Daria Pašalić
Department of Medical Chemistry, Biochemistry and Clinical Chemistry
Zagreb University School of Medicine
Šalata ul 2.
10 000 Zagreb, Croatia
Phone +385 (1) 4590 205; +385 (1) 4566 940
E-mail: dariapasalic [at] gmail [dot] com

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P09-1 (Oral presentation)

Fijačko M1, Fijačko V2, Kristek J3, Dobrošević B1, Pavela J1, Cetina N1. PO9-1: Standardization of cytokine concentrations in bronchoalveolar lavage fluid (BALF) with albumin. Biochemia Medica 2009;19(Suppl 1):S144.
1Institute for Clinical Laboratory Diagnostics, Osijek University Hospital, Osijek, Croatia
2Institute for Pulmonary Disease, Osijek University Hospital, Osijek, Croatia
Corresponding author:fijacko [dot] mirjana [at] kbo [dot] hr
Background: The technique of BAL is based on the concept that aliquots of sterile normal saline solution infused through the bronchoscope mix with epithelial lining fluid (ELF). When the saline solution is recovered by aspiration, the ELF and its components are recovered along with it. However, the recovered BALF is a variable mixture of saline solution, ELF and ELF components. The interpretation of BALF findings is still hindered because the procedure cannot be precisely standardized. In particular, there is still no satisfactory method of determining the dilution factor during lavage. Albumin is widely used as an internal marker as reference standard to assess dilution.
Material and methods: We dosed the BALF concentrations of IL-1 beta, IL-6, IL-8 and albumin in 24 patients (19 female and 5 male, age 36-65) affected by pulmonary sarcoidosis, using the ELISA and turbidimetric method. We also presented our data as ratios of the concentration of cytokines to the concentration of albumin in BALF, i.e. the crude BALF cytokine levels directly measured by ELISA were divided by the BALF albumin levels, and date were given as ratios of the amount of cytokine per miligram of albumin (standardized BALF cytokine levels).
Results: BALF cytokine levels correlated positively with standardized BALF cytokine levels (for IL-1 beta r = 0.999, P < 0.001; for IL-6 r = 0.7969, P < 0.05, and for IL-8 r = 0.976, P < 0.001). Moreover, these standardized cytokine levels were strongly mutually correlated.
Conclusion: This standardization method removes the variable of dilution and allows comparison between data from different subjects and investigators.
Klasić A1, Beljan B2, Kozić-Dokmanović S3, Oršolić Lj4, Bukovec Megla Ž5, Getaldić B6. PO9-2: Eosinophil Cationic Protein (ECP), IgE and eosinophils in asthmatic children. Biochemia Medica 2009;19(Suppl 1):S145.
1Special Hospital for Medical Rehabilitation Krapinske Toplice, Krapinske Toplice, Croatia
2Rijeka Clinical Hospital Center, Rijeka, Croatia
3University Hospital for Infection Deseases Fran Mihaljević, Zagreb, Croatia
4Slavonski Brod General Hospital, Slavonski Brod, Croatia
5Laboratory for Endocrinology, Sestre milosrdnice University Hospital, Zagreb, Croatia
6University Department of Chemistry, Sestre Milosrdnice University Hospital, Zagreb, Croatia
Corresponding author:anita [dot] klasic [at] gmail [dot] com
Background: Eosinophil cationic protein is a basic protein located in eosinophil matrix. During variety of inflammatory conditions, including asthma, ECP is released through the degranulation process and can be determined in blood. ECP level is proportinal to the intensity of allergic inflammatory process. IgE is a marker of atopy in the serum of asthmatics.
Aims of this research were: to compare the serum ECP concentracion in asmathic patients and in the patients with non-specific respiratory diseases (NSRD) with total eosinophil count in peripheral blood and with IgE level in serum, and to compare those parameters within both groups.
Materials and methods: Quantitative measurement of ECP and IgE in serum was performed with an automated commercial FEIA method (ImmunoCAP100,Sweden) in the group of 42 asthmatic children and in 45 children with NSRD. In blood specimens eosinophils were counted on Abbott CD 3200.
Results: We found higher concentrations of all three parameters in asthmatic children than in NSRD diagnosis but difference was not significant (P > 0.05). In the group of asthmatic patients concentrations of ECP and IgE showed weak to acceptable correlation (r = 0.384; P = 0.012), until the ECP concentration and eosinophil count showed moderate correlation (r = 0.522; P < 0.01). No correlation was found between concentrations of ECP and IgE (r = 0.025; P = 0.87) nor between eosinophil count and ECP (r = 0.06; P = 0.68) in the group with NSRD.
Conclusion: Our results do not sugest that ECP is criterion for distinction among patients with asthma and NSRD and ECP level is not associated with IgE level and total eosinophil count in both groups.
Kozmar A, Škaričić A, Rnjak L, Rudolf M, Radić N, Malenica B. PO9-3: Evaluation of multiplexed fluorescent microspheres immunoassay for detection of autoantibodies to nuclear antigens. Biochemia Medica 2009;19(Suppl 1):S146.
Department of Immunology, Institute for Laboratory Diagnosis, Zagreb Clinical Hospital Center, Zagreb, Croatia
Corresponding author:akozmar [at] kbc-zagreb [dot] hr
Introduction: Anti-nuclear autoantibodies (ANA-ENA) directed against various cell nuclear autoantigens characterize systemic rheumatic diseases. Some of them have assigned in the classification for systemic lupus erythematosus (SLE), mixed connective tissue disease (MCTD), systemic sclerosis (ScS) and Sjogren syndrome (SS). Because ANA testing is a critical part of diagnosis in systemic rheumatic diseases, it is important to detect them very carefully. Indirect immunofluorescences (IIF) on HEp-2 cells, ELISA and polystyrene microsphere-based fluorescent assay have been used for this purpose.
Materials and methods: The aim of our study was to compare sensitivity and concordance of these tests for detection of ANA-ENA and determination of nine different target autoantigens in 42 patients with SLE and 164 patients with suspicious systemic autoimmune diseases.
Results: Comparison of ANA screening results by IIF with ELISA and AtheNa Multi-Lyte ANA test system, showed a high rate of concordance (Kappa value from 0.289–0.793 and agreement from 88-89%). Positive discrepant results were found for ds-DNA specificity in 12.5% (25/200) specimens by AtheNa technology, while all tested sera were negative for this antibody by ELISA. Negative discrepant results were observed by AtheNa system for anti-ds-DNA (1.5%; 3/200). In 7 serum samples (3.5%; 7/200) we found completely different target antigens by ELISA (ds-DNA) and by AtheNa technology (SS-A, RNP or SS-A, SS-B). Very good concordance in the detection of individual target autoantigens was observed between ELISA and microsphere-based assay (Kappa value from 0.306-0.708 and agreement from 90-98%).
Conclusion: Our results suggest that the AtheNa Multi-Lyte ANA test system may be a useful diagnostic tool for ANA-ENA determination.
Pavela J, Dobrošević B, Fijačko M, Majetić-Cetina N. PO9-4: Anti–Golgi antibodies: an early sign of autoimmune disease: Case report. Biochemia Medica 2009;19(Suppl 1):S147.
Institute for Clinical Laboratory Diagnostics, Osijek University Hospital, Osijek, Croatia
Corresponding author:pavela [dot] jasna [at] kbo [dot] hr
Introduction: Anti-Golgi antibodies (AGAs) are a type of anticytoplasmic autoantibody rarely found during routine examination of pathological samples.Altough AGAs are primarily associated with Systemic lupus erythematosus and Sjogren syndrome several reports described AGAs in rheumatoid arthritis and HIV infection.
Case report: A 52 years old women presented with morning stifness with symetrical inflamatory polyarthritis involving PIP of 15 years duration. Laboratory findings show: ANA test = negative; RF = negative; CCP = 24.8 IU/mL (reference value < 25 IU/mL); CRP = 8.2 mg/L; Anti-nuclear fluorescence test = positive, showed distinct perinuclear crescent shaped structure in Hep-2 cells AGAs antibodies. The titer was 1:3200. One year later laboratory findings were similar exept rise CCP = 46.4 IU/mL. CCP. The diagnosis is Polyarthritis rheumatoides.
Materials and methods: Anti-Golgi antibodies were detected by indirect immunofluoroscence test (IIFT) utilizes Hep-2 cell lines purchased from Euroimmun. Cyclic citrulinated peptide (CCP) antibodies were detected by ELISA test manufactured by IMUNOSCAN.
Conclusion: Altough the detection of anti-Golgi antibodies is rare, and may represent a transitory epiphenomenon on patients with a viral infection, their presence in high titre in the absence of a clear clinical picture may constitute an early sign of systemic autoimmune disease.
Šiftar Z1, Kardum Paro MM1, Kardum-Skelin I2, Dominis M3, Sokolić I1, Flegar-Meštrić Z1. PO9-5: CD20+ T Non-Hodgkin lymphoma; Case report. Biochemia Medica 2009;19(Suppl 1):S147-S148.
1Institute of Clinical Chemistry, Merkur University Hospital, Zagreb, Croatia
2Laboratory for Cytology and Haematology, Merkur University Hospital, Zagreb, Croatia
3Institute for Clinical Pathology and Cytology, Merkur University Hospital, Zagreb, Croatia
Corresponding author:zoran [dot] siftar [at] hdmb [dot] hr
Introduction: T Non-Hodgkin lymphoma (T-NHL) is part of lymphoproliferative disease with appearance less than 15%, in which is very rarely described positivism of CD20 marker. Imunophenotyping of cells using flow cytometry is necessary for his undoubtedly proving.
Material and methods: A 67 years old man, with an enlarged neck lymph nodes, the normal number of leukocytes, platelets and erythrocytes, and elevated sedimentation erythrocytes rate, was received at the haematological department “Merkur” University Hospital, for treating by FED scheme and extending diagnosis of changes in lymph node on the molecular analysis of cells clonality and flow cytometry imunophenotyping. Morphological analysis was done according to Pappenheim (MGG staining), and immunocytochemistry procedures alkaline phosphatase anti-alkaline phosphatase (APAAP) staining. Molecular diagnostics of cells clonality was made by Van Dogen (Leukemia, 2003) using multiplex PCR method. Imunophenotyping is made by using flow cytometry according to Rothe et al. (Leukemia, 1996). The analysis was conducted on isolated mononuclear cells through density gradient, after stained with directly conjugated antibodies from Beckman-Coulter and Dako. Measurement results were in accordance and within the target value of internal and external quality assurance programme UKNEQAS Leukaemia Immunophenotyping.
Results: Cytological finding of lymph node aspirates speaks about monomorphous population of lymphatic cells that are CD20+ (72%) and CD3+ (59%), pathological findings are: CD20-CD3+/-CD2+CD4+. Molecular diagnostics proved clonally TCRb and TCRg gene arrangement with polyclonal IgH finding. Cell phenotype using flow cytometry was: CD2+ (99.6), CD3+ (96.9), CD5+ (97.5), CD4+ (96.9) with positive CD20 on T lymphocytes (CD3+ CD20+ from 88.0%).
Conclusion: Found phenotype results of applied morphological diagnostics are contradictory, unlike flow cytometry, which alone can prove with certainty the presence of unusual marker from one lineage at the others.
Romić Ž, Unić A, Đerek L, Živković M, Marijančević D, Serdar T. PO9-6: Anti-citrullinated protein antibody and rheumatoid factor in patients with end-stage renal disease on hemodialysis. Biochemia Medica 2009;19(Suppl 1):S148-S149.
Dubrava University hospital, Zagreb, Croatia
Corresponding author:adrianaunic [at] gmail [dot] com
Introduction: Hemodialysis as a treatment for end-stage renal disease (ESRD) may eliminate some concurrent disorders, but it leads to the development of new ones. Rheumatoid arthritis (RA) is an autoimmune disorder which causes chronic joint inflammations, and affects 1% of the adult population. Patients with ESRD on hemodialysis are at increased risk of developing this condition, as they have defective immunity.
Aim: To examine whether ESRD as well as the procedure and the length of hemodialysis impair the importance of determinating anti-CCP, as an early predictive marker, and RF as the only laboratory criteria for RA.
Materials and methods: Study included: 37 healthy volunteers (age: 50 ± 7years) and 57 ESRD patients (age: 54 ± 13years) who had been undergoing hemodialysis from 1-12 years. Hemodialysed patients were divided into two groups depending on the lenght of hemodialysis (N1: 1-5 years; N2: > 5 years). Anti-CCP was determined using the MEIA on Abbott AxSym (IL, USA). RF was determined by immunoturbidimetry on Olympus AU2700 (Tokyo, Japan). Kolmogorov-Smirnov test was used to test normal distribution. Chi square test and ANOVA were used for data comparison, P < 0.05 was considered statistically significant. MedCalc9.2.0.0 (MedCalc, Mariakerke, Belgium) has been used for statistical analysis.
Results: ANOVA has not shown significant difference in the values of anti-CCP (P = 0.11) and RF (P = 0.98) among the investigated groups. The values were within the reference range (anti-CCP < 3 U/mL; RF < 14 IU/mL).
Conclusions: Our results indicate that ESRD and HD, as well as the lenght of the procedure, do not impair the values of anti-CCP nor RF, and that hemodialysis is not likely the trigger of RA development.
Krajnović-Tomašić V, Pipić-Kitter A. PO9-7: Anti-CCP – diagnostic marker for rheumatoid arthritis. Biochemia Medica 2009;19(Suppl 1):S149-S150.
Dr. Josip Benčević General Hospital, Slavonski Brod, Croatia
Corresponding author:vera [dot] krajnovic [at] vip [dot] hr
Introduction: RA is a chronic autoimmune disease, with progressive damage of joints. RA often affects the small joints of hands and feet. It is difficult to distinguish it from other forms of arthritis. Early diagnosis is very important because of introducing effective therapy which will minimize irreversible damage of joints. RF (rheuma factor) is the marker for rheumatoid arthritis, which can be positive (seropositive RA), but it can also be negative, because of that today anti-CCP is the marker of choice. Anti-CCP are the antibodies against citruline (amino acid generated by posttransitional modification of arginine residues by the enzyme peptidyl arginine deiminaze). Multiple studies showed great diagnostic value of anti-CCP because of its great sensitivity (60-80%) and specificity (98%) for RA. The studies also showed that anti-CCP can be elevated up to 10 years before manifestation of RA.
Materials and methods: Testing was performed with anti-CCP test from Abbott on AxSYM analyzer with MEIA technology. We classified our results for anti-CCP with diagnosis to see in which proportion are the results in agreement with these diagnosis.
Results: For seropositive RA in most cases the results for anti-CCP were very high, in other cases of arthritis the results were lower or normal, with some exceptions (the patients who were not fully clinically examined).
Conclusion: Higher results for anti-CCP are of great help for physicians evaluating indistinguishing poliyarthritis because they can predict the possibility of RA.
Vrdoljak Gudasić J, Bokulić A, Džaja N, Vrane V. PO9-8: The most frequently requested and the most frequently positive allergens in Karlovac region. Biochemia Medica 2009;19(Suppl 1):S150-S151.
Department of Laboratory Diagnostics, Karlovac General Hospital, Karlovac, Croatia
Corresponding author:adriana [dot] bokulic [at] gmail [dot] com
Background: This study was designed to determine the specific immunoglobulin E (sIgE) to the most common allergens in samples routinely received by the Department of Laboratory Diagnostics in General Hospital Karlovac.
Materials and methods: During a six-month period, 223 samples were examined. Serum concentrations of sIgE for Dermatophagoides pteronyssinus, Dermatophagoides farinae, Ambrosia elatior (common ragweed), Dactylis glomerata (cocksfoot), Corylus avellana (hazel), cat dander and dog dander were measured by ImmunoCAP technology (UniCAP). Results of < 0.35 kU/L were considered to be of a negative value and results of > 0.35 kU/L a positive value.
Results: From the total number of patients, 54.7% were women and 45.3% men. Also, 64.6% were adults and 35.4% were children under 18 years. The most requested allergens are: D. pteronyssinus (80.7%), D. farinae (71.2%), common ragweed (36.3%), hazel (29.1%), cat dander (27.8%), cocksfoot (26.5%) and dog dander (26.0%). The prevalence of positive results is: D. farinae (40.0%), D. pteronyssinus (39.8%), cocksfoot (32.2%), common ragweed (32.1%), hazel (24.6%), dog dander (15.5%) and cat dander (14.5%).
Conclusions: On the basis of this study, it can be concluded that the most frequently requested allergens are D. pteronyssinus and D. farinae. At the same time, D. pteronyssinus and D. farinae are tests with the highest number of positive results. For the overview of the most frequently requested and the most frequently positive allergens to be more reliable, study should include more patients over longer period of time. That way we could include influence of allergens distribution during a year in Karlovac region.