[CROATIAN GUIDELINES FOR IN VITRO DIAGNOSIS OF IgE MEDIATED HYPERSENSITIVITY].

In vitro diagnostic procedure in allergology includes determination of serum levels of total and allergen specific IgE antibodies, allergen specific 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 specific IgE antibodies, together with positive medical history, are indicative of clinically relevant allergy. A recommended laboratory method for total and specific IgE concentration measurement is the sandwich-type fluoroimmunoassay ImmunoCAP, considered as an ideal immunoassay. Serum levels of allergen specific 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 ß) level is an indication of mast cell activation caused by specific 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 inflammatory 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 flow 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 specific 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 specific allergen.

Calcium and magnesium in exhaled breath condensate of children with endogenous and exogenous airway acidification.

BACKGROUND AND AIMS: 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). MATERIAL AND METHODS: 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). RESULTS: 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)]. CONCLUSION: 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.

Gene frequencies of platelet-specific antigens in Croatian population.

The human platelet antigens (HPA) are genetically defined polymorphisms expressed on platelet membrane glycoproteins. As platelet antigens are very important in several clinical situations and in population genetics, we used the polymerase chain reaction with sequence-specific primers (PCR-SSP) to investigate HPA-1, -2, -3 and -5 allele frequencies in the Croatian population. The HPA frequencies obtained in 219 Croatians were: 1a-0.854, 1b-0.146, 2a-0.890, 2b-0.110, 3a-0.575, 3b-0.425, 5a-0.895 and 5b-0.105. These data are similar to the frequencies reported in most European studies with some significant differences in HPA-2 when compared with the Dutch and German population, in HPA-3 when compared with the Swiss population and in HPA-5 when compared with the Finnish population. The three most common condensed HPA genotypes in the Croatian population were: HPA-1a/a, -2a/a, -3a/b, -5-a/a (0.283), HPA-1a/a, -2a/a, -3a/a, -5-a/a (0.137) and HPA-1a/b, -2a/a, -3a/b, -5-a/a (0.087). Data obtained in this study can be used for better understanding and treatment of immune-mediated platelet disorders in our population.

Markers of systemic and lung inflammation in childhood asthma.

AIM: To compare different biomarkers of inflammation in children with controlled and uncontrolled asthma and to investigate their relationship with other clinical indices of asthma control (symptoms, lung function, serum IgE, and prn beta-agonist use). MATERIALS AND METHODS: A total of 62 consecutive asthmatic children (age 11 +/- 3.3 years, 32 girls) with controlled ([C], n = 19) and uncontrolled asthma ([NC], n = 43) were studied. Measured lung function and inflammatory biomarkers included: spirometry, exhaled NO (F(E)NO), high-sensitivity C-reactive protein (hs-CRP), peripheral blood white blood cells (WBC) counts, and differentials. RESULTS: Hs-CRP was significantly higher in uncontrolled than in controlled asthma (hs-CRP, median [IQR], mg/L; 0.56 [0.60] vs 0.25 [0.34], p = 0.008). Discriminant analysis (backward stepwise) depicted hs-CRP and lymphocytes (as Z-score for absolute count) as significant discriminative factors for asthma control (F = 8.319, p = 0.0007) with 82.3% diagnostic accuracy. Divided into quartiles hs-CRP showed the significant inverse association with F(E)NO (F = 7.359, p = 0.003, ANOVA) with no significant difference for asthma control (F = 1.032, p = 0.386). Post-hoc analysis revealed that F(E)NO values were significantly lower in the third and the fourth quartile of hs-CRP in comparison to the first and the second one (p < 0.05 for all). CONCLUSION: In asthmatic children with uncontrolled asthma serum hs-CRP was increased compared to children with controlled asthma. Although F(E)NO values were also increased (insignificantly) and inversely correlated with hs-CRP they were probably reflecting different etiology underlying the loss of control. The role of peripheral blood biomarkers in asthmatics is still poorly investigated so new studies are required.

Iron and ferritin concentrations in exhaled breath condensate of children with asthma.

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.

Superoxide dismutase, copper and zinc concentrations in platelet-rich plasma of pneumonia patients.

BACKGROUND: The aim of this study was to analyse platelet superoxide dismutase (SOD) activities (total SOD, manganese SOD and copper zinc SOD) and copper (Cu) and zinc (Zn) concentrations during the course of community-acquired pneumonia (CAP), and to compare them between patients with normal platelet count and those who have developed reactive thrombocytosis (RT). METHODS: Platelet count, SOD activities and Cu and Zn concentrations in platelet-rich plasma were measured in patients with CAP on admission and at discharge. RESULTS: Post-therapeutic platelet count increased significantly from the value recorded on admission. By the end of treatment, 42% of patients developed RT. All platelet SOD activities as well as Cu concentration were significantly lower in CAP patients than in control subjects. The initial Zn concentration was greater in CAP patients compared with controls and showed a decrease at discharge. On admission, there was no difference in all SOD activities between either subgroup with normal platelet count or subgroup with RT. At discharge all SOD activities were significantly lower in patients with RT. Also, catalytic activities of those enzymes were significantly lower in both subgroups in comparison with the initial values. Post-therapeutic Cu value was lower in patients with RT in comparison with patients having normal platelet count. Zn concentration decreased significantly at discharge when compared with the initial values only in patients with RT. CONCLUSION: The pattern of changes might be indicative of a certain role of platelets in antioxidant response during treatment in CAP patients.

Magnesium concentration in plasma, leukocytes and urine of children with intermittent asthma.

BACKGROUND: Magnesium (Mg) is involved in numerous physiological functions, including protein folding, intracellular signalling and enzyme catalysis. It acts as a smooth muscle relaxant. We decided to test changes of total Mg concentration in plasma, leukocytes and urine of 16 healthy children and 26 patients with intermittent asthma aged 3 to 14 years. METHODS: Samples were taken on the first day of broncho-obstruction and five days after the acute attack. During this period, patients were under salbutamol therapy. RESULTS: Plasma Mg concentration in patients increased by about 40% (first day 0.58+/-0.05 mmol Mg/l and five days later 0.64+/-0.04 mmol Mg/l) compared with healthy children (0.42+/-0.04 mmol Mg/l). Leukocyte Mg concentrations showed significant changes. On the first day of broncho-obstruction, Mg in leukocytes significantly decreased by about 60% (1.16+/-0.31 mmol Mg/g protein) compared to healthy children (3.04+/-0.68 mmol Mg/g protein). Five days later, Mg values significantly increased (3.28+/-1.09 mmol Mg/g protein) and almost reached the values of the healthy group. Mg concentration in urine statistically decreased by about 30% (0.55+/-0.06 mmol Mg/mmol creatinine) on the first day of broncho-obstruction compared to healthy children (0.75+/-0.05 mmol Mg/mmol creatinine). Five days after the acute attack, Mg concentration in patients' urine (0.73+/-0.07 mmol Mg/mmol creatinine) was close to values of healthy children. CONCLUSION: The results obtained indicate that the intracellular measurement of Mg concentration is relevant for estimation of magnesium concentration in the human organism. Hence, determination of Mg concentration in leukocytes may be used in evaluation of asthmatic pathology.

Bisalbuminemia in two Croatian families.

BACKGROUND: Bisalbuminemia is a dysproteinemia characterized by the occurrence of two albumin fractions on serum protein separation by electrophoresis on cellulose acetate sheets. Bisalbuminemia may occur as a hereditary trait or as analytical interference with some drugs, especially penicillin. METHODS: Two patients with the finding of bisalbuminemia are presented. Both patients (patient 1 was a 4-1/2-month-old male infant, and patient 2 was a 15-year-old boy) were admitted for respiratory infection. RESULTS: Bisalbuminemia was detected by serum protein electrophoresis and confirmed by isoelectric focusing in pH gradient gel (pH range 4.0-6.5). This finding was supported by simultaneous detection of abnormal albumin in the mother of patient 1, while the father had normal albumin. The abnormal fast albumin in both patients had an increased relative mobility of 1.08 when measured from the sample application position. CONCLUSIONS: The results presented are the first description of albumin mutations in Croatia (that according to the CISMEL group could be classified as ZC/HZ), and present the first step in identification prior to determination of structural change and amino acid sequence in the albumin molecule.

[Relation between month of birth and the manifestation of atopic diseases in children and adolescents]. / Ispitivanje povezanosti mjeseca rodenja i ocitovanja atopijskih bolesti u djece i mladezi.

A total of 354 children with atopic diseases (asthma, pollenosis, dermatitis) were examined. Less than half of them (46.6%) had atopy in their families. Children of atopic parents often had two atopic diseases. Hypersensitivity to single allergen occurred in 66% (234/354) of patients and to more than one allergen in 34% (120/354) of patients. It seems that the season of birth might be important for children sensitized to pollens (born on the eve of pollen season). For the children sensitized to Dermatophagoides pteronyssimus season of birth was not decisive.

[Foreign bodies in the airways in children]. / Strana tijela u disnim putovima djece.

From 1984 to 1998 we treated 22 children (15 male, 7 female) with aspirated foreign bodies in their tracheobronchial tree. Most patients (17/22, 77%) were aged up to 3 years (two children were seven months old). Only 12 (54%) patients had history of aspiration. Most common clinical findings were prolonged expiration and impaired respiration on one side (12 patients, 54%) or impaired respiration on one side (7 patients, 32%). Chest x-rays mostly showed one-sided hyperinflation (64% of cases). 50% of children aspirated walnut into their airways. Foreign bodies in most children were installed tracheobronchially on the left and right side and in one child the foreign body was found in the larynx. The foreign bodies were successfully removed in 19 (85%) patients. In three children foreign bodies were not found on time, bronchial obliteration occurred, so pulmectomy of the left lung, lobectomy of the left lower lobe, or bilobectomy (lower and middle right lobe) had to be done.

[BCG dissemination in a child with normal immunity]. / BCG diseminacija u djeteta s normalnom imunosti.

A fourteen month old girl with BCG dissemination and BCG lymphadenitis is presented. Mycobacterium bovis BCG strain was isolated from axillary fistula and from gastric contents. Pathohystological examination of the lymph mode and cytological examination of the bronchial mucus confirmed granulomatous inflammation. All tests for humoral and cellular immunity were with reference ranges. Antituberculous therapy successfully eliminated Mycobacterium bovis in gastric contents, and healed the wound in the left armpit. As the patient had tuberculoid form of BCG dissemination, the therapy was successful.

[Primary ciliary dyskinesia]. / Primarna cilijarna diskinezija.

The immotile cilia syndrome is a genetic disease characterized by chronic disease of the upper and lower respiratory tract, male infertility and situs viscerum inversus in 50% of affected patients. Abnormalities of ciliary structure are the cause of abnormalities in mucociliary clearance and chronic inflammation consecutively. The boy aged 13 years with clinical manifestations (chronic rhinorrhea, sinuitis, recurrent otitis media, recurrent bronchial inflammation, pneumonia and finally bronchiectasis) has been presented. The boy had a normal visceral status. The diagnosis was confirmed by electron microscopy analysis of cross section of bronchial cilia: numerous abnormalities in microtubules positioning and lack of radiar arms have been found.

Antioxidants, trace elements and metabolic syndrome in elderly subjects.

OBJECTIVE: To examine whether concentrations of several trace elements and activities of several antioxidant enzymes are modified in metabolic syndrome, and to evaluate their possible association with metabolic syndrome components. Additionally, concentration of CRP, as a marker of inflammation, was measured. DESIGN: Cross-sectional study. PARTICIPANTS: The study group consisted of 100 subjects, aged 71-88 years. MEASUREMENTS: Anthropometric measurements and biochemical analyses of fasting blood samples were performed by standardized methods. According to the International Diabetic Federation (IDF) criteria, metabolic syndrome was diagnosed in 64 subjects. Whole blood glutathione peroxidase (GPx), erythrocyte superoxide dismutase (SOD) and catalase (CAT), serum selenium (SSe), copper (SCu) and zinc (SZn), glucose, lipoprotein profile and C-reactive protein (CRP) were determined in all subjects. RESULTS: No clear influence of metabolic syndrome on SSe, SZn and SCu concentration and SOD and CAT activity was found. However, significantly higher GPx was found in subjects with metabolic syndrome than in subjects without metabolic syndrome (p=0.029), as well as in subjects with hypertriglyceridemia than in control subjects (p=0.038). After adjusting for potentially confounding variables by multiple regression, significant positive relationship between SCu and CRP was found, indicating that elevated levels of Cu could have influence on inflammatory mechanisms. CONCLUSION: Our results suggest that GPx and CRP, as biomarkers of oxidative stress and chronic inflammation, respectively, have significant role in the pathogenesis of metabolic syndrome.