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Introduction: The aim of this study was to examine whether the Canadian Laboratory Initiative on Paediatric Reference Intervals (CALIPER) could be applied to Croatian children and adolescents. Materials and methods: A total of 295 outpatient healthy children and adolescents of age 1 to 18 were selected using the direct a posteriori sampling method. According to current guidelines, 20 samples were tested for each of a total of 51 reference intervals for ferritin, cortisol, dehydroepiandrosterone sulfate, follicle stimulating hormone, lutein stimulating hormone, prolactin, progesterone, sex hormone binding globulin, thyroid stimulating hormone, total testosterone, total thyroxine and total triiodothyronine. Serum samples were analysed on the Beckman Coulter DxI600 immunoassay analyser by chemiluminescence immunoassay method. A reference interval was adopted if < 10% of the results fall outside CALIPER reference interval range. For analytes in which this criterion is not met in the first set of samples, a new set of 20 samples were collected. Results: After the first set of measurements, 96% of all tested reference intervals were adopted for use. The additional sets of 20 reference subjects were tested for only two reference intervals; follicle stimulating hormone for female aged 1 to 9 years, and irrespective of the gender, sex hormone binding globulin for children aged 8 to 11 years. All results of additional samples were within the specified interval limits. Conclusions: CALIPER reference intervals for ferritin and 11 hormones defined for Beckman Coulter DxI600 immunoassay analyser can be implemented into the Croatian laboratories and clinical practice.

Introduction: The study aimed to investigate the prevalence and titres of anti-SARS-CoV-2 antibodies in children treated at the Children’s Hospital Zagreb in the first and the second wave of the COVID-19 pandemic. Statistical significance of difference at two time points was done to determine how restrictive epidemiological measures and exposure of children to COVID-19 infection affect this prevalence in different age groups.Materials and methods: At the first time point (13th to 29th May 2020), 240 samples and in second time point (24th October to 23rd November 2020), 308 serum samples were tested for anti- SARS-CoV-2 antibodies by enzyme-linked immunosorbent assay (ELISA) and electrochemilumines-cence immunoassay (ECLIA). Confirmation of results and titre determination was done using virus micro-neutralization test. Subjects were divided according to gender, age and epidemiological history. Results: Seroprevalence of anti-SARS-CoV-2 antibodies differs significantly in two time points (P = 0.010). In first time point 2.9% of seropositive children were determined and in second time point 8.4%. Statistically significant difference (P = 0.007) of seroprevalence between two time points was found only in a group of children aged 11-19 years. At the first time point, all seropositive children were asymptomatic with titre < 8. At the second time point, 69.2% seropositive children were asymptomatic with titre ≥ 8. Conclusions: The prevalence of anti-SARS-CoV-2 antibodies was significantly lower at the first time point than at the second time point. Values of virus micro- neutralization test showed that low titre in asymptomatic children was not protective at the first time point but in second time point all seropositive children had protective titre of anti-SARS-CoV-2 antibodies.

Despite the identification of a wide range of inherited and acquired risk factors for arterial ischemic stroke (AIS) in children, genetic risk factors are incompletely characterized and may vary among different populations. We investigated the role of individual and combined inherited prothrombotic and intermediate-risk factors in 73 children with perinatal (n = 35) and childhood AIS (n = 38) and 100 age- and sex-matched controls. Ten polymorphisms in 8 candidate genes encoding coagulation and fibrinolytic proteins (factor V [FV] Leiden, FV HR2, factor II [FII] G20210A, β-fibrinogen [β-FBG]-455G>A, factor XIII [FXIII]-A p.Val34Leu, plasminogen activator inhibitor 1 4G/5G), homocysteine metabolism (methylenetetrahydrofolate reductase [MTHFR] C677T, MTHFR A1298C), and intermediate-risk factors (angiotensin-converting enzyme I/D, apoE ∊2-4) were detected using a multilocus genotyping assay. Allele-specific polymerase chain reaction was used for the determination of human platelet alloantigens (HPA-1, HPA-2, HPA-3, and HPA-5). Factor V Leiden was associated with an increased risk of AIS (odds ratio [OR]: 4.72, 95% confidence interval [CI]: 1.22-18.27) and perinatal AIS (OR: 8.29, 95% CI: 1.95-35.24). Human platelet antigen-3b allele carriers had a 2-fold lower risk of AIS (OR: 0.51, 95% CI: 0.26-0.98) and perinatal AIS (OR: 0.40, 95% CI: 0.18-0.92). A 2.21-fold increased risk of childhood AIS (95% CI: 1.03-4.73) was identified in FXIII-A Leu34 allele carriers. Combined FV Leiden/FV HR2, FV Leiden/MTHFR A1298C, FV Leiden/MTHFR C677T/MTHFR A1298C, and FV Leiden/FV HR2/MTHFR A1298C heterozygosity was identified in children with AIS but not in controls, which revealed a statistically significant difference. This case–control study shows that besides already documented association between FV Leiden and AIS, other previously unreported polymorphisms (FXIII-A p.Val34Leu, HPA-3) and several genotype combinations that always include heterozygous FV Leiden can be related to AIS in Croatian population.

Introduction: Respiratory syncytial virus (RSV) infection is the most common cause of hospitalization in infants and small children. The aim was to present a 13-months old boy diagnosed with acute airway infection, acute otitis media (AOM) and hepatitis during the RSV-infection. Material and methods: Serum catalytic activities of alkaline phosphatase (ALP), aspartate aminotranspherase (AST), alanine aminotranspherase (ALT), gamma glutamyl transpherase (GGT), lactate dehydrogenase (LD), and concentrations of bilirubin were monitored during hospitalization and at control examination. Results: The child had clinical signs and symptoms of respiratory failure, AOM, and laboratory findings of virus infection and liver disease. On admission, catalytic activities of enzymes were markedly increased, especially the activity of ALP (10333 U/L, i.e. 24 fold increase in comparison with the upper reference limit). The highest increased in AST (339 U/L, 4, 5 fold), ALT (475 U/L, 10, 3 fold) and LD (545 U/L, 1, 5 fold) were registered on the 3rd day, and the highest increase in GGT (68 U/L, 3, 1 fold) occured on the 11th day. Seven weeks after discharge AST, ALT, GGT and LD decreased into reference range, and ALP remain mildly increased (478 U/L, 1, 1 fold increase). RSV was confirmed in nasal lavage fluid. Conclusion: Laboratory results in patient with RSV infection needs to be interpreted in the light of both, respiratory and extrapulmonary manifestations of the infection, respectively.

There are limited data available on interferon-γ release assay (IGRA) performance in children up to 5 years of age, with documented exposure to active tuberculosis (TB). The aim of this study was to evaluate (1) the influence of infectivity of adult source cases on test results, (2) the impact of age, and (3) the level of agreement, between IGRA and tuberculin skin test (TST) results. A total of 142 Bacille Calmette-Guerin-vaccinated children up to 5 years of age were investigated because of a history of exposure to active TB. QuantiFERON-TB Gold In-Tube IGRA (QFT) and TST assays were performed. Test results were significantly influenced by positive finding of cavitary lesions (QFT, odds ratio [OR] = 6.15 ; TST, OR = 7.48) and positive acid-fast bacilli (QFT, OR = 4.01 ; TST, OR = 4.47) in active TB contacts. QFT resulted in 1 indeterminate response (0.7%), attributable to low mitogen. There was no evidence for age having any effect on QFT performance. The 2 tests showed a moderate overall concordance (89% ; κ = 0.591) at a TST cutoff value of ≥10 mm. Association of positive QFT and TST results with risk factors for infection in child contacts (presence of cavitary lesions and acid-fast bacilli smear positivity in index cases) suggests that both the tests have good diagnostic accuracy. However, there was significant discord between results of the 2 tests that could not be definitively resolved. Thus, in a high-risk population of children up to 5 years of age, both tests (QFT and TST) should be performed and the child should be considered infected if either or both tests are positive.

Interferon-γ (IFN-γ) release assay (IGRA) is used for diagnosis of latent tuberculosis infection (LTBI), and for serial testing of active tuberculosis (TB). The aim of this study was to evaluate the results of IGRA for diagnosis and treatment monitoring of children with LTBI and children with TB. IGRA was performed in BCG vaccinated children before and six months after the beginning of treatment. A total of 59 BCG vaccinated children aged 4-18 years were investigated due to exposure to active TB. The participants were divided into two groups: Group 1, children with LTBI (N = 41), and Group 2, children with TB (N = 18). IGRA (QuantiFERON-TB Gold In-Tube) was performed twice, i.e., before treatment and at the end of prophylaxis and therapy. There was no significant difference in IFN-γ concentrations between Group 1 and Group 2 subjects either before or after the treatment. Difference between pre-treatment and post-treatment IFN-γ concentrations compared in either Group 1 or Group 2 was not statistically significant. During follow-up, children with LTBI did not develop active TB. In addition, in children with TB, signs and symptoms of TB improved with anti-TB therapy. This study showed that the concentrations of IFN-γ did not differ in children with LTBI and TB either before or at the end of treatment. IGRA may remain positive over a long period of time. It seems that IGRA is not useful for monitoring treatment of children with LTBI and children with TB. Pediatr Pulmonol.

Regulation of pH in the airways is of physiological importance. As acidification of the airways causes bronchoconstriction, the aim of the present study was to find out whether there is any difference in calcium and magnesium in exhaled breath condensate (EBC) of children with uncontrolled asthma (i.e., with endogenous acidification) and children with gastroesophageal reflux disease, GERD (i.e., with exogenous acidification). A total of 142 children were included in the study: children with uncontrolled asthma (N = 51), children with GERD (N = 61), and healthy controls (N = 30). In addition, according to the pH cut-off value children with asthma and GERD were divided into two subgroups, that is, patients with pH ≤ 6.93 (subgroup A) and patients with pH > 6.93 (subgroup B). The mean EBC pH was significantly lower in children with asthma than in children with GERD (6.791 ± 0.374 vs. 7.002 ± 0.361, p = .006). Concentration [median and interquartile range-M (IQR)] of total magnesium, but not calcium, was lower in both asthmatic [(10 (10-40) μmol/L, p = .016)] and GERD children [(20 (10-40) μmol/L, p = .012)] in comparison with controls (47 ± 27 μmol/L). There was no statistically significant difference in EBC calcium and magnesium concentrations between asthmatic and GERD children. In asthmatic children a positive correlation was confirmed between forced expiratory volume in the first second (FEV1) and magnesium in EBC (r = 0.307 ; p = .030), and negative correlation was found between FEV1 and calcium/magnesium ratio (r = -0.290 ; p = .047). In addition, positive correlation was confirmed between fractional concentration of exhaled NO and calcium/magnesium ratio (r = 0.360 ; p = .018). In GERD patients a negative correlation (r = -0.404 ; p = .003) was found between magnesium and pH values. Concentration of calcium was higher in the GERD subgroup A children [(50 (30-90) μmol/L)] than in the subgroup B children [(30 (20-45) μmol/L, p = .031)]. In addition, concentration of magnesium was higher in the GERD subgroup A children [(30 (20-70) μmol/L)] than in the subgroup B children [(10 (10-20) μmol/L, p < .001)]. The present study indicates that decreased total magnesium concentration may be found in EBCs, irrespective of whether the acidification is the result of endogenous pathomechanisms or reflux-induced mechanisms. In children with GERD, EBC pH-metry should be performed after acute coughing episode. Future research is needed to investigate the mechanisms of onset and dynamics of these changes. Scientific significance: Lower concentration of magnesium may indicate its role in bronchoconstiction

Background: To assess the differences in serum eosinophilic cationic protein (ECP) concentrations between treated and untreated children with asthma, children with rhinitis, and children with both and possible influence of seasonal exposure to sensitizing allergens on ECP levels. Methods: The study included treated (n=156) and untreated (n=55) children with asthma, children with rhinitis, and children with asthma and rhinitis. Serum ECP was measured in serum collected between 8 am and 12 pm under standardized pre-analytical conditions (regarding the type of blood collection tube, time, and incubation temperature during blood clotting). Results: Untreated children had significantly higher ( P

Background: Urate levels may be a marker of oxidative stress. The aim of the present study was to find out are there any differences in urate concentrations in exhaled breath condensate (EBC) between children with obstructive sleep apnea (OSA) and healthy children. Materials and methods: EBC was collected in children with obstructive sleep apnea (OSA) and clinically healthy children. Urate measurements in EBC and serum were performed by enzymatic co-lor test. Results: The higher concentration of urates in EBC of children with OSA than clinically healthy chil-dren indicate the oxidative stress in their airways. Since there was no significant difference in serum concentration of urates between children with OSA and healthy children, it could be considered that urates are sintetized in the airways of children with OSA. Conclusions: The present study indicated that urates in EBC (but not in serum) may be used as a marker of local synthesis of antioxidant compounds, but definitive conclusion must be supported by investigations involving larger number of participants.

Introduction: In childhood, bronchoobstruction is mostly caused by respiratory viral infections. The aim of this study was to find out possible changes in serum concentration of magnesium and calcium (participants in bronchoconstriction), and concentration of C-reactive protein (marker of inammation) in children with moderate and severe bronchoconstriction, caused by viral respiratory infections. Materials and methods: The study included 32 children with acute bronchoconstriction caused by a viral respiratory infection. Inhalation of salbutamol was administered according to the severity of bronchoconstriction. Blood sampling was done on admission and the third day of salbutamol administration. Results: Therapy with salbutamol led to a relief of dyspnea within 36 hours, and symptoms of viral infection have relieved. During follow-up period, magnesium concentration was higher in children with moderate (but not in severe) bronchoconstriction than in healthy children, with consequently higher magnesium to calcium ratio. Concentration of C-reactive protein decreased spontaneously by gradual disappearance of signs and symptoms of viral infection. Conclusion: Determination of serum magnesium and calcium concentrations and determination of their ratio are not sufficient enough to follow-up on effects of therapy. Additional studies of ionized forms of magnesium and calcium, their intracellular content, as well as concentration in exhaled breath condensate are needed.

Uvod: Povećane koncentracije antitireoidnih autoantitijela nalaze se u upalnim bolestima te u autoimunim bolestima štitnjače. Cilj ovog članka jest prikazati povećane vrijednosti IgA u ispitanice s pozitivnim antitireoidnim autoantitijelima. Navode se rezultati trinaestomjesečnog praćenja. Materijali i metode: Koncentracije tireoidnih hormona i antitijela u uzorcima seruma izmjerene su primjenom enzimske imunoanalize na mikročesticama. IgA je određen pomoću dvije imunokemijske metode, tj. imunoturbidimetrijskom metodom i radijalnom imunodifuzijom. Rezultati: Početne pretrage funkcije štitnjače dale su normalne vrijednosti T3, T4 i hormona koji stimulira štitnjaču (TSH) te povećane vrijednosti autoantitijela na tireoidnu peroksidazu (anti-TPO), tireoglobulin (anti-Tg) i IgA. Ispitanica je liječena jednokratnim dnevnim dozama levotiroksina. Povećane početne koncentracije anti-TPO stabilizirane su primjenom levotiroksina, dok su anti-Tg ostala povećana. IgA je mjeren imunoturbidimetrijskom metodom i bio je značajno povećan tijekom razdoblja praćenja ispitanice. Ponovljenim određivanjem IgA radijalnom imunodifuzijom otkrivene su manje vrijednosti IgA. Zaključak: Moguće lažno povećane vrijednosti IgA mogu biti rezultat, kako interferencija kozjih anti-IgA antitijela iz reagensa, tako i križne reaktivnosti s endogenim anti-Tg autoantitijelima u serumu. Potrebna su daljnja istraživanja kako bi se utvrdio uzrok povećanih vrijednosti IgA u bolesnice s pozitivnim antitireoidnim autoantitijelima., Introduction: Increased concentrations of antithyroid autoantibodies are found in inflammatory diseases as well as in thyroid autoimmune disorders. The aim of this report is to present increased IgA concentrations in a female patient with positive anti-thyroid autoantibodies. Results throughout 13 months of follow-up are presented. Materials and methods: The levels of thyroid hormones and antibodies in the serum samples were measured using microparticle enzyme-immunoas-say. IgA was determined by two immunochemical methods, immuno-turbi-dimetric method and radial immunodiffusion, respectively. Results: Initial thyroid function tests showed normal values of T3, T4 and thyroid stimulating hormone (TSH), and increased values of thyroid peroxi-dase autoantibodies (anti-TPO), anti-thyroglobulin (anti-Tg) autoantibodies and IgA. The patient underwent treatment with single daily dose of levot-hyroxine. The elevated initial levels of anti-TPO stabilized with the administration of levothyroxine, while anti-Tg remained increased. IgA determined by immuno-turbidimetric method was significantly increased during the follow-up period. Repeated determination of IgA by radial immunodiffusion revealed lower IgA concentrations. Conclusion: Possible false increased IgA values could be a result of both, interferences of goat anti-IgA antibodies from reagents, and cross-reactivity with endogenous anti-Tg autoantibodies in serum, respectively. Further investigations are needed to ascertain the cause of increased IgA values in patient with positive anti-thyroid autoantibodies.

Background and Aims The aim of the present study was to investigate the occupational risk of tuberculosis in health care workers (HCW) at a children's hospital in a middle-income country with an intermediate prevalence of tuberculosis and compulsory bacille Calmette-Guerin vaccination and to assess the association of latent tuberculosis infection (LTBI) with the concentration of immunoglobulins (IgG, IgA, IgM), C-reactive protein and hematological changes. Methods Fifty four HCW were divided into groups according to their exposure to tuberculous patients and interferon (IFN-γ) findings. IFN-γ determination and tuberculin skin test (RT23) were performed in parallel. Results Positive IFN-γ results were recorded in 31% of study HCW. Higher exposure to tuberculous patients did not lead to a statistically significant increase in the rate of positive IFN-γ findings but did increase the number of hyperreactors. HCW with positive IFN-γ findings were 5 years older than IFN-γ negative subjects. The median IgA concentration was 29% higher ( p = 0.0233) in IFN-γ positive subjects (2.44 g/L) as compared with IFN-γ negative subjects (1.89 g/L). Conclusions Study results showed the prevalence of LTBI in HCW at a children's hospital to be comparable to the prevalence recorded in the general population. IFN-γ proved to be a more reliable test to determine LTBI in a population of bacille Calmette-Guerin-vaccinated HCW. The new diagnostic approach will hopefully contribute to more rational use of x-rays and prevent unnecessary administration of chemoprophylaxis. The higher concentration of IgA in IFN-γ-positive HCW pointed to the possible protective role of IgA antibodies in LTBI.

Background: Magnesium and calcium physiologic functions are closely related. Magnesium is primarily an intracellular cation, the action of which also involves maintenance of cellular ionic balance, while influencing calcium homeostasis by blocking calcium channels. The aim of this study was to compare the concentrations of magnesium and calcium in exhaled breath condensate (EBC) of children with asthma and gastroesophageal reflux disease (GERD). Subjects and methods: EBC was collected from 66 children aged 7–14 years (23 children with acute asthma, 17 children with GERD, and 26 healthy children).Determination of magnesium and calciumconcentrations was preceded by optimization and validation for low concentrations. Results: No difference was recorded for either magnesium or calcium concentration between study groups. However, the magnesium to calcium ratio was statistically significantly lower in both GERD and asthma children as compared with control group. Conclusion: Study results showed the magnesium to calcium ratio to be a statistically significantly better indicator of certain pathologic changes than absolute concentration of either ion. J. Clin. Lab. Anal. 23:34–39, 2009. © 2009 Wiley‐Liss, Inc.

Cilj: U posljednje se vrijeme hsCRP (engl. high sensitive CRP) primjenjuje kao prognostički biljeg kronične upale. Cilj ovoga rada bio je ispitati dolazi li do promjene serumske koncentracije hsCRP, C3 i C4 u djece s latentnom tuberkuloznom infekcijom nakon dva mjeseca profilakse izonijazidom. Ispitanici i metode: Ukupno je ispitano 79-ero djece podijeljene u tri skupine: 1) ispitanici s latentnom tuberkuloznom infekcijom (LTBI); 2) ispitanici s tuberkulozom pluća; 3) klinički zdravi ispitanici upućeni na sistematski pregled, s biokemijsko-hematološkim pokazateljima unutar referentnih vrijednosti za dob - kontrolna skupina. Krv je uzorkovana dvaput: prije započinjanja terapije i poslije dvomjesečne terapije tijekom koje su lijekovi primjenjivani svakodnevno. Imunokemijskim metodama određena je koncentracija hsCRP, C3 i C4. Rezultati: Koncentracija hsCRP, C3 i C4 u ispitanika s LTBI prije profilakse izonijazidom bila je statistički značajno veća nego u kontrolnoj skupini. Nakon profilakse izonijazidom u osoba s LTBI koncentracija hsCRP bila je statistički značajno manja nego prije primjene izonijazida. Specifičnost je za sve odabrane analite bila veća nego osjetljivost. Granične vrijednosti za hsCRP imale su bolju dijagnostičku učinkovitost nego granične vrijednosti za C3 odnosno C4. Zaključak: Koncentracija hsCRP može se primijeniti za praćenje bolesnika s LTBI u svrhu procjene odgovora na profilaksu izonijazidom i stupnja aktivnosti bolesti., Aim: Recently, hsCRP (high sensitive CRP) concentration has been used as a prognostic marker of chronic inflammation. The aim of the study was to determine whether two-month isoniazid prophylaxis induced changes in serum concentrations of hsCRP, C3 and C4 in children with latent tuberculosis infection (LTBI). Subjects and methods: The study included 79 children divided into three groups: 1) subjects with LTBI; 2) subjects with lung tuberculosis; and 3) control group of clinically healthy subjects, referred for systematic examination, with biochemistry-hematology parameters within reference range for age. Blood sampling was performed twice: before drug administration and after a two-month period during which drugs were administered on a daily basis; hsCRP, C3 and C4 were determined by use of immunoassays. Results: Before prophylactic isoniazid therapy induction, the concentrations of hsCRP, C3 and C4 were significantly higher in LTBI group as compared with control group. After prophylactic therapy, the concentration of hsCRP in LTBI was lower than before isoniazid administration. The specificity was greater than sensitivity for all study analytes. The cut-off value of hsCRP showed higher optimum diagnostic efficiency than the cut-off values of C3 and C4. Conclusion: The concentration of hsCRP can be used in the follow up of LTBI patients to evaluate response to isoniazid prophylaxis and the level of disease activity.

Uvod: Opisuje se slucaj prolazne hiperfosfatazemije u 21-mjesecnog djecaka s ponavljajucom sipnjom tijekom akutnog respiracijskog infekta. Materijali i metode: Određivanje kataliticke aktivnosti alkalne fosfataze provedeno je preporucenom fotometrijskom metodom, a elektroforetsko razdvajanje izoenzima alkalne fosfataze na agaroznom gelu uz obradu seruma lektinom. Rezultati: Tijekom bolnickog lijecenja dijete je imalo povecanu kataliticku aktivnost alkalne fosfataze: prvog dana 4109 U/L, sestog dana 2156 U/L. Pri kontrolnom pregledu 27. dana od prijema u bolnicu aktivnost se smanjila na 505 U/L. Elektroforetskim razdvajanjem izoenzima alkalne fosfataze dokazana je elektroforetska vrpca brža od jetreno-kostanog izoenzima, tzv. brza anodna vrpca. Zakljucak: Pojava izrazito povecane kataliticke aktivnosti alkalne fosfataze uz tipicnu elektroforetsku razdiobu izoenzima u trajanju od nekoliko tjedana ukazuje na prolaznu hiperfosfatazemiju. Pravodobnim prepoznavanjem izbjegava se daljnja nepotrebna dijagnosticka obrada.

In vitro dijagnostika alergijskih bolesti temelji se na određivanju koncentracije ukupnih i speciičnih IgE antitijela u serumu, koncentraciji IgG antitijela, plazmatske triptaze, eozinoilnog kationskog proteina te testu aktivacije bazoilnih leukocita. Upotreba in vitro dijagnostičkih testova mora biti temeljena na korelaciji anamneze, kliničke slike te in vivo provokativnih testova. Kliničko značenje povišene serumske koncentracije ukupnih IgE antitijela u dijagnostici alergijskih bolesti jest ograničena, budući da se ona susreće i u drugim nealergijskim bolestima. Povišene koncentracije speciičnih IgE protutijela, zajedno s pozitivnom anamnezom, indikativne su za klinički značajnu alergijsku bolest. Preporučena laboratorijska metoda za određivanje koncentracije IgE protutijela u serumu jest luoroenzimimunokemijska metoda. Povišene serumske koncentracije IgG protutijela nemaju dokazanu ulogu u dijagnostici alergije na hranu, a mogu poslužiti u procjeni uspješnosti speciične imunoterapije na alergene kukaca te procjeni rizika razvoja anailaksije uzrokovane ovom skupinom alergena. Povišena koncentracija serumske triptaze (podtip ), indikator je degranulacije mastocita uzrokovane speciičnim alergenom. Porast koncentracije eozinoilnog kationskog proteina (EKP) nastupa tijekom kasne faze alergijske reakcije te se može koristiti u praćenju alergijskih i drugih upalnih stanja u kojima eozinoili imaju glavnu ulogu. Test aktivacije bazoilnih (BAT) leukocita temelji se na određivanju staničnih razlikovnih obilježja, CD 63 i CD203c metodom protočne citometrije. BAT je koristan u dijagnostici alergija na alergene kukaca, nutritivnih te medikamentnih alergija. Alergološka dijagnostika temeljena na ekstraktima alergena može se koristiti u monosenzibiliziranih bolesnika s jasnom kliničkom slikom te u bolesnika kojima se planira prepisati samo simptomatska terapija. U suvremenoj su kliničkoj praksi razvijenih zemalja mnogo češći polisenzibilizirani bolesnici s kompleksnom simptomatologijom te sumnjom na križnu reaktivnost. Kod ovih se bolesnika preporuča koristiti molekularnu alergološku dijagnostiku., In vitro diagnostic procedure in allergology includes determination of serum levels of total and allergen speciic IgE antibodies, allergen speciic IgG antibodies, plasma tryptase, eosinophil cationic protein (ECP) and basophil activation test (BAT). In vitro tests should be used according to clinical history, physical examination, and in vivo methods for allergy testing. Clinical relevance of elevated total IgE in allergy diagnosis is modest, since it can be caused by other conditions. Elevated serum levels of allergen speciic IgE antibodies, together with positive medical history, are indicative of clinically relevant allergy. A recommendedlaboratory method for total and speciic IgE concentration measurement is the sandwich-type luoroimmunoassay ImmunoCAP, considered as an ideal immunoassay. Serum levels of allergen speciic IgG antibodies have no proved clinical relevance in food allergy diagnosis. They can be useful to monitor venom immunotherapy success, as well as to estimate the risk of venom induced anaphylaxis. Elevated plasma tryptase (subtype ) level is an indication of mast cell activation caused by speciic allergen. It should be obtained within 4 hours after an anaphylactic episode. Elevated level of ECP can be detected in patient blood during late phase of allergic reaction. It can be used to monitor patients with chronic allergenic and inlammatory conditions in which eosinophils play a central role. BAT includes measurement of CD 63 (cluster of differentiation) and CD 203 antigens of the molecular surface by low cytometry. It is useful in the diagnosis of venom, food and drug allergy, estimation of severity of allergic disease and natural tolerance to allergens. In vitro tests based on allergen extracts can be used for in vitro diagnosis in monosensitized patients with clear medical history and symptomatic treatment. Molecular allergy diagnosis should be performed in special clinical indications such as diagnosis of cross reactivity, prescription of speciic immunotherapy (especially in polysensitized patients with complex symptoms), diagnosis of idiopathic or cofactor induced anaphylaxis, latex allergy, and assessment of the risk of allergic reaction to speciic allergen.

Background: To determine whether urates as a marker of oxidative stress could be determined in exhaled breath condensate (EBC), and if there are any differences in urate concentrations between asthmatic and healthy children. Methods: Exhaled breath condensate was collected using an EcoScreen condenser (Erich Jaeger GmbH, Hoechberg, Germany). Urate measurements were performed by enzymatic color test. Asthmatic children were divided into 2 groups: children with controlled asthma and children with acute exacerbation of asthma. Results: Exhaled breath condensate urate concentration was statistically significantly higher in children with controlled asthma compared with control group and acute exacerbation group. Seven children with acute exacerbation of asthma had unmeasurable urate concentrations. Conclusion: Study results showed EBC to contain a measurable urate concentration, which was significantly higher in children with controlled asthma compared with clinically healthy children and children with acute exacerbation of asthma.

The aim is to present a case of serial use of interferon-γ release assay (IGRA) test in the evaluation of an infant with a latent tuberculosis infection (LTBI) accompanied with a positive finding of Mycobacterium (M.) fortuitum in gastric juice. In addition to routine diagnostic evaluation (tuberculin skin test (TST), culture of gastric juice, chest X-rays), a 12-month-old female infant with a history of close family contact and a case of active tuberculosis was investigated with IGRA (QuantiFERON-TB Gold In-Tube, Cellestis, Carnegie, Australia). Tuberculin skin tests were positive. The first result of IGRA (on admission) was negative; the second (day 16) and third (day 57) results of IGRA were positive. M. fortuitum was cultured from gastric juice samples. Positive IGRA finding confirmed LTBI, and prophylaxis with isoniazid was administered. Since M. fortuitum does not express antigens that could be confirmed by the QuantiFERON-TB Gold In-Tube test, isolation of M. fortuitum could be interpreted as a possible contamination.

Latent tuberculosis infection (LTBI) is a subclinical infection with Mycobacterium (M.) tuberculosis. Children and immunosuppressed individuals due to pathologic conditions or therapeutic procedures are at a high risk of reactivation of LTBI to active disease compared to general population and require timely identification. The diagnosis of LTBI is based on the measurement an adaptive immune response against M. tuberculosis. The use of tuberculin skin test (TST) has been associated with a number of interfering factors that cause false-positive or false-negative test results. The new ex vivo whole blood tests determining interferon-gamma (IFN-γ) released from T-lymphocytes (interferon-gamma release assays, IGRAs) upon stimulation with M. tuberculosis specific antigens. The introduction of IGRAs to routine clinical practice has improved the diagnosis of LTBI, but recommendations and guidelines for the diagnosis of LTBI are not consistent between different European countries. Therefore, European centre for disease prevention and control developed guidance document based on the up-to-date scientific evidence. Novel concepts of IGRAs and new studies should be designed so as to provide answers to all open questions., Latentna tuberkulozna infekcija (LTBI) definira se kao supklinička infekcija bakterijom Mycobacterium (M.) tuberculosis. Kod djece i osoba oslabljenog imunološkog sustava, zbog pojedinih patoloških stanja ili terapijskih postupaka, postoji veći rizik za aktivaciju LTBI-ja u aktivnu tuberkulozu u odnosu na opću populaciju, te je takvu infekciju potrebno pravovremeno otkriti. Dijagnostika LTBI-ja temelji se na određivanju imunološke reakcije usmjerene protiv M. tuberculosis. Primjena tuberkulinskog testa otežana je brojnim čimbenicima koji mogu uzrokovati lažno pozitivne i lažno negativne rezultate testa. Novi dijagnostički pristup temelji se na ex vivo testovima iz pune krvi kojima se određuje IFN-γ oslobođen iz T-limfocita (interferon-gamma release assays, IGRAs) nakon podražaja specifičnim antigenima za M. tuberculosis. Primjena IGRAs-a u rutinskoj kliničkoj praksi unaprijedila je dijagnostiku LTBI-ja, ali preporuke i smjernice za dijagnostiku LTBI-ja nisu podudarne u pojedinim europskim zemljama. Zbog toga je Europski centar za prevenciju bolesti i kontrolu pripremio smjernice utemeljene na najnovijim znanstvenim podatcima. Unaprjeđenja IGRAs-a i nova istraživanja treba kreirati tako da se mogu dati odgovori na sva otvorena pitanja.

Eosinophil cationic protein (ECP) is a heterogeneous molecule originating from activated eosinophil granulocytes. Biological activity and the cellular content of ECP are determined by genetic and posttranslational factors. Several single nucleotide polymorphisms (SNPs) in human ECP gene (RNASE3) have been described so far. ECP is a mediator in host immune response to parasites, bacteria and viruses. By its cytotoxic and non-cytotoxic activity, ECP may also cause side-effects in the host's own tissues. The largest number of clinical studies is focused on the role of ECP in eosinophil-related disorders, particularly in asthma. Although present in numerous body fluids, difficult bioavailability of biological material, invasive sampling methods and complex sample management prior to ECP level determination are the reasons that serum is most commonly used in routine laboratory practice. As numerous biological and methodological preanalytical factors (the type of collection test-tube, temperature and duration of blood clotting, centrifugation, hemolysis) may affect test result, the sample for serum ECP determination should be collected under standardized conditions. Regarding interpretation of results, it is necessary, along with absolute ECP concentration values, to monitor changes in ECP concentration during the duration of disease or after implemented therapy, and interpret ECP test result in combination with other laboratory and clinical findings. Rational approach to selection of new tests is indeed one of important requirements that medical workers meet today. To enable them to determine the clinical significance of ECP with better certainty, further studies on a large number of specific patient groups are needed.

Laboratory diagnosis of type I hypersensitivity reaction is based on the determination of serum concentrations of total immunoglobulin E (tIgE) and allergen specific IgE (sIgE) antibodies. Serum concentration of tIgE varies throughout the lifespan. 1 Physiologic changes are mainly noticeable in the first decade of life. In spite of defined reference intervals for tIgE, as many as 5% of healthy children might have serum tIgE concentrations above the age-specific reference range, whereas 10% of children with clinical signs of hypersensitivity might have serum tIgE concentrations within the age-specific reference range. 1 As the concentration of circulating IgE antibodies might not always correlate with local tissue IgE synthesis, screening of sensitized individuals based on serum tIgE concentrations may prove inadequately reliable in some cases. 2 An increased concentration of sIgE indicates sensitization to a particular allergen. In sensitized individuals, only 5% of total sIgE production is found in the circulation, indicating current immune activity, whereas the rest of the sIgE is bound to tissue mastocytes and mediates local bioactivity. 3

QuantiFERON-TB Gold In Tube (QFT-IT) assay is a highly sensitive and specific test for the diagnosis of latent tuberculosis infection. Data on the use of QFT-IT assay in children are scarce and contradictory. The aim of the study was to assess the rate of indeterminate test results and to identify the factors contributing to indeterminate results on routine use of QFT-IT assay in non-immunosuppressed children. This retrospective study included 2173 children, age range from 1 month to 18 years. Determination of interferon-gamma (IFN-γ) in peripheral blood was performed by commercial QFT-IT assay. Indeterminate test results were recorded in ten (0.46%) subjects with ages ranging from 15 months to 15 years. The value of negative control was >8.0 kIU/L in one subject, whereas in the remaining nine subjects indeterminate results were consequential to positive control (

Maintenance of iron homeostasis is of utmost importance for the respiratory system physiology and pathophysiology. Local iron deficiency or accumulation may result in particular respiratory function impairment. The aim of the present study was to find out whether iron and ferritin could be determined in exhaled breath condensate (EBC) of healthy children and children with asthma. Oxidative stress was verified by determination of EBC superoxide dismutase (SOD) activity, and the airway inflammatory process by determination of exhaled nitric oxide (F(E)NO). EBC was collected from 39 children (22 healthy children as a control group and 17 asthmatics) using an EcoScreen condenser. Iron, ferritin, and SOD were determined on optimization and validation for low concentrations. In comparison with a control group, asthma patients had a statistically significantly lower iron concentration (p = 0.0001) and higher SOD catalytic activity (p = 0.0160), with no significant difference in ferritin levels (p = 0.5252), although percentile values indicated elevated ferritin concentration in about half of asthma patients. F(E)NO values were significantly higher in the asthma group (p = 0.0047). This preliminary study demonstrated the possibility of determining iron and ferritin concentrations and SOD activity in EBC, and a significant difference in EBC iron and SOD between asthma patients and healthy children.

Objective: To assess (a) the effect of ambient and exhaled gases on water gas analysis and (b) the impact of argonization on the results of oxygen (Po 2 ) and carbon dioxide (Pco 2 ) determination in exhaled breath condensate. Methods: Samples of deionized water exposed to atmosphere and exhaled air were analyzed to assess the effect of ambient gases on the respective analyte results. Exhaled breath condensate (EBC) samples were argonized by 2 methods. Also, Po 2 and Pco 2 were determined in EBC obtained from healthy children. Results: Deionized water samples exposed to air showed similar Po 2 and Pco 2 values in ambient air. The values of Po 2 were higher, and those of Pco 2 lower in water after exhaled air bubbling. The values of Po 2 and Pco 2 were significantly higher in EBC samples protected by an argon layer. Conclusion: Ambient/exhaled gases influence gas analysis in water and EBC, respectively. Additional validation of the method is needed.

Uvod: Bolesnici sa šećernom bolesti tipa 1 često imaju narušenu staničnu imunost i podložni su zaraznim bolestima. Cilj je ovog rada prikazati slučaj latentne tuberkulozne infekcije (LTBI) u 46-godišnje zdravstvene djelatnice (medicinske sestre na odjelu za tuberkulozu male djece) koja 20 godina boluje od šećerne bolesti tipa 1. Materijali i metode: Tuberkulinski kožni test učinjen je s 2 tuberkulinske jedinice (Tuberkulin RT-23, Statens Serum Institute, Kopenhagen, Danska). Krv za ispitivanje oslobađanja interferona gama, IFN-γ, (engl. Interferon Gamma Releasing Assay, IGRA) uzorkovana je u epruvete koje sadrže specifične antigene mikobakterija tuberkuloze (ESAT-6, CFP10 i TB7.7). Nakon inkubacije (22 sata) na 37°C u plazmi je određena koncentracija IFN-γ metodom ELISA (Qu-antiFERON-TB Gold In Tube - Cellestis Ltd., Victoria, Australia). Pozitivna kontrola bila je krv uzorkovana s mitogenom fitohemaglutininom, a negativna kontrola krv uzorkovana s heparinom. Rezultati: Tuberkulinski kožni test bio je negativan (induracija 6 mm), a nalaz otpuštanja IFN-γ iz aktiviranih limfocita (≥ 0,35 kIU/L) pozitivan. Normalna stanična imunost potvrđena je pozitivnom kontrolom. Ispitanica nema znakova aktivne tuberkuloze. Zaključak: U medicinske sestre koja boluje od šećerne bolesti tipa 1, a godinama radi s tuberkuloznim bolesnicima LTBI je dokazana testom oslobađanja IFN-γ, ali ne i tuberkulinskim kožnim testom. Unatoč dugogodišnje šećerne bolesti tipa 1, reakcija oslobađanja IFN-γ prema specifičnim tuberkuloznim antigenima je očuvana, a aktivna se tuberkuloza nije razvila., Introduction: Patients suffering from diabetes mellitus type 1 often have impaired cell-mediated immunity and are prone to infectious diseases. The aim of this paper is to present a case study on latent tuberculosis infection (LTBI) in a 46 year-old female health care worker (a nurse at the department for tuberculosis in young children) who has been having type 1 diabetes for 20 years. Materials and methods: Tuberculin skin test was performed using 2 tuberculin units (Tuberkulin RT-23, Statens Serum Institute, Copenhagen, Denmark). Blood for interferon gamma (IFN-γ) release assay, (IGRA) was sampled in test tubes containing specific antigens of mycobacterium tuberculosis (ESAT-6, CFP10 and TB7.7). After incubation (22 hours) at 37°C, IFN-γ concentration in plasma was determined by ELISA assay (QuantiFERON-TB Gold In Tube - Cellestis Ltd., Victoria, Australia). For positive control, blood was sampled by mitogene phytohemagglutinin and for negative control by heparin. Results: Tuberculin skin test was negative (induration 6 mm) and test result of IFN- γ release from activated lymphocytes (≥ 0.35 kIU/L) positive. Normal cellular immunity was confirmed by positive control sample. The subject showed no signs of active tuberculosis. Conclusion: In a nurse suffering from type 1 diabetes who has worked for years with tuberculosis patients, LTBI was detected by IFN-γ release assay rather than by tuberculin skin test. In spite of the long lasting diabetes, reaction of IFN-γ release toward specific tuberculosis antigens has been preserved and active tuberculosis has not developed.

Background and Purpose: Based on the facts that the blockade of adrenergic receptors can alter lipid profile in the serum and that it has been suggested that butyrylcholinesterase (BuChE) is involved in lipid metabolism, different adrenergic blocking agents were administered to rats to modify lipid concentrations in serum. The activity of BuChE was examined under such conditions and correlations with serum lipids were investigated. The purpose of this study was to evaluate the effects of different adrenergic antagonists on BuChE activity and to investigate the correlation between BuChE activity and serum lipids. Materials and Methods: Six groups of male Fischer 344 rats (9 animals/ group) were treated orally with adrenergic antagonists (mixed in commercial diet) during 6 weeks: oxprenolol, atenolol, doxazosin, oxprenolol and doxazosin, atenolol and doxazosin, and guanethidine. A control group (9 rats) received only commercial diet. BuChE activity in serum was determined with kinetic color test using butyrylthiocholine as a substrate. Concentrations of serum lipids (total cholesterol, triglycerides and HDL cholesterol) were determined by enzymatic colorimetric tests. Data were analyzed by Kruskal-Wallis test and Spearman’s correlation coefficient. Results: The results revealed that oxprenolol and doxazosin (given alone or in combination with atenolol or oxprenolol) increased (>30 %) BuChE activity. BuChE activity correlated with different serum lipids, and correlation depended on the type of adrenergic blockade. Conclusion: Although the examined adrenergic antagonists did not influence serum lipid concentrations, the increase of BuChE activity and correlation with serum lipid concentrations suggested that the increase of this enzyme’s activity might be the first sign of altered lipid metabolism.

Uvod: Opisuje se slučaj prolazne hiperfosfatazemije u 21-mjesečnog dječaka s ponavljajućom sipnjom tijekom akutnog respiracijskog infekta. Materijali i metode: Određivanje katalitičke aktivnosti alkalne fosfataze provedeno je preporučenom fotometrijskom metodom, a elektroforetsko razdvajanje izoenzima alkalne fosfataze na agaroznom gelu uz obradu seruma lektinom. Rezultati: Tijekom bolničkog liječenja dijete je imalo povećanu katalitičku aktivnost alkalne fosfataze: prvog dana 4109 U/L, šestog dana 2156 U/L. Pri kontrolnom pregledu 27. dana od prijma u bolnicu aktivnost se smanjila na 505 U/L. Elektroforetskim razdvajanjem izoenzima alkalne fosfataze dokazana je elektroforetska vrpca brža od jetreno-koštanog izoenzima, tzv. brza anodna vrpca. Zaključak: Pojava izrazito povećane katalitičke aktivnosti alkalne fosfataze uz tipičnu elektroforetsku razdiobu izoenzima u trajanju od nekoliko tjedana ukazuje na prolaznu hiperfosfatazemiju. Pravodobnim prepoznavanjem izbjegava se daljnja nepotrebna dijagnostička obrada., Introduction: A case of transient hyperphosphatasemia in a 21-month-old boy diagnosed with recurrent wheezing during acute respiratory infection is described. Materials and methods: Determination of alkaline phosphatase catalytic activity was performed by the recommended photometric method, and elec-trophoretic separation of alkaline phosphatase isoenzymes on agarose gel by serum processing with lectin. Results: During hospital treatment, increased catalytic activity of alkaline phosphatase was recorded (4109 U/L on day 1 and 2156 U/L on day 6). On control examination on day 27 of hospital admission, the value decreased to 505 U/L. Electrophoretic separation of alkaline phosphatase isoenzymes demonstrated a band faster than the hepatic-osseous isoenzyme, a fast anodal form. Conclusion: A marked increase in the alkaline phosphatase catalytic activity with typical electrophoretic isoenzyme pattern persisting for several weeks points to transient hyperphosphatasemia. Additional unnecessary diagnostic work-up can be obviated by timely recognition of the phenomenon.

We employed the analysis of single-strand conformation polymorphisms to identify mutations in exon 4 of the low density lipoprotein receptor gene causing familial hypercholesterolemia. Three familial hypercholesterolemia heterozygotes had abnormal single-strand conformation polymorphism patterns. DNA sequencing revealed that the abnormal pattern of exon 4A was due to heterozygosity (T/C) at nucleotide 442. Nucleotide 442 is the first base of codon 127, and the T→C mutation (C127R) changes this codon from Cys TGT to Arg CGT . Abnormal patterns of exon 4B were due to heterozygosity (A/G) at nucleotide 662: nucleotide 662 is the second base of codon 200, and the A→G mutation (D200G) changes this codon from Asp-GAC to Gly GGC . Mutation D200G was previously described as FH Padova, but mutation C127R (FH Zagreb) has not been reported previously. This novel mutation was confirmed by restriction endonuclease analysis with Dsa I. The screening of 420 familial hypercholesterolemia heterozygotes suggests that C127R and D200G account for about 0.7% of mutations causing familial hypercholesterolemia in Croatia.

The aim of this report is to present results of serial interferon-gamma (IFN-γ) determination (QuantiFERON-TB Gold In-Tube assay) in a 57-year-old BCG-vaccinated female health care worker with a latent tuberculosis infection. Increased IFN-γ, IgA, and anti-thyroglobulin (anti-Tg) autoantibodies were recorded throughout a 13-month follow-up. After 13 months the subject was clinically healthy and free from any signs and symptoms of active tuberculosis. The increased levels of anti-Tg autoantibodies may have been a concurrent process that did not impair the patient’s immune status. Since false-positive IgA could be found in patients suffering from autoimmune thyroid diseases, it could be assumed increased IgA in this subject is a result of the positive interferences of goat anti-IgA antibodies. Although isoniazid prophylaxis was not used, tuberculosis did not develop during the follow-up period. Serial determination of IFN-γ is necessary due to occupational exposure to tubercle bacilli.