stanisic N (3) (1) Special Hospital "Codra", Podgorica, Montenegro and Faculty of Natural Sciences, University of Montenegro, Podgorica, Montenegro (2) Faculty of Natural Science, University of Montenegro, Podgorica, Montenegro (3) Special Hospital "Codra", Department for Gynecology, Podgorica Corresponding author: [emailprotected]
Introduction: The triple test is a screening test and not a diagnostic test. This test only notes that a mother is at a possible risk of carrying a baby Biochemia Medica 2012;22(3):A54-A204
A182
with a genetic disorder The triple screening test is performed between the 14th and 22nd week of pregnancy. This test is recommended for women who have a family history of birth defects, are 35 years or older. Materials and methods: These results represent three years study of screening for genetic disorders (trisomy 21, trisomy 18 or another tip of chromosome abnormality). Triple screening test is a combination of maternal age, weight, ethnicity, gestation of pregnancy, biparietal diametar (BPD) and serum total HCG, AFP and unconjugated estriol. We analysed 168 serum samples from pregnant women at the second trimester by Immulite 1000 (Siemens, risk assessment software PRISCA 4). The distribution of estimated risks for trisomy 21 was determined and sensitivity and false-positive rate for a risk cut-off 1 in 250 were calculated. Results: The average maternal age was 30 (range 16-44) years and in 26 (15%) the age was 35 years or more, the average gestation at screening was 15.9 (14-22) weeks and average of BPD was 35 (range 29-56) mm. The estimated risk for trisomy 21 based on mentioned parameters was 1 in 250 or greater in 3.0% (4 of 168) of pregnancies. Conclusion: Our result of screening for chromosome abnormality by measuring of fetal BPD and maternal biochemical parameters are similar to those reported in literature.
2nd European Joint Congress of EFLM and UEMS
P19-06 Analysis of biochemical parametars in the perinatal screening
nificant shift in the diagnosis and prediction of when it comes to certain complications of pregnancy such as gestational diabetes and pregnancy induced hypertension.
Vujovic S (1), Popovic D (2), Ajanovic E (1), Jovanovic M (3) (1) Faculty of Science, University of Montenegro, Biology, Podgorica, Montenegro (2) Special Hospital "Codra", Biochemistry, Podgorica, Montenegro (3) Primary Health Care Centre, Gynecology, Podgorica, Montenegro Corresponding author: [emailprotected]
Introduction: The pregnancy is specific condition of the body followed by a number of changes and different physiological states, so that a phenomenons that are the subject of this study is gestational diabetes and pregnancy induced hypertension. Aim of this study was to determine whether the deviations from the reference values of β-humanchorion gonadothropin and pregnancy-associated plasma protein-A are correlated with certain disorders of pregnancy such as pregnancy-induced hypertension, or gestational diabetes, and whether these parameters can be used in the prediction these complications in pregnancy. Materials and methods: In these study are analised 50 pregnant women. In the control group were 20 physiologically healthy pregnant women; in the study group of fifteen pregnant women (50%) had established PIH, and the other fifteen (50%) were diagnosed with gestational diabetes. Biochemical analysis was done by testing the imunofluoroscency (Wallac DELFIA Xpress). Results are expressed as multiples of median (MoM) specific gestational age. Results: In these research found no discrepancy between the values of biochemical parameters in the study and control group. The value of βhCG and PAPP-A at physiologically normal pregnant women and pregnant women diagnosed with PIH and gestational diabetes had no significant deviation. Conclusions: Based on analysis of data in the form of biochemical screening of pregnant women age and number of deliveries we could not see a sig-
P19-07 SNP’s in LEP gene and LEPR gene are associated with recurrent spontaneous abortions (RSA) Wagner J (1), Muller A (2), Škrlec I (1), Heffer M (1), Maver A (3), Peterlin B (3) (1) Faculty of Medicine, Department of Medical Biology, Osijek, Croatia (2) University Hospital Centre Osijek, Clinic for Gynecology and Obstetrics, Osijek, Croatia (3) University Medical Center Ljubljana, Institute of Medical Genetics, Ljubljana, Slovenia Corresponding author: [emailprotected]
Aim: Single nucleotide polymorphisms (SNP) in leptin (LEP) and leptin receptor (LEPR) genes were compared between a group of female patients with more than three recurrent spontaneous abortions (RSA) and women with two or more successful pregnancies (SP). Materials and methods: In a cohort study 145 women with SP and 178 women with RSA were tested. Genotype of four SNP’s in LEP (rs7799039, rs2122627, rs11761556, rs10244329) and four SNP’s in LEPR (rs1137101, rs7516341, rs1186403, rs12062820) were determined using KASP SNP Genotyping system and ABI Prism 7000 SDS instrument. Statistical comparison was done using Chi−square statistic. The haplotype frequencies and haplotype−disease associations were estimated using haplo.stats package. Results: The genotype frequencies did not deviate from HWE, except in the case of one LEP and two LEPR SNPs. In the case of rs7799039 P value for RSA and all examinees was 0.03. The recessive model (AG + GG/AA) revealed significant association between 2548A genotype and RSA (OR = 1.58). Also, two SNPs from intron region of LEPR Biochemia Medica 2012;22(3):A54-A204
A183
2nd European Joint Congress of EFLM and UEMS
(rs7516341 and rs1186403) deviated from normal distribution. In dominant model (CC + TC/TT) of the first SNP allele C decreases risk of RSA (P = 0.034, OR = 0.61). The second SNP was significantly different for SP group (P = 0.008) where T allele is of limited protective effect in the recessive model of inheritance (Chi-square P = 0.082, OR = 0.51).
spectively. After this period of time, epididymal sperm were collected and the indexes of sperm quality were determined in all groups. The lipid peroxidation levels and the reduced glutathione contents of sperm were measured. The Diameter of seminiferous tubules (µm) and germinal cell layer thickness (µm) of testis were determined.
Conclusion: It is known that mother’s BMI during pregnancy influence maternal and newborn health. LEP and LEPR are candidate genes for RSA and therefore their influence on mother’s BMI during pregnancy and final outcome of pregnancy deserves further investigation.
Results: A significant increase was found in the percentage of forward progressive sperm motility and a significant decrease was found in the percentage of sperms with slow movement in rats treated with different doses of POMo. The epididymal sperm concentration in rats that eat POMo was higher than control group. The biochemical investigation showed that POMo increased the level of reduced glutathione and decreased the level of lipid peroxidation in the sperm. The Diameter of seminiferous tubules (µm) and germinal cell layer thickness (µm) were higher in treatment rats.
P19-08 The effect of pomegranate seed oil on histological features of testis and sperm quality in male rats Nikseresht M (1), Mahmoudi R (1), Sharifi A (2), Rahimipour Sisakht F (3), Sane F (4), Jafari M (5) 1) Yasuj University of Medical Sciences, Cellular and Molecular Research Center, Yasuj, Iran, Islamic Republic of (2) Yasuj University of Medical Sciences, Microbiology, Yasuj, Iran, Islamic Republic of (3) Jahrom Branch, Islamic Azad University, Biology, Jahrom, Iran, Islamic Republic of (4) Kazerun Branch, Islamic Azad University, Biology, Kazerun, Iran, Islamic Republic of (5) Yasuj University of Medical Sciences, Histology, Yasuj, Iran, Islamic Republic of Corresponding author: [emailprotected]
Conclusion: This study shows potential effect of POMo on sperm quality and may use for increasing the fertilizing potency of sperm.
P20 - Renal replacement P20-01 Monitoring serial creatinine results in kidney transplant patients using the StatSensor POCT device Cobbaert C (1), Romijn F (1), van Lint C (2), Schenk P (1), van der Boog P (2)
Background: Pomegranate fruit extracts have been commonly marketed as dietary supplements in recent years because its health benefits have been shown in many studies. Many investigators have shown that the pomegranate possesses antioxidant activity and may act as a free radical scavenger.
(1) Leiden University Medical Center, Department of Clinical Chemistry, Leiden, Netherlands (2) Leiden University Medical Center, Department of Nephrology, Leiden, Netherlands
Materials and methods: POMo (pomegranate seed oil) was extracted by petroleum ether. Male Wistar rats were divided into four groups (six per group). One milliliter corn oil, 200, 500 and 1000 mg/kg body weight POMo were given daily for seven weeks by gavage to first- 4th groups, re-
Background: Reference Change Values (RCVs) are helpful to interpret changes in serial diagnostic results. After renal transplantation creatinine is monitored frequently to detect rejection. To judge the usefulness of a device for home monitoring of creatinine, StatSensor POCT device (Nova Biomedical)
Biochemia Medica 2012;22(3):A54-A204
A184
Corresponding author: [emailprotected]
2nd European Joint Congress of EFLM and UEMS
and central laboratory creatinine RCVs were determined, using a split sample comparison approach. Materials and methods: Finger pricks were taken from 38 stable post-transplant patients (creatinine 50-450 µmol/L), and whole blood StatSensor measurements were performed. In addition, venous blood was sampled in lithium-heparin tubes and in serum separation tubes. Heparinised whole blood was used for replicate creatinine measurements on the StatSensor, and serum creatinine was determined using an IDMS-traceable enzymatic method on the Roche Modular P800. RCVs were calculated from: 2.8 x (CVa2 + CVb2)1/2 (P < 0.05, CVb = 5.3%). Results: Mean creatinine levels on the StatSensor were 161 ± 86 and 154 ± 81 µmol/L in finger prick and venous whole blood, respectively. Serum creatinine levels on the Modular were 172 ± 82 µmol/L. Mean overall StatSensor CVa was 10.4% for finger prick and 5.2-6.6% for venous blood. Overall CVa for laboratory serum creatinine was < 1.5%. StatSensor RCVs were 35% and 23% for finger prick and venous whole blood, respectively, compared to 15.5% for the laboratory method. Conclusions: Our data illustrate that RCVs are highly affected by overall CVa of POCT devices. Insufficient precision of the StatSensor causes a 2.3-fold increase in RCV as compared to the central laboratory method. A quality mark for POCT devices is recommended to guarantee desirable analytical performance.
P20-02 Falsely decreased ionized calcium in a dialysis patient
vent thrombosis of the haemodialysis catheter. With patients suffering from heparin induced thrombocitopenia, heparinization is contraindicated and therefore, citrate is used as an alternative. Materials and methods: A 72-year old female patient suffering from kidney failure attending haemodialysis for 25 years has been chosen for this report. Last year the patient was diagnosed with heparin induced thrombocitopenia and therefore catheter-locking in the interdialytic period was performed with citrate as the anticoagulant. Before the haemodialysis and immediately after the removal of the catheter-locking solution, blood sampling for routine laboratory tests was performed. Concentration of ionized calcium was determined potentiometrically using the point-ofcare analyzer RapidLab 1265 (Siemens, Germany). Results: The concentration of ionized calcium was measured prior to two haemodialysis treatments and the results were 0.33 mmol/L and 0.37 mmol/L, respectively. The patient, however, did not suffer from any symptoms of hypocalcemia. After the haemodialysis, ionized calcium was determined again, and its values were within the reference range (1.18–1.32 mmol/L). Conclusion: The patient not suffering from any clinical symptoms of hypocalcemia and the concentration of ionized calcium being within the reference range after haemodialysis, indicates that a preanalytical error has occured. It is highly probable that the removal of the citrate solution from the catheter was not complete, which led to falsely decreased values of ionized calcium due to the capacity of citrate to chelate calcium ions.
Lukić I (1), Lapić I (1), Fressl Juroš G (1), Brunetta Gavranić B (2), Rogić D (1) (1) Clinical Hospital Center Zagreb, Clinical Institute for Laboratory Diagnostics, Zagreb, Croatia (2) Clinical Hospital Center Zagreb, Department for Nephrology, Arterial Hypertension and Dialysis, Zagreb, Croatia Corresponding author: [emailprotected]
Background: Administration of an anticoagulant is required with patients on haemodialysis to preBiochemia Medica 2012;22(3):A54-A204
A185
2nd European Joint Congress of EFLM and UEMS
P20-03
P20-04
High-sensitive cardiac troponin T predicts survival in patients on chronic hemodialysis
Interpretation of cardiac markers in end stage renal disease patients in hemodialysis (HD)
Rajdl D (1), Racek J (1), Eiselt J (2), Trefil L (1), Malánová L (3)
Aakre K (1), Røraas T (2), Petersen P (2), Svarstad E (3), Sandberg S (1)
(1) Charles University, Medical Faculty in Pilsen and University Hospital in Pilsen, Institute of Clinical Biochemistry and Hematology, Pilsen, Czech Republic (2) Charles University, Medical Faculty in Pilsen and University Hospital in Pilsen, 1st Department of Internal Medicine, Pilsen, Czech Republic (3) B. Braun Avitum, Dialysis center, Pilsen, Pilsen, Czech Republic
(1) Haukeland University Hospital, Laboratory of Clinical Biochemistry, Bergen, Norway (2) University of Bergen, The Norwegian Quality Improvement of Laboratory Services in Primary Care (NOKLUS), Department of Public Health and Primary Health Care , Bergen, Norway (3) University of Bergen, Renal Research Group, Institute of Medicine, Bergen, Norway
Corresponding author: [emailprotected]
Corresponding author: [emailprotected]
Background and aims: Cardiac troponins and natriuretic peptide type B (BNP) use is well established in acute myocardial infarction and heart failure diagnosis. Recently, these markers (and especially cardiac troponins measured by high-sensitive assays) are emerging as new prognostic markers in wide variety of diseases. The goal of this study was to compare the prognostic utility of cardiac Trpoponin I (cTnI, Access 2, Beckman-Coulter), BNP (AxSYM 2, Abbott) and high-sensitive cardiac troponin T (hsTnT, Cobas e411, Roche) in hemodialysed (HD) patients.
Background: The aim was to calculate the withinsubject variation (CVi) and reference change value (RCV) for TnT and pro-BNP in HD patients.
Materials and methods: We analyzed cTnI, BNP and hsTnT levels in blood samples of 83 chronically HD patients (31 females; median age [interquartile range] = 65 [56-71] years). There were 37 deaths in our study population (median follow-time was 38.3 [17.0-54.0] months). We used Cox proportional hazard model to reveal the possible prognostic role of measured markers. Results: Our results indicate that the best prediction of overall survival can be obtained from hsTnT (relative risk (RR) of overall mortality with its 95 % confidence intervals (CI): 1.022 [1.03 to 1.042], P = 0.03, followed by BNP (RR [CI]: 1.0001 [to 1.00004 to 1.001], P = 0.05. cTnI was not significantly associated with survival of HD patients in this setting. Conclusion: High-sensitive troponin T and BNP can help in risk stratification of hemodialysed patients. Biochemia Medica 2012;22(3):A54-A204
A186
Materials and methods: 19 patients (treated with HD for median 16.5 month) and 20 healthy voluntaries were included. Samples were collected once a week (immediately before dialysis and during morning hours, respectively) for ten weeks. Results below the detection limit of the high sensitive TnT and NT-proBNP assay (Modular Roche Diagnostics; 3 ng/L and 1 pmol/L) were excluded. CVi and RCVs were calculated after ln-transformation of the data. Results: Half of the healthy controls were female and median age was 61 years. Median age of patients was 71 years, four patients were female. Eight patients had nephrosclerosis and six glomerulonephritis as main cause for renal disease. Four patients had clinical events during the ten week observation period and were excluded from calculations. Mean TnT and pro-BNP concentrations ranged from 18 to 189 ng/L and 22 to 6189 pmol/L, respectively in the HD patients. Most control subjects had TnT values below 3 ng/L (CVi could not be calculated) whilst mean NT-proBNP concentrations ranged from 2-63 pmol/L. In HD patients TnT CVi was 8.2%; TnT RCVs were -18% and +22%; NTproBNP CVi was 26% and RCVs were -51% and +105%. In healthy controls NT-proBNP CVi was 55% and RCVs were -77% and +336%.
2nd European Joint Congress of EFLM and UEMS
Conclusion: In cardiac stable HD patients TnT show less variation compared to NT-proBNP. The NT-proBNP variation in HD patients is lower as compared to healthy controls.
P20-05 The role of NGAL as predictor of delayed graft function after kidney transplantation Glisic B (1), Lalic N (1), Naumovic R (2), Durutovic O (3), Majkic-Singh N (1) (1) Clinical Center of Serbia, Center of Medical Biochemistry, Belgrade, Serbia (2) Clinical Center of Serbia, Clinic for Nephrology, Belgrade, Serbia (3) Clinical Center of Serbia, Clinic for Urology, Belgrade, Serbia Corresponding author: [emailprotected]
Background: Delayed graft function (DGF) is very common complication after kidney transplantation which can increase the risk of early or late graft loss. Commonly, it is monitored by measuring the serum creatinin levels, unreliable parameter for assessing acute changes in graft function. Recently, NGAL has emerged as a novel biomarker for predicting the onset of DGF. We performed a pilot study to assess the role of NGAL, measured 3 hours after the transplantation, as a potential predictor of DGF. Materials and methods: Our study included 20 kidney transplanted patients, divided into two groups: IGF (immediate graft function, 6 patients) and DGF (14 patients). NGAL was measured using the ARCHITECT® Urine NGAL assay (Abbott Diagnostics, Illinois, USA) in urine samples collected exactly 3 hours after the transplantation. Results: The mean uNGAL values in IGF and DGF groups were 1215.3 ± 766.7 ng/mL and 1630.0 ± 1023.9 ng/mL, respectively. We found no statistically significant differences between the groups. After expressing the results as uNGAL to urinary creatinin ratio (NGAL/Cr), the differences were statistically significant (P < 0.01) with levels of 143.6 ±
79.4 ng/mg for IGF and 905.6 ± 656.7 ng/mg for DGF. Conclusions: Our results have shown that NGAL measured in urine samples collected three hours after the transplantation can be used as a predictor of DGF, if expressed as NGAL-to-creatinine ratio.
P20-06 Nutritional status assessment in patients undergoing hemodialysis Crnokrak S, Mujagić R, Honović L General Hospital Pula, Clinical laboratory, Pula, Croatia Corresponding author: [emailprotected]
Introduction: Approximately 40 to 70 percent of patients with end-stage renal disease are malnourished. The assessment of nutritional status should be a routine care of dialysis patients to permit early recognition and the institution of appropriate therapy. Most of the standard methods of assessing nutritional status can be applied to patients with renal failure; however, some of these parameters are altered by uremia. There is no single measurement that can be used to determine the presence of malnutrition. Materials and methods: Seventy male hemodialysis patients were included in this study. Nutritional parameters (body mass index, cholesterol, triglyceride, transfferin, albumin, pseudocholinesterase, uric acid, blood lymphocyte count) were measured by standard biochemical tests. Results were evaluated by a Forward stepwise multiple linear regression method. Results: There were revealed an standard equation; body mass index (kg/m2) = 1.795 × cholesterol (mmol/L) + 2.657 × transferrin (g/L); that shows a significant association of body mass index, especially, with cholesterol and transferrin (multiple R = 0.675; P < 0.001). However, body mass index were not correlated (P > 0.05) with zinc, albumin and pseudocholinesterase in serum and blood lymBiochemia Medica 2012;22(3):A54-A204
A187
2nd European Joint Congress of EFLM and UEMS
phocyte count. Thereafter, there were revealed an improved equation; body mass index (kg/m2) = 0.016 × uric acid (µmol/L) + 0.756 × triglyceride (mmol/L) + 0.955 × cholesterol (mmol/L) + 1.875 × transferrin (g/L); that shows a higher correlation of body mass index (multiple R = 0.727; P < 0.001) with uric acid, triglyceride, cholesterol and transferrin.
-0.05, P = 0.152); PAI-1 (r = -0.08, P = 0.558) and fibrinogen (r = 0.04, P = 0.772)). Statistically significant correlation between visfatin level and fibrinogen (r = 0.51; P = 0.008) and the time on dialysis (r = 0.70; P < 0.001) was observed in female patients. In conclusion, visfatin is not associated with CRP, fibrinogen and PAI-1 in patients on hemodialysis.
Conclusion: We were suggested equation that may confidentially predict the nutritional status in patients undergoing hemodialysis.
P20-08
P20-07
Dobrošević B (1), Bilandžija M (2), Barbić J (3), Šerić V (1)
Visfatin is not associated with inflammatory markers in patients on hemodialysis Ćelap I (1), Šimundić AM (1), Nikolac N (1), Miler M (1), Altabas K (2), Šefer S (2) (1) University Hospital Center Sestre milosrdnice, Clinical Institute of Chemistry, Zagreb, Croatia (2) University Hospital Center Sestre milosrdnice, Center for Hemodialysis, Department of Nephrology, Zagreb, Croatia Corresponding author: [emailprotected]
Background: Patients with chronic renal disease often suffer from diverse cardiovascular complications. Endothelial dysfunction has been involved in development of different forms of the cardiovascular diseases, including chronic renal disease. In recent years, ubiquitous adipokine called visfatin has been considered as a novel marker of endothelial dysfunction and inflammation. Thus, the aim of our study was to investigate the association of serum visfatin concentrations and well-established markers of cardiovascular risk in the patients on hemodialysis. Materials and methods: Serum and plasma samples from 66 patients (40 males and 26 females) treated by hemodialysis were analysed for visfatin, fibrinogen, CRP and PAI-1 levels. Visfatin was determined by ELISA method while CRP, fibrinogen and PAI-1 were obtained by standard laboratory methods. Results and conclusion: Visfatin did not correlate with the studied markers of inflammation (CRP (r = Biochemia Medica 2012;22(3):A54-A204
A188
Effect of hemodialysis on the expression of TLR-4 on monocytes
(1) Clinical Hospital Centre Osijek, Department of Clinical Chemistry, Osijek, Croatia (2) Clinical Hospital Centre Osijek, Department of hemodialysis, Osijek, Croatia (3) University of Osijek, Medical School, Osijek, Croatia Corresponding author: [emailprotected]
Background: Infections are frequent complications in patients undergoing hemodialysis. Endstage renal disease is simultaneously associated with inflammation. It was recently shown that family of Toll-like receptors (TLR) play a critical role in innate immunity. It has been reported that expression of TLR-4 was lower in patients on dialysys. The purpose of this study was to determine the effect of hemodialysis on the expression of TLR-4 on CD14+ monocytes. Materials and methods: In 33 hemodialysis patients, we measured expression of TLR-4 on CD14+ monocytes at the beginning (0 min.) and following (180 min.) hemodialysis. The hemodialysis procedure was performed by using polysulphone dialysers. Expression of TLR-4 on CD14+ monocytes was determined by staining with PE labeling antiTLR-4 monoclonal antibody (eBioscience) and analyzed by flow cytometry (FACSCalibur, BD). Results: The percentage of TLR-4 on CD14+ monocytes was significantly lower 180 min. after hemodialysis (21.7 ± 6.5) compared with beginning ( 24.8 ± 6.4 ; P < 0.01).
2nd European Joint Congress of EFLM and UEMS
Conclusions: The expression of TLR-4 on monocytes becomes down-regulated in uremic patients. The hemodialysis procedure may suppress the expression of TLR-4.
in patients with metabolic syndrome (P < 0.001) whereas leptin and resistin concentrations did not differ between groups (P = 0.115 and P = 0.569, respectively). BMI was lower in group of patients on hemodialysis regarding to patients on peritoneal dialysis and with metabolic syndrome (P < 0.001).
P20-09
Conclusions: CRP is increased in dialyzed patients and that probably indicates inflammatory response in dialysis. Since leptin and resistin concentrations are not increased in dialyzed patient, we hypothesize that those markers share some other regulatory mechanisms.
Markers of the obesity and inflammation in patients with metabolic syndrome and on dialysis Miler M (1), Segulja D (2), Nikolac N (1), Celap I (1), Kackov S (3), Simundic AM (1) (1) Sestre milosrdnice University Hospital Center, University Department of Chemistry, Zagreb, Croatia (2) University Hospital Centre Zagreb, Department of Laboratory Diagnostics, Zagreb, Croatia (3) Polyclinic Bonifarm, Medical biochemistry laboratory, Zagreb, Croatia Corresponding author: [emailprotected]
Background: Dialysis is an invasive treatment in chronic kidney failure associated with increased activation of inflammatory system. The aim of this study was to examine the difference between concentrations of classic inflammatory marker (C-reactive protein, CRP) and new markers from adipokine family (leptin and resistin) in patients on dialysis and with metabolic syndrome. Materials and methods: Total of 140 patients were included in the study, 55 of them with metabolic syndrome (according to NCETP ATP III criteria), 66 on hemodialysis and 18 on peritoneal dialysis. For all patients body mass index (BMI) was provided. CRP concentration was determined with turbidimetric method on Beckman Coulter AU2700 analyzer (Beckman Coulter, Brea, USA). Concentrations of leptin and resistin were determined with fluorescent bead immunoassay (Bender MedSystems GmbH, Vienna, Austria) on the flow cytometer (Beckman Coulter, Brea, USA). Differences between 3 groups were tested with Kruskal-Wallis test. Results: According to the results, CRP concentration was higher in group of dialyzed patients than
P21 - Toxicology and TDM P21-01 Analytical validation of valproic acid on the Abbott Architect c8000 clinical chemistry system Čepić K, Dujmov I, Mladina B, Šupe Domić D, Stanišić L Split University hospital, Department of Clinical Chemistry, Split, Croatia Corresponding author: [emailprotected]
Background: Valproic acid is a broad-spectrum anticonvulsant drug. High concentration has been associated with hepatic toxicity and acute toxic encephalopathy. The aim of this study was to evaluate analytical performance of Abbott Architect c8000 analyzer for therapeutic drug monitoring (TDM) of valproic acid. Materials and methods: Analytical validation of valproic acid determination by particle enhanced turbidimetric inhibition immunoassay (PETINIA) on Abbott Architect c8000 system included: inaccuracy (bias), within-run imprecision, between-run imprecision and method comparison with analytical system Architect i1000, CMIA method, for 66 human samples. Results: Inaccuracy (bias) result was -0.24% to 2.4%. The highest coefficient of variation (CV) for Biochemia Medica 2012;22(3):A54-A204
A189
2nd European Joint Congress of EFLM and UEMS
within run imprecision was 3.01% and beetwenrun imprecision was 2.82%. Linearity was confirmed with calibration curve in 6 points in concentration range from 12.5 to 150 μg/mL. Passing-Bablock regression analysis of valproic acid comparison on two analyzers showed statistically significant, but clinically insignificant deviation in slope of regression equation (b = 0.9410; 95%CI = 0.90420.9766). The Cusum linearity test proved that there was a linear relationship between two methods. Conclusion: Analytical validation of valproic acid by PETINIA method on Abbott Architect c8000 system fulfilled all previously established criteria and could be implemented in a routine laboratory work.
effect of synthesized Pt-nps on the ferroxidase activity of HSA was investigated. Finally, their potential anti-cholinesterase or neurotoxic properties were also studied. Synthesized Pt-nps significantly increased the ferroxidase activity of HSA by up to 9-fold. Results showed further, no significant inhibition on AChE activity compared to about 80% inhibition earlier reported with chemically synthesized nanoparticles. Pt-nps are well known to be efficient catalyst and may explain the rapid increase in the ferroxidase activity of HSA observed. We also believe the protein shell of HSA shielded the Pt-nps from eliciting any significant effect on the activity of AChE.
P21-02
P21-03
The possible anti-cholinesterase properties of biologically synthesized platinum nanoparticles
Recreational drugs in clinical toxicology: new challenges in the everyday routine
Arowolo A, Whiteley C
Lakatos A (1), Lajtai A (1), Porpaczy Z (2), Mayer M (2), Kovacs G (1)
Rhodes University, P.O. Box 94, Grahamstown, South Africa., Biochemistry, Microbiology and Biotechnology, Grahamstown, South Africa
(1) University of Pécs, Med. School, Dept. of Laboratory Medicine, Pécs, Hungary (2) University of Pécs, Med. School, Dept. of Forensic Medicine, Pécs, Hungary
Corresponding author: [emailprotected]
Corresponding author: [emailprotected]
Biomineralization, using protein cavity (cage) as a limiting growth field for the synthesis of uniformed sized and shaped nanoparticles (nps), is fast becoming a common approach in biological synthesis of nps. This is due to the fact that this method of production is by far simple, environmentally friendly and cost effective with great potential application in drug-delivery. However, little is known on the possible effect of these synthesized nps on the natural biological function of this protein cages. Also, the toxic effect of these nps on various biomedical target is unknown. In this work, spherical platinum nanoparticles (Pt-nps) of relatively uniform sized (3-5 nm) were biologically synthesized within the cavity of horse spleen apoferritin (HSA). Nanoparticles were characterized using UV, Transmission electron microscopy (TEM) and Electron dispersion analysis of X-rays (EDAX). The
In clinical laboratory toxicology we use biological samples (mostly urine) of the poisoned patient to confirm the consumed dangerous substances. The anamnesis in this field is usually inefficient. The patients are often unconscious or don’t want to tell or don’t even know what was the substance they used for suicide or abuse. The spectra of most often used poisonous agents are changing in the time. It is also true for the medical drugs but in the field of the recreational drugs weekly appearing and explosively spreading new substances result everyday challenge for the clinical toxicologists. Not to be illegal for a while helps diffusing the usage of them. The lack of experience of the application of emerging designer drugs often draw the user to run into serious status and need emergency medical treatment. Neither the widespread immunological rapid tests, nor the medical experi-
Biochemia Medica 2012;22(3):A54-A204
A190
2nd European Joint Congress of EFLM and UEMS
ence can help the diagnosis of them. We use HPLCDAD (Shimadzu TOX.I.S) system for toxicological screening and identifying the poisonous basic drugs. Its library can be expanding by the user. To validate the „reference materials” we use MALDI TOF method. We have managed to analyze mephedrone, flephedrone, 4-MEC, MDPV, Benzo-fury (5APB), 4FA, pentedrone and methoxetamine in the last 2 years in increasing number (about 300) of clinical cases. The HPLC method is convenient for the detection and identification of a broad spectrum of drugs it can be either well known or new.
P21-04 Cannabimimetics – A new challenge in routine clinical toxicology Grobosch T, Binscheck T Labor Berlin - Charité Vivantes GmbH, Laboratoriumsmedizin & Toxikologie, Berlin, Germany Corresponding author: [emailprotected]
Background: A 24-yr-old woman with a history of anorexia nervosa/bulimarexia was found unresponsive, breathless and asystolic in bed. After 30 min reanimation attempts of the emergency doctor the patient was defibrillated and transported to hospital. On admission, the EGC showed a prolonged QT interval and developed a significant metabolic acidosis with severe hypokalaemia. Materials and methods: Systematic toxicological analysis: CEDIA, HPLC-DAD, HS-GC. Qualitative Screening (XLC-QQTOF). Quantitative analysis: Cannabimimetics, fentanyl, midazolam (LC-MS/ MS), amiodarone (LC-MS). Results: Urine: Drug screening by CEDIA was positive for benzodiazepines. Additional analytics, including diuretics, laxantia, etc. resulted all negative. XLCQQTOF analysis identified midazolam, fentanyl, and the N-(5-hydroxypentyl) metabolite of JWH018 (110 µg/L). The quantitative LC-MS/MS analysis
for cannabimimetics identified another metabolite of JWH-018, N-pentanoic acid (43 µg/L). Plasma: STA revealed midazolam (640 µg/L). XLCQQTOF analysis identified midazolam, amiodarone and fentanyl. Additional quantitative analysis of the plasma sample identified JWH-018 (58 µg/L), the N-pentanoic acid metabolite (40 µg/L). Three days after reanimation, the neuron specific enolase in serum was significantly increased (80 µg/L) - indicating hypoxic brain damage. Conclusions: Clinicians, and the users need to be aware of the severe clinical effects following consumption of synthetic cannabinoid preparations marketed as partly legal cannabis alternatives. The cannabinomimetics cannot be detected by commonly used immunoassays for THC, therefore mass spectrometry is essential for identification. It is not clear whether the disturbance of myocardial repolarisation in this case was specifically induced by JWH-018 or might be facilitated by other specific circumstances in this case.
P21-05 Extreme case of designer drug abuse Lajtai A (1), Lakatos A (1), Benkő A (2), Mayer M (2), Porpáczy Z (2), Kovács L. G (2) (1) University of Pécs, Medical School, Department of Laboratory Medicine, Pécs, Hungary (2) University of Pécs, Medical School, Department of Forensic Medicine, Pécs, Hungary Corresponding author: [emailprotected]
The identification of new designer drugs means the largest problem in the clinical and forensic toxicology examinations; reference materials are not available, their metabolisms are unknown, their versatility is unlimited. The medical condition of a drug user is frequently critical, requiring intensive care, not to mention the possibility of long-term adverse reactions. Since January 2012 nine designer drugs became illegal in Hungary (e.g. MDPV, 4- Fluroamphetamin, some of the Biochemia Medica 2012;22(3):A54-A204
A191
2nd European Joint Congress of EFLM and UEMS
JWH compounds). Illicit marketing and abuse of these compounds seem to be reduced, in contrast to those drugs which are still legal but we haven’t met them before in our diagnostics practice (e.g. tryptamine derivates, 3-FA, pentedrone). In the near past, we analyzed serum and urine samples of a 27 year old chronic male drug user (GC, Abbott Axsym, HPLC-DAD: Shimadzu TOX.I.S). In the urine sample we detected several kinds of drugs in large quantities. The main component was pentedrone. However, the interpretation of the results was not simple. The complication was caused by some unknown peaks that appeared on the chromatogram. These peaks are likely to be the products of the metabolic reduction of pentedrone. Spectra of these metabolites are very similar to those of ephedrine, but their retention times are different. In conclusion, consumers of various designer drugs might be viewed as the participants of a pharmaceutical study, who voluntarily accept to take part in a dangerous and uncontrolled experiment and help us to discover new drugs,metabolic routes and adverse effects.
P21-06 Identification of acidic compounds in acute intoxications by on-line SPE-HPLC-DAD Mut L (1), Grobosch T (1), Binscheck T (1), Frenzel W (2) (1) Labor Berlin - Charité Vivantes GmbH, Laboratory Medicine and Toxicology, Berlin, Germany (2) Berlin Institute of Technology, Department of Environmental Technology, Berlin, Germany Corresponding author: [emailprotected]
Background: Most therapeutic drugs relevant in clinical toxicology are characterized by basic and neutral properties, but a number of acidic medications (NSAID’s, barbiturates) can cause lifethreatening poisonings. Therefore, an on-line SPE-HPLC-DAD screening method was developed to identify acidic compounds in poisonings. An Biochemia Medica 2012;22(3):A54-A204
A192
in-house library was generated to support this method. Materials and methods: To reduce matrix effects and to deproteinate the plasma, precipitants (acetone, methanol), ratios (1/2, 1/3, 1/4, 2/3, 3/4 plasma/precipitant), buffers (pH: 6, 2.3, 9), injection volumes (0.25 mL, 0.50 mL, 1.0 mL) were tested for maximum performance. Four SPE cartridges were examined in terms of extraction efficiency of acidic compounds. Six HPLC-columns were studied for optimum peak intensity and symmetry. Gradients for an on-line extraction and chromatographic separation of acidic compounds were created. Data of analyzed substances and poisonings were used to establish an inhouse library. The method was applied to acute intoxications. Results: Optimal results for sample preparation were precipitation of plasma with acetone (1/2), dilution of supernatant in buffer (pH = 6) and injection volume: 1.0 mL. Only the SPE-column StrataX-A (pH = 6), allowed the extraction of acids. The most appropriate column for analytical separation and peak symmetry was a 150 x 4.6 mm, 3 µm C6-Phenyl column. The analysis time was 44 min., including on-line extraction (12 min). An in-house library included > 150 entries of acidic compounds and metabolites. Such substances as salicylic acid, or ibuprofen were identified in intoxications. Conclusion: The described method proved to be efficient and sensitive for screening of acidic therapeutic drugs in human plasma in case of acute poisonings.
2nd European Joint Congress of EFLM and UEMS
P21-07 Automated online solid phase extraction UPLC/MS/MS for the analysis of mycophenolic acid Eastwood M (1), Chusney G (2), Calton L (1) (1) Waters Corporation, Clinical, Manchester, United Kingdom (2) Hammersmith Hospital, Imperial College Renal & Transplant Centre, London, United Kingdom
tion monitoring of phospholipids and post-column infusion of analytes. Conclusions: We have successfully quantified mycophenolic acid utilising automated online SPE with UPLC/MS/MS. The assay demonstrates good linearity, precision and accuracy with minimal ion suppression. *NOTE: The MassTrak Online SPE Analyzer is under development
Corresponding author: [emailprotected]
Background: The recent consensus report evaluating therapeutic drug monitoring of mycophenolic acid (MPA) highlighted the need for accurate drug dosing strategies to minimize the incidence of drug-related toxicity while maintaining efficacy. Here we evaluate the potential of a new online solid phase extraction (SPE) system coupled to UltraPerformance liquid chromatography tandem mass spectrometry (UPLC/MS/MS) for the automated sample preparation and analysis of MPA in human plasma. Materials and methods: Commercially available kits were used for calibrators and QC material. Method comparison was carried out using anonymized patient samples quantified using a validated LC/MS/MS assay. All samples were pre-treated with zinc sulphate and methanol. Automated online extraction was carried out with a Waters® ACQUITY UPLC coupled to a Waters MassTrak Online SPE Analyzer* and analysed using a Waters ACQUITY® TQD mass spectrometer. Results: Following CLSI-EP6-A, the assay was shown to be linear from 0.01–50 µg/mL (N = 5). Coefficients of variation for inter- and intra-assay imprecision for low (1.94 µg/mL), mid (2.35 µg/mL), high (5.5 µg/mL) QC samples were all < 10% (N = 25, days = 5). Method comparison using patient samples previously analyzed with a validated LC/ MS/MS assay (N = 50) was described by the Deming equation y = 0.99x-0.01. Compared with conventional one-dimensional chromatography, matrix effects were reduced by the online SPE as determined qualitatively by targeted multiple reac-
P21-08 Automated online solid phase extraction UPLC/MS/MS for simultaneous analysis of immunosuppressants Eastwood M, Balloch S, Cooper D, Calton L Waters Corporation, Clinical, Manchester, United Kingdom Corresponding author: [emailprotected]
Background: Therapeutic drug monitoring of immunosuppressants is an important requirement for the management of transplant patients. To streamline workflow there is a demand for the simultaneous measurement of multiple analytes. Here we evaluate the potential of online solid phase extraction (SPE) coupled to UltraPerformance liquid chromatography tandem mass spectrometry (UPLC/MS/MS) for the automated sample preparation and simultaneous analysis of cyclosporin A (CsA), tacrolimus, sirolimus and everolimus. Materials and methods: Commercially available kits were used for calibrators and QC material. Samples obtained from the ASI Ltd International Proficiency Testing Scheme (IPT) were used to assess accuracy. All samples were pre-treated with zinc sulphate and acetonitrile. Automated online extraction was carried out with a Waters® ACQUITY UPLC coupled to a Waters MassTrak Online SPE Analyzer* and analysed using a Waters ACQUITY® TQD mass spectrometer. Biochemia Medica 2012;22(3):A54-A204
A193
2nd European Joint Congress of EFLM and UEMS
Results: The assay was linear from 24.8–1515 ng/ mL for CsA, 1.0–31.6 ng/mL for tacrolimus, 0.9–30.1 ng/mL for everolimus and 0.9–27.7 ng/mL for sirolimus, with r2 values > 0.997 (N = 5). Inter- and intraassay imprecision for low, mid, high QCs were all < 10% CV (N = 5 per analyte). IPT samples were all within 10% of expected values (N = 5 per analyte). Compared with conventional one-dimensional chromatography, matrix effects were reduced as determined qualitatively by targeted multiple reaction monitoring of phospholipids and post-column infusion of analytes. Conclusions: We have successfully quantified CsA, tacrolimus, sirolimus and everolimus simultaneously utilising automated online SPE with UPLC/ MS/MS. The assay demonstrates good linearity, precision and accuracy with minimal ion suppression. *NOTE: The MassTrak Online SPE Analyzer is under development
P21-09 Development of a LC-MS/MS method for the measurement of propofol and propofol glucuronide
precipitated using acetone. Supernatant was heated for 10mins at 60 °C with dansyl chloride (2.7 mg/mL) and ammonium hydroxide (0.03N). The reaction mixture was injected on to a Waters Acquity UPLC and Quattro Premier XE tandem mass spectrometer. Gradient elution was followed by quantification by electrospray ionisation mass spectrometry in multiple reaction monitoring mode. Results: Dansyl chloride derivatisation significantly increased the detection of propofol. Propofol glucuronide did not react with dansyl chloride and was quantified in its underivatised state. The run time was 11.5 mins. Standard curves were linear (r2 > 0.99) across the calibration ranges. The intraand inter-assay coefficients of variation were < 15% (N = 9-10) and the lower limits of quantitation for propofol and propofol glucuronide were 0.1 µg/mL and 0.25 µg/mL respectively. No ion suppression or enhancement or carry-over was observed. The parent drug and its metabolite were detectable in whole blood from patients receiving propofol infusions. Conclusions: A novel LC-MS/MS method for the simultaneous measurement of propofol and propofol glucuronide was developed, which will be of use in the further investigation of PRIS.
Pitkin S, Zaman M, Benton S Barts Health NHS Trust, Department of Clinical Biochemistry, London, United Kingdom Corresponding author: [emailprotected]
Background: Propofol is an intravenous hypnotic agent used for sedation in intensive care units. A potentially fatal adverse effect is ‘propofol-related infusion syndrome’ (PRIS). There is interest in whether risk factors for PRIS relate to changes in metabolism of propofol. The aim was to develop and validate an LC-MS/MS method for the measurement of propofol and propofol glucuronide in whole blood, suitable for pharmacokinetic studies. Materials and methods: Freeze-thawed whole blood was spiked with internal standards (propofol-d17; propofol glucuronide-d17) and proteins Biochemia Medica 2012;22(3):A54-A204
A194
P21-10 Oxidative stress/serum acetylcholinesterase in Nigeria organophosphate pesticide exposed farmers Akinosun O (1), Adewole T (2) (1) College of Medicine, University of Ibadan, Chemical Pathology Department, Ibadan, Nigeria (2) University College Hospital, Chemical Pathology Department, Ibadan, Nigeria Corresponding author: [emailprotected]
Background: Organophosphate agents constitute about half of all pesticides used globally and they appear to pose the greatest risk among all the pes-
2nd European Joint Congress of EFLM and UEMS
ticides. Despite significant advances in the understanding of the potential mechanism of toxicity in intentional exposures, the precise health effects following occupational exposures are yet to be completely defined. Materials and methods: Oxidative stress status and Acetylcholinesterase activities were studied in blood samples obtained from 25 farmers in Idi Ayunre, Oluyole local government area of Oyo State, using organophosphate (OP) pesticides in spraying their cash crops (cocoa and cola nut trees) with a minimum work history of 10 years, in the age range of 35-75 years. 20 age-matched workers, who never had any exposure to OP pesticides were selected as controls in Ibadan, the capital of Oyo State. Total Plasma Peroxide (TPP) levels using FOX-2 reagent, Total Anti oxidant potential (TAP) using the ferric reducing antioxidant power (FRAP) assay were determined and oxidative stress index (OSI) an indicator of oxidative stress status was calculated. Blood acetylcholinesterase activity was measured using HPLC. Results: Statistically significant decrease in the mean blood levels of acetylcholinesterase (IU/L) in the farmers (43.35 ± 9.07) compared to the controls(65.28 ± 7.66). TPP (umol H2O2/L) increased significantly in the farmers (14.32 ± 5.18) than in the controls (10.25 ± 3.60) (P < 0.05), while depletion of TAP (umolTroloxequiv/L) was observed in the farmers (915.65 ± 130.16) than the controls (975.80 ± 142.70). OSI(%) in farmers (1.65 ± 0.69) increased significantly than controls (1.08 ± 0.39). Conclusion: OP pesticides users are exposed to increased oxidative stress. Assay of acetylcholinesterase activities could be a good biomonitoring index.
P21-11 Blood mercury concentrations in 3 cities in Spain Trasobares E (1), González -Estecha M (1), Martínez García M (2), Herbello Hermelo P (3), Bermejo Barrera P (3), Guillén Pérez J (4) (1) Hospital Clínico San Carlos, Laboratory Medicine, Madrid, Spain (2) Universidad Politécnica de Cartagena, Ingeniería Química y Ambiental, Cartagena, Spain (3) Universidad de Santiago de Compostela, Química Analítica, Santiago de Compostela, Spain (4) Dirección General de Salud Pública, Región de Murcia, Dirección General de Salud Pública, Región de Murcia, Cartagena, Spain Corresponding author: [emailprotected]
Background: There is increasing concern about the effects of exposure to methylmercury in adults. The aim of this multicenter study is to measure blood mercury concentrations in an adult population in 3 cities in Spain. Materials and methods: We recruited 792 employee volunteers from 2 hospitals and one University in Madrid, Cartagena and Santiago de Compostela. Blood mercury concentration (μg/L) was measured in Madrid by cold vapour atomic absorption spectrometry in a Perkin Elmer FIMS 400 and in Cartagena in a direct manner in a DMA-80 Millestone based on the EPA 7473 method. Evaluation of concordance by Bland Altman plot was performed between these two methods. Blood mercury in Santiago de Compostela was determined by Cold Vapour Atomic Absorption Spectrometry in a Perkin Elmer 4100 equipped with a FIA Perkin Elmer 400. Results: Upon evaluation of concordance between Madrid and Cartagena methods, almost all the measurements concorded and were included in the 95% confidence interval of the mean of the differences. The medians of blood mercury (µg/L) obtained were: Madrid (7.9; IQR: 5.2-11.5); Cartagena (8.95; IQR: 6.7-13.8) and Santiago de Compostela (15.1; IQR: 10.2-19.9). A statistically significant difference was observed among blood mercury concentrations in the 3 cities (P < 0.001). We also observed statistically significant differences between Madrid Biochemia Medica 2012;22(3):A54-A204
A195
2nd European Joint Congress of EFLM and UEMS
and Cartagena (P = 0.004); Madrid and Santiago de Compostela (P < 0.001) and between Cartagena and Santiago de Compostela (P < 0.001). Conclusions: Higher blood mercury concentrations were found in Spain than those previously reported in other European countries, probably due to the higher fish consumption in Spain.
P21-12 Influence of immunosuppressive regimen change on renal function in liver transplant recipients Ožvald I (1), Mrzljak A (2), Radisic Biljak V (1), Perkov S (1), Flegar Mestric Z (1) (1) University Hospital Merkur, Institute of Clinical Chemistry and Laboratory Medicine, Zagreb, Croatia (2) University Hospital Merkur, Department of Gastroenterology, Zagreb, Croatia Corresponding author: [emailprotected]
Background: Liver transplantation is accepted treatment of choice for many liver diseases. Long– term survival is limited by toxicity of immunosuppressive agents, sub-clinical as well as cronic rejection. Newer classes of immunosuppressive agents, including calcineurin inhibitors CNI (cyclosporine and tacrolimus) and mammalian target of rapamycin (mTOR) inhibitors (sirolimus and everolimus) have potential to improve long-term outcomes. Many long-term survivors face a considerable risk of renal dysfunction due to CNI. The aim of the study was to determine benefits of sirolimus compared to CNI towards kidneys toxicity. Materials and methods: We have monitored nine orthotopic liver transplantation patients (OLT) who underwent conversion of immunosuppressive regiment from CNIs to sirolimus. Creatinine concentrations were measured with the creatinine enzymatic assay. Measured concentrations of serum creatinine were used to estimate renal function, after dosing CNIs and converting to sirolimus, each one, three and six months after the dose. Biochemia Medica 2012;22(3):A54-A204
A196
Results: Referring to the long-term outcomes of OLT patients following results could be seen: Among all patients switched to sirolimus, the levels of serum creatinine (80.3 µmol/L ± 12.8 vs. 76.0 µmol/L ±12.7) remained stable in three of them, whereas the levels of serum creatinine started to decrease (119.4 µmol/L ± 29.8 vs. 83.2 µmol/L ± 26.3) after administration of sirolimus in six of the patients. Conclusion: These preliminary results have shown that sirolimus is effective in preventing rejection in OLT recipients and it is associated with improved renal function. mTOR inhibitors might have a role as an early alternative to CNIs in patients with CNI nephrotoxicity.
P21-13 Multi-drug intoxication fatality involving atorvastatin: a case report Cibicek N (1), Caran T (2), Pribyl P (3), Dobias M (4), Ondra P (4), Vorisek V (5) (1) Palacky University Olomouc, Faculty of Medicine and Dentistry, Department of Medical Chemistry and Biochemistry, Olomouc, Czech Republic (2) Hospital Hranice, Department of Anaesthesiology and Resuscitation, Hranice, Czech Republic (3) Hospital Hranice, Department of Internal Medicine, Hranice, Czech Republic (4) University Hospital Olomouc, Institute of Forensic Medicine and Medical Law, Olomouc, Czech Republic (5) Charles University in Prague, Faculty of Medicine and University Hospital in Hradec Kralove, Institute of Clinical Biochemistry and Diagnostics, Hradec Kralove, Czech Republic Corresponding author: [emailprotected]
Background: Mixed antihypertensive drug intoxication poses a significant risk for patient mortality. In tandem to antihypertensives, hypolipidemic medicines (especially statins) are often prescribed. Among their adverse effects belongs rhabdomyolysis. Case description: We report a case of fatal multidrug overdose in a 65-year-old female alcoholic. The woman was admitted to a municipal hospital unconscious. Empty blister packs indicated the abuse of 250 tablets of urapidil, 42 tablets of vera-
2nd European Joint Congress of EFLM and UEMS
pamil/trandolapril, 50 tablets of moxonidin, 80 tablets of atorvastatin and 80 tablets of diacerein. Standard measures (gastric lavage, mechanical ventilation, massive doses of vasopressors, volume expansion, diuretics and alkalinisation) failed to provide adequate drug elimination and hemodynamic support. The patient deceased on the fourth day. Results: Dramatic elevations of serum myoglobin (34880 ug/l) and creatine kinase (281 ukat/l) were accompanied by rise in cardiac troponin I and creatinine. Gas chromatography revealed ethanol 1.17 g/kg (blood) and 2.81 g/kg (urine). Thin layer chromatography and gas chromatography of gastric content and urine verified verapamil, moxonidin and urapidil fragment (diacerein method was unavailable). Atorvastatin and trandolapril concentrations (LC-MSn) equaled 277.7 ug/L and 57.5 ug/L, resp. (serum) and 8.15 ug/L and 602.3 ug/L, resp. (urine). Histology confirmed precipitates of myoglobin with acute necrosis of proximal renal tubules in association with rhabdomyolysis of striated muscle and myocardial dystrophy. Conclusions: Distributive and cardiogenic shock in conjunction with acute renal failure due to the combined self-poisoning with vasoactive agents and atorvastatin were determined to be this decedent's immediate cause of death. The manner of death was assigned to be suicidal.
P21-14 Pharmacokinetics of mycophenolic acid in renal allograft recipients: role of ABCC2 polymorphisms Lalić Z (1), Božina N (1), Nađ Škegro S (2), Trkulja V (3), Lovrić M (1), Granić P (1)
netic variability. MPA is metabolized by UGTs to inactive 7-O-MPA-glucuronide (MPAG). MPA and MPAG are subject to enterohepatic recirculation. Biliary and kidney excretion of MPA/MPAG involves several transporters, including multidrug resistant protein-2 (MRP-2) coded by polymorphic ABCC2, which can influence MPA pharmacokinetics. The objective of this study was to perform MPA pharmacokinetics et steady state conditions during one dosing interval (12 h), in 68 renal allograft recipients. Pharmacokinetic variability in relation to donor and recipient ABCC2 genotypes is estimated. Patients and methods: Blood samples were drawn at 0, 0.5, 1, 2, 3, 8, and 12 h after the morning dose. Genotyping of ABCC2 C-24T and G1249A was performed using TaqMan-based allele-specific PCR assay. Plasma concentrations of MPA were determined using validated HPLC method. Results: Pharmacokinetic parameters: Cmax,ss (mg/L) 12.3 ± 6.7 ; Tmax (hrs) 2 (0.2-12); AUCt,ss (mg*h/L) 39.9 ± 20.7; Cmin,ss (mg/L) 1.3 ± 1.2; Trough 1 (time 0) (mg/L) 2.7 ± 2.1; Trough 2 (time 12) (mg/L) 1.9 ± 1.9. Considering ABCC2 genotypes associations were found between: donor C-24T and lower through1 concentrations in T allele carriers (P = 0.003); G1249A variants and lower Cmax/ dose in A allele carriers (P < 0.05), -24T allele carriers and % of concentration swing (OR 1.88, 95%CI 1.09-3.23). For recipient genotypes correlations were between: 1249A allele and lower Cmin and trough2 concentrations, and higher % of concentration swing (OR 1.85 95%CI 1.05-3.28). Conclusion: The pharmacokinetics of MPA is affected by the ABCC2 polymorphisms.
(1) University Hospital Center Zagreb, Department of Laboratory Diagnostics, Zagreb, Croatia (2) University Hospital Center Zagreb, Department of Urology, Zagreb, Croatia (3) Zagreb University School of Medicine, Department of Pharmacology, Zagreb, Croatia Corresponding author: [emailprotected]
Background: Mycophenolic acid (MPA) displays large between- and within-subject pharmacokiBiochemia Medica 2012;22(3):A54-A204
A197
2nd European Joint Congress of EFLM and UEMS
P21-15
P22 - Vitamin D - PTH
HPLC of carbohydrate-deficient transferin and more sialylated transferrin glycoforms in children
P22-01
Bianchi V (1), Raspagni A (2), Arfini C (3), Vidali M (4) (1) SSS Antonio e Biagio Hospital, Toxicology Laboratory, Alessandria, Italy (2) Insubria University, School of Medicine, Varese, Italy (3) SS Antonio e Biagio, Clinical Laboratory, Alessandria, Italy (4) Maggiore della Carità Hospital, Clincal Chemistry Unit, Novara, Italy Corresponding author: [emailprotected]
Background: The evaluation of age-specific distribution of transferrin glycoforms in paediatric patients may help in defining reference intervals which are critical for an improved and earlier diagnosis. Materials and methods: Serum samples from 224 children (age: 2 months-14 years) were analyzed by HPLC (CDT by HPLC kit, Bio-Rad, Munich, Germany) and glycoforms expressed as percentage of the total area of transferrin (Tf). Results: Asialo- and Monosialo Tf were not detectable in any patient. Median (IQR) were respectively 0.92% (O.80-1.04%) for DisialoTf; 3.47% (2.694.18%) for Trisialo-Tf; 82.54% (81.32-83.53%) for Tetrasialo-Tf; 12,73% (11.91-14.09%) for PentasialoTf. Statistically significant differences in Trisialo-Tf (P < 0.001), Tetrasialo-Tf (P = 0.001), Pentasialo-Tf (P 0.05). The coefficient of variation described by the commercial insert of the method (also verified by our laboratory) is 4.17 and the ob-
tained according to the duplicate samples of the study between two groups was 4.30, so no differences were observed. Conclusions: According to results, in the iPTH cuantification is unnecessary the immediate centrifugation of the sample. The determination may be made in the same serum tube used to analyze the different biochemical parameters, whose centrifugation is usually done between 2 and 3 hours after collection; this protocol can avoid preanalytical errors, saving time and expenditure rationalization.
P22-03 Evaluation of automated method for measurement of serum 25-hydroxyvitamin D Vogrinc Ž, Lovrić M, Sertić J Clinical Hospital Center Zagreb, Clinical Institute of Laboratory Diagnostics, Zagreb, Croatia Corresponding author: [emailprotected]
Background: The most reliable indicator of vitamin D status in organism is measurement of circulating 25-hydroxyvitamin D (25(OH)D) in serum or plasma. The measurement is challenging because 25(OH)D is highly lipophylic, bound strongly to protein, present in low concentrations and exsists in two structurally similar forms, 25(OH)D3 and 25(OH)D2. The aim of the study was to evaluate a new automated assay for quantitative determination of serum total 25(OH)D (Roche Diagnostics) and compare it with selective and sensitive HPLC method with UV detector. Materials and methods: 25(OH)D from human sera was measured using two methods: fully automated competitive electrochemiluminiscence method (Roche Elecsys Vitamin D total assay) and Chromsystems HPLC method for 25(OH) D3/D2. The evaluation protocol consisted of within-run imprecision (10 sequential runs) and between-run imprecision (10 consecutive working days, 2 sequential runs) with commercial controls PreciControl Biochemia Medica 2012;22(3):A54-A204
A199
2nd European Joint Congress of EFLM and UEMS
Bone 1 and 2, inaccuracy (N = 20), and method comparison (routine serum samples, N = 37). Methods were compared by Passing and Bablok regression and Bland-Altman analyses. Results: Within-run imprecision for Roche Elecsys Vitamin D total assay was 2.56%, and between-run imprecision was 2.99% and 3.79%. Quality requirement for inaccuracy was fulfilled. The comparison with HPLC method demonstrated strong correlation (r = 0.9581; y=0.918x-4.5082) and good agreement (bias = ± 1.96 SD). Conclusion: Roche Elecsys Vitamin D total assay showed good correlation and agreement with HPLC-UV method and represents an accurate and precise automated tool for serum total 25(OH)D determination.
P22-04 Vitamin D status in heart failure patients is dependent on the assay method used Silva N (1), Martins S (2), Tuna D (2), Lourenço P (3), Bettencourt P (4), Guimaraes J (1) (1) Faculty of Medicine, University of Porto & Sao Joao Hospital, Biochemistry Department & Clinical Pathology Department, Porto, Portugal (2) Sao Joao Hospital, Clinical Pathology Department, Porto, Portugal (3) Sao Joao Hospital, Internal Medicine Department, Porto, Portugal (4) Faculty of Medicine, University of Porto & Sao Joao Hospital, Internal Medicine Department, Porto, Portugal Corresponding author: [emailprotected]
Background: Heart failure (HF) is a prevalent public health problem. Studies indicate a beneficial role for Vitamin D (VTD) on cardiovascular health. Knowing that VTD measurement is highly method dependent, our aim was to evaluate how two different automated assays influence VTD status classification in HF patients. Materials and methods: Serum samples from 134 patients (65 males and 69 females with 75 ± 13 Biochemia Medica 2012;22(3):A54-A204
A200
years old) were evaluated using two chemiluminescent immunoassays (Roche Cobas®- routine method, and Abbott Architect®). Three groups were set: ≤ 10 ng/mL for deficient (A); 11-20 ng/mL for insufficient (B) and > 20 ng/mL for optimal (C). The statistical analysis was performed in Medcalc® software. Results: For Architect® the values ranged from 5.2 ng/mL to 29.8 ng/mL with a mean value of 14.3 ng/mL. For Cobas® ranged from 3.0 ng/mL to 23.1 ng/mL with a mean value of 8.2 ng/mL. The correlation coefficient was 0.8295, with a mean difference of 6.0 ng/mL, (95%Cls, [5.5; 6.7], P < 0.001). When evaluated on Architect®, 73 (54%) of the patients changed VTD group status: 59 from A to B and 14 from B to C. From the 32 samples which had a value of < 3.0 ng/mL (LOD) on Cobas®, 21 patients continued in A group while 11 changed to B. The mean difference for each group was 6.1, 6.1 and 6.4 ng/mL, respectively. Conclusion: Pathologists and clinicians must be aware of the clinical consequences that method selection has on patients Vitamin D status classification.
P22-05 Comparison of vitamin D3 and total vitamin D values Županić D, Crnokrak S, Juričić G, Jedrejčić K, Honović L Pula General Hospital, Department of Laboratory Diagnostics, Pula, Croatia Corresponding author: [emailprotected]
Introduction: Vitamin D is a fat-soluble vitamin, which is mainly produced in the skin from sun exposure. To become biologically active, vitamin D undergoes two hydroxilations in the liver and kidney. There are two important forms of vitamin D (D2 and D3). While vitamin D3 is produced in the body, D2 is derived from food and supplements.
2nd European Joint Congress of EFLM and UEMS
Objective: We wanted to show the value of vitamin D3 and total vitamin D (D2 and D3) in the same patients. Materials and methods: The study included 40 patients (30 women and 10 men). The values of vitamin D3 and total vitamin D were determined by the immunoassay on analyzer COBAS e601 (Roche, USA).
tions by National Osteoporosis Foundation. First group – vitamin D concentration below 25 nmol/L (50 serums), second group -concentration between 25-75 nmol/L (48 serums) and third group – concentration 75 nmol/L or higher (50 serums). All the serums of three groups were re-measured with both methods in one batch at the same day.
Results: The study group value of vitamin D3 < 10 nmol/L was found in 6 patients, 10-30 nmol/L in 10 patients, 30-75 nmol/L in 22 patients and > 75 nmol/L in 2 patients. Total vitamin D was determined for the same patients; the value of total vitamin D < 10 nmol/L was not detected in any patient, 10-30 nmol/L in 5 patients, 30-75 nmol/L in 23 patients and > 75 nmol/L in 11 patients.
Results: The coefficient of determination (R2) was 0.577 (P < 0.001) for the first, 0.859 (P < 0.001 ) for the second and 0.669 (P < 0.001) for the third group. The linear regression between methods was ELISA (y) = 1.28*ECLIA (x) + 24.66 (SE = 5.59) for the first, ELISA(y) = 0.94*ECLIA (x) + 17.73 (SE = 5.96) for the second and ELISA (y) = 0.89*ECLIA (x) + 25.46 (SE = 11.15) for the third group.
Conclusion: Based on the results we can conclude and confirm that the method for total vitamin D determines both vitamin D3 and vitamin D2 because the values are higher than those from the method that determines only vitamin D3.
Conclusions: There was no good correlation between two methods used in this study. The correlation between two assays was best for the second group containing serums with 25-OH vitamin D concentrations between 25-75 nmol/L. Additional studies containing more methods are required to evaluate and compare the methods used for measurement of 25-OH vitamin D.
P22-06 Comparison of two immunoassays for 25-OH vitamin D Mägi M
P22-07 25-hydroxi-vitamin-D levels in clinical conditions with low plasma albumin
East-Tallinn Central Hospital, Central Laboratory, Tallinn, Estonia Corresponding author: [emailprotected]
Background: There are several analytical methods used for measuring the concentration of 25OH vitamin D in serum. The purpose of this study was to compare the concentrations of vitamin D using two different immunoassays: electrochemiluminescence immunoassay (ECLIA, Roche Cobas e411) and enzyme-linked immunosorbent assay (ELISA, Euroimmun AG). Material sand methods: First the vitamin D concentrations in serums were measured with ECLIA and according to this results were divided into three groups based on vitamin D recommenda-
Kovacs G (1), Horvath D (2), Viragh E (2), Kovacs L (3), Locsei Z (2), Toldy E (4) (1) University of Pecs, Institute of Laboratory Medicine, Pecs, Hungary (2) Markusovszky Teaching Hospital, 1st Department of Medicine, Szombathely, Hungary (3) Markusovszky Teaching Hospital, B. Braun Avitum Dialysis Centre, Szombathely, Hungary (4) University of Pecs, Institute of Diagnostics, Szombathely, Hungary Corresponding author: [emailprotected]
Introduction: Majority of circulating total 25(OH) D is bound to proteins. 90% is bound to vitamin D binding protein (DBP), an alfa-2globulin (a-2-Gl) Biochemia Medica 2012;22(3):A54-A204
A201
2nd European Joint Congress of EFLM and UEMS
fraction, and 10% to albumin (ALB). Nowadays the total t25(OH)D level is considered to be the most widely accepted marker of vitamin D supply in physiological states. Under pathological circumstances (e.g. in diseases with low ALB), however, the situation might be different. Our aim was to investigate the t25(OH)D concentration in clinical conditions with low ALB levels. Materials and methods: 95 patients (48 men, 47 women; mean age: 68.2 ± 14.4 years) with low ALB (31.9 ± 5.9 g/L) were studied. 58 patients had chronic renal failure, 8 nephrosis, 17 cirrhosis and 12 malnutrition. 58 healthy adults (30 men, 28 women; mean age: 65.9±15.8 years) were in the control group. 25(OH)D, intact parathormon (PTHi), calcium, TP, ALB, DBG, a-2-Gl were measured. Results: 90% of the patients with low ALB had vitamin D deficiency (< 50 nmol/L). Low vitamin D, however, also occurs among healthy people. Vitamin D deficiency is much more frequent in cases with low DBP (< 272 mg/L), than in those with normal DBP. There was a correlation between vitamin D and DBP. This was much stronger in the group of patients with renal failure. Conclusion: Our results suggest that in clinical conditions with low ALB levels - especially if hypalbuminaemia is associated with low DBP and excess of a-2-Gl - t25(OH)D levels may not only depend on vitamin D supply, but also on the presence and capacity of the binding proteins.
Biochemia Medica 2012;22(3):A54-A204
A202
P22-08 Intraoperative parathyroid hormon measurements in femal with parathyroid adenoma Mayer L (1), Gaće M (1), Dobrijević S (1), Špacir-Prskalo Z (1), Janušić R (2), Ramljak V (3) (1) Sestre Milosrdnice University Hospital, University Hospital for Tumors, Clinical Institute of Chemistry, Zagreb, Croatia (2) Sestre Milosrdnice University Hospital, University Hospital for Tumors, Department of Head and Neck Surgery, Zagreb, Croatia (3) Sestre Milosrdnice University Hospital, University Hospital for Tumors, Department of Cytology, Zagreb, Croatia Corresponding author: [emailprotected]
Primary hyperparathyroidism (PHPT) is disorder characterized by increased and uncontrolled parathyroid hormone secretion, cause of hyperfunction of one or more parathyroid glands. In 80-85% cases of PHPT is caused by parathyroid adenoma. Persistent hyperparathyroidism leads to altered osseous metabolism involving bone resorption and tissue changes. In rare cases, approximately in every thirteenth patient with PHPT, the bone mass is suspected of being a neoplastic lesion - brown tumor induced by primary hyperparathyroidism. The only way of PHPT correction is surgical elimination hyperactive parathyroid glands. In this article we report the first case of intraoperative parathyroid hormone measurements for primary hyperparathyroidism in Republic of Croatia. The possibility of intraoperative PTH monitoring provides an additional patient and operator safety. Intraoperative PTH becomes an exact instructor (navigator) to operator –surgeon. On the basis of decreasing value of this peptide with very short half-life time, surgeon makes immediate decision if the operation is completed or he requires further excision parathyroid glands because of hyperplasia. Guarantee of successful diagnosis, which is a prerequisite for the correct treatment, is a multidisciplinary, continuous, systematic and synchronized cooperation of whole and heterogeneous medical team, which includes clinicians, radiologists, cytologists, pathologists and medical biochemists.
2nd European Joint Congress of EFLM and UEMS
P22-09 Patients with colorectal cancer have profound deficiency of vitamin D Greplova K (1), Valik D (1), Obermannova R (2) (1) Masaryk Memorial Cancer Institute, Department of Laboratory Medicine, Brno, Czech Republic (2) Masaryk Memorial Cancer Institute, Department of Comprehensive Cancer Care, Brno, Czech Republic Corresponding author: [emailprotected]
Background: Vitamin D plays an important role in a number of physiological functions including calcium absorption, bone metabolism, immune function, muscle function and cellular regulation. Numerous clinical studies have shown that vitamin D has significant protective effect against the development of cancer. We studied changes in 25(OH)D serum concentrations at different temperature storage conditions. Subsequently we measured 25(OH)D serum concentration in 100 healthy individuals and 281 patients with colorectal cancer. Materials and methods: Blood samples were taken in sample tube without anticoagulant (Sarstedt, catalog number 01.1728.001). After centrifugation (1500 x g, 15˚C, 20 min), separated serum was pipetted into 3 test tubes. 25(OH)D was measured 1) immediately after serum separation 2) after 3 weeks of freezing at -80 ˚C 3) after 3 weeks of freezing at -30˚C. 25(OH)D serum concentrations were measured using Architect i2000sr (Abbott) analyzer. Results: We didn’t find any significant concentration changes of 25(OH)D in 100 samples at various temperature storage conditions. In the group of healthy individuals median of 25(OH)D concentration was 55.35 nmol/L (range within 21.7-116.4 nmol/L), yielding reference range 30-90 nmol/L (95% confidence interval). In the group of colorectal cancer patients median of 25(OH)D was 26.1 nmol/L (range within 0-84.7 nmol/L). Conclusions: The CMIA method proved to be robust and stable for clinical determination of serum
25(OH)D levels. In colorectal cancer patients we observed profound deficiency of vitamin D. This study was funded by European Regional Development Fund and State budget of the Czech Republic (RECAMO:CZ 1.05/2.1.00/03.0101) and by Ministry of Education, Youth and Sports (BBMRI:LM2010004).
P22-10 Vitamin D mediated inhibition of cancer: Do cytokines play a role? Mohapatra S (1), Saxena A (1), Gandhi G (2), Singh T (3), Koner BC (1), Ray PC (1) (1) Maulana Azad Medical College, Department of Biochemistry, New Delhi, India (2) Maulana Azad Medical College, Department of Obstetrics and Gynecology, New Delhi, India (3) Maulana Azad Medical College, Department of Pathology, New Delhi, India Corresponding author: [emailprotected]
Background: There exists an inverse relationship between vitamin D levels in blood and incidence of many cancers. Vitamin D suppresses pro-inflammatory Th1 cytokines (TNF-α) and promotes Th2 subtype differentiation (as marked by rise in IL-4). In ovarian cancer, the tumour microenvironment is enriched with a broad spectrum of pro-inflammatory cytokines which helps in tumour progression. Role of vitamin D in ovarian cancer has not yet been clearly defined. Materials and methods: A case control study was conducted recruiting fifty ovarian cancer patients and fifty controls. Serum vitamin D, TNF-α and IL-4 were measured in fasting blood sample of the subjects. Results and conclusions: Serum vitamin D levels were significantly (P < 0.033) lower in ovarian cancer cases [20.1 ng/mL (61.8-6.93)] as compared to controls [4.6 ng/mL (47-7.3)] which was more evident in post-menopausal group of ovarian cancer patients. TNF-α levels were significantly higher in ovarian cancer patients [cases: 12.2 pg/mL (21.0Biochemia Medica 2012;22(3):A54-A204
A203
2nd European Joint Congress of EFLM and UEMS
5.1); controls: 6.2 pg/mL (12.0-2.0); P < 0.001] and IL-4 levels were significantly lower as compared to those of controls (cases: 2.22 ± 0.51 pg/mL; controls: 2.99 ± 0.68 pg/mL; P < 0.001). Vitamin D levels were negatively correlated (R2 = 0.092, P < 0.034) with TNF-α and positively correlated (R2 = 0.227, P < 0.001) with IL-4. None of the ovarian cancer patients had serum vitamin D level in highest
Biochemia Medica 2012;22(3):A54-A204
A204
tertile of the study group. Our study provides evidence that increased Th1 cytokines release and decreased Th2 response are associated with low serum vitamin D which might be a risk factor for ovarian cancer. This indicates that supplementing vitamin D might be protective against ovarian cancer (especially in postmenopausal women) by modulating the cytokine environment.
