Antiphospholipid antibodies in patients with antiphospholipid syndrome.

Antiphospholipid syndrome (APS) is a rare systemic autoimmune disease characterized by recurrent pregnancy morbidity or thrombosis in combination with the persistent presence of antiphospholipid antibodies (aPLs) in plasma/serum. Antiphospholipid antibodies are a heterogeneous, overlapping group of autoantibodies, of which anti-ß2-glycoprotein I (aß2GPI), anticardiolipin (aCL) antibodies and antibodies that prolong plasma clotting time in tests in vitro known as lupus anticoagulant (LAC) are included in the laboratory criteria for the diagnosis of APS. The presence of LAC antibodies in plasma is indirectly determined by measuring the length of coagulation in two tests - activated partial thromboplastin time (aPTT) and diluted Russell's viper venom time (dRVVT). The concentration of aß2GPI and aCL (immunglobulin G (IgG) and immunoglobulin M (IgM) isotypes) in serum is directly determined by solid-phase immunoassays, either by enzyme-linked immunosorbent assay (ELISA), fluoroimmunoassay (FIA), immunochemiluminescence (CLIA) or multiplex flow immunoassay (MFIA). For patient safety, it is extremely important to control all three phases of laboratory testing, i.e. preanalytical, analytical and postanalytical phase. Specialists in laboratory medicine must be aware of interferences in all three phases of laboratory testing, in order to minimize these interferences. The aim of this review was to show the current pathophysiological aspects of APS, the importance of determining aPLs-a in plasma/serum, with an emphasis on possible interferences that should be taken into account when interpreting laboratory findings.

Divergent Trends in the Prevalence of Children's Asthma, Rhinitis and Atopic Dermatitis and Environmental Influences in the Urban Setting of Zagreb, Croatia.

BACKGROUND: Previous studies have reported that the allergy epidemic in developed countries has reached its plateau, while a rise is expected in developing ones. Our aim was to compare the prevalence of allergic diseases among schoolchildren from the city of Zagreb, Croatia after sixteen years. METHODS: Symptoms of asthma, allergic rhinitis (AR) and atopic dermatitis (AD) and risk factors were assessed using the International Study of Asthma and Allergies in Childhood (ISAAC) questionnaire. An allergic profile was determined by a skin prick test. RESULTS: The prevalence of current, ever-in-a-lifetime and diagnosed AR of 35.7%, 42.5% and 14.9% and AD of 18.1%, 37.1% and 31.1% demonstrated a significant increase. The asthma prevalence has remained unchanged. The allergen sensitivity rate has remained similar, but pollens have become dominant. Mould and dog exposure are risks for asthma (OR 14.505, OR 2.033). Exposure to cat allergens is protective in AR (OR 0.277). Parental history of allergies is a risk factor in all conditions. CONCLUSION: Over sixteen years, the prevalence of AR and AD, but not of asthma, have increased. The proportion of atopy has remained high. The AR/AD symptom rise is probably a consequence of increased pollen sensitisation united with high particulate matter concentrations. The stable asthma trend could be a result of decreasing exposures to indoor allergens.

Current status of the lateral flow immunoassay for the detection of SARS-CoV-2 in nasopharyngeal swabs.

Early detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and diagnosis of coronavirus disease 2019 (COVID-19) are priorities during the pandemic. Symptomatic and suspected asymptomatic individuals should be tested for COVID-19 to confirm infection and to be excluded from social interactions. As molecular testing capacity is overloaded during the pandemic, rapid antigen tests, such as lateral flow immunoassays (LFIAs), can be a useful tool as they allow greater test availability and obtain results in a very short time. This short review aims to present the analytical properties of LFIAs in the detection of SARS-CoV-2 in nasopharyngeal swabs. Lateral flow immunoassay is a method that combines thin-layer chromatography and indirect immunochemical sandwich method and allows the detection of a specific SARS-CoV-2 antigen in nasopharyngeal swabs. Swab specimens should be adequately collected and tested as soon as possible. Users should pay attention to quality control and possible interferences. Antigen tests for SARS-CoV-2 show high sensitivity and specificity in cases with high viral loads, and should be used up to five days after the onset of the first symptoms of COVID-19. False positive results may be obtained when screening large populations with a low prevalence of COVID-19 infection, while false negative results may happen due to improper specimen collection or insufficient amount of antigen in the specimen. So as to achieve reliable results, a diagnostic accuracy study of a specific rapid antigen test should be performed.

Ferroptosis: regulated cell death.

Ferroptosis is a recently identified form of regulated cell death that differs from other known forms of cell death morphologically, biochemically, and genetically. The main properties of ferroptosis are free redox-active iron and consequent iron-dependent peroxidation of polyunsaturated fatty acids in cell membrane phospholipids, which results in the accumulation of lipid-based reactive oxygen species due to loss of glutathione peroxidase 4 activity. Ferroptosis has increasingly been associated with neurodegenerative diseases, carcinogenesis, stroke, intracerebral haemorrhage, traumatic brain injury, and ischemia-reperfusion injury. It has also shown a significant therapeutic potential in the treatment of cancer and other diseases. This review summarises current knowledge about and the mechanisms that regulate ferroptosis.

SARS-CoV-2 - a new challenge for laboratory medicine.

The new corona virus SARS-CoV-2 (Severe Acute Respiratory Syndrome Corona Virus 2) causes a disease called COVID-19 (coronavirus disease 2019), that develops mostly in subjects with already impaired immune system function, primarily in the elderly and in individuals with some chronic disease or condition. The reasons for this should be sought in the processes of aging and chronic latent inflammation, i.e. immunosenescence and inflammaging. Laboratory medicine specialists are currently focused on proving the presence of the virus and defining biomarkers that would enable the prediction of disease progression. For now, it has been shown that useful biomarkers can include general biomarkers of inflammation (parameters of complete blood count, C-reactive protein, interleukin-6, procalcitonin), biomarkers of myocardial damage (high sensitivity troponin I/T, B-type natriuretic peptide, and N-terminal B type natriuretic peptide), and vascular biomarkers (D-dimer, prothrombin time, fibrinogen). Their actual diagnostic specificity, sensitivity and predictive value need to be tested on a larger number of subjects. In addition, it is important to find and evaluate specific biomarkers of immunosenescence.

Hallmarks of senescence and aging.

The complex process of biological aging, as an intrinsic feature of living beings, is the result of genetic and, to a greater extent, environmental factors and time. For many of the changes taking place in the body during aging, three factors are important: inflammation, immune aging and senescence (cellular aging, biological aging). Senescence is an irreversible form of long-term cell-cycle arrest, caused by excessive intracellular or extracellular stress or damage. The purpose of this cell-cycles arrest is to limit the proliferation of damaged cells, to eliminate accumulated harmful factors and to disable potential malignant cell transformation. As the biological age does not have to be in accordance with the chronological age, it is important to find specific hallmarks and biomarkers that could objectively determine the rate of age of a person. These biomarkers might be a valuable measure of physiological, i.e. biological age. Biomarkers should meet several criteria. For example, they have to predict the rate of aging, monitor a basic process that underlies the aging process, be able to be tested repeatedly without harming the person. In addition, biomarkers have to be indicators of biological processes, pathogenic processes or pharmacological responses to therapeutic intervention. It is considered that the telomere length is the weak biomarker (with poor predictive accuracy), and there is currently no reliable biomarker that meets all the necessary criteria.

Filaggrin and atopic march.

There is an increasing number of experimental, genetic and clinical evidence of atopic dermatitis expression as a pre-condition for later development of other atopic diseases such as asthma, food allergy and allergic rhinitis. Atopic dermatitis is a heterogeneous, recurrent childhood disease, also present in the adult age. It is increasingly attributed to systemic features and is characterized by immunological and skin barrier integrity and function dysregulation. To maintain the protective function of the skin barrier, in particular the maintenance of pH, hydration and antimicrobial functions, the filaggrin, among others, plays a significant role. Filaggrin is a multifunctional, histidine-rich, insoluble protein. The lack of filaggrin is associated with various cutaneous (e.g. ichthyosis vulgaris, allergic contact dermatitis) and non-cutaneous (e.g. diabetes, inflammatory conditions of the gastrointestinal tract) diseases and may be a result of genetic, immunological factors combined with environmental factors. In this review we summarised (emphasized) recent findings in understanding the role of filaggrin in atopic dermatitis and other diseases, participants in the atopic march.

Corrigendum to: The potential of component-resolved diagnosis in laboratory diagnostics of allergy.

[This corrects the article DOI: 10.11613/BM.2018.020501.].

The potential of component-resolved diagnosis in laboratory diagnostics of allergy.

The initial laboratory approach in the diagnosis of allergies is to detect the type of allergic reaction, i.e. whether the patient's allergy is mediated by immunoglobulin E (IgE) or not. For this purpose, the concentration of total serum IgE (tIgE) and specific IgE (sIgE) are determined. Progress in laboratory diagnostics is the use of component-resolved diagnosis (CRD) which implies determination of sIgE against purified native and recombinant allergenic molecules. Component-resolved diagnosis is used in laboratory practice as singleplex and multiplex assays. The choice of allergen for singleplex assay is based on anamnesis, clinical findings of a patient and on skin prick test results. Multiplex-microarray assays simultaneously determine multiple sIgE's against numerous allergens. The goal of CRD is to distinguish the true allergens from the cross-reactive allergen molecules. Component-resolved diagnosis allows predicting the risk of severe symptoms, as well as anticipating the development of allergies. Thus, determination of sIgE against allergenic components may significantly improve current diagnostics of allergy. Since this method is applied in laboratory practice just a few years, it is necessary to acquire new knowledge and experience, to establish good co-operation between specialist in medical biochemistry and laboratory medicine and the specialist allergologist, so that the method can be applied in a rational manner. Component-resolved diagnosis will significantly improve the diagnostics of IgE-mediated allergy in the future. The aim of this article is to present potentials of CRD in the laboratory diagnostics of allergy mediated by IgE.

Contribution to diagnostics/prognostics of tuberculosis in children. I. New methods of assaying zinc and simultaneously copper and zinc in diluted sera by flame atomic-absorption spectrometry.

In an attempt to provide a reliable status of metal ions in children, new methods of analysis of children's sera are proposed. New flame atomic-absorption spectrometric (FAAS) methods are simple, cost- and time-effective and, above all, labor-, reagent- and sample-saving. Two methods were suggested: method A for simultaneous determination of Cu and Zn from 5-fold diluted sera, and method B, for assaying zinc alone in 10-fold diluted samples. Both methods are based on a single-step sample pretreatment (deproteinization with 3 mol dm-3 HCl). Method A uses a single-step calibration with a mixed standard. The main advantage of method B is an additional reduction in sample consumption. Both methods were fully validated against reference methods. Accuracy, sensitivity and precision have proven them to be comparable to the reference methods in terms of analytical performance, and applicable to analyses of children's sera.

Contribution to diagnostics/prognostics of tuberculosis in children. II. Indicative value of metal ions and biochemical parameters in serum.

Newly introduced methods of assaying simultaneously copper and zinc and zinc alone in serum by flame atomic-absorption spectrometry are simple and economical, especially in saving the consumption of serum material. Along with biochemical parameters, they have been successfully applied to diagnostics/prognostics of tuberculosis in children, through analyses of sera from pediatric patients with lung tuberculosis or suspected tuberculosis, enabling the follow-up of therapeutic efficiency. The prognostic strength of Cu and Cu/Zn ratio together with C-reactive protein, complement components C3 and C4, and erythrocyte sedimentation rate have been documented.

Discordance Between Tuberculin Skin Test and Interferon-γ Release Assay in Children Younger Than 5 Years Who Have Been Vaccinated With Bacillus Calmette-Guérin.

BACKGROUND: Interferon-γ release assays (IGRAs) offer the possibility of improved detection of latent tuberculosis infection (LTBI). OBJECTIVE: To analyze discordant tuberculin skin testing (TST) and IGRA results in ethnic Croatian children as old as 5 years for whom there is documented exposure to an adult with active tuberculosis (TB) and who have been vaccinated with Bacillus Calmette-Guérin. METHODS: In specimens from our cohort individuals, we tested the performances of the QuantiFERON-TB Gold In-Tube (QFT-GIT) test and TST and analyzed discordant results. RESULTS: At the TST cutoff value of 10 mm or greater, the estimated prevalence of M. tuberculosis infection was 18.1% (31/171) using TST and 15.2% (26/171) using QFT-GIT. The results of these 2 tests showed an overall concordance of 87.7%. There was no evidence that subjects' age correlated with discordant results. CONCLUSIONS: The reasons for discordant results in young children are still unclear, which highlights the importance of further longitudinal studies to better understand the interpretation and any possible clinical implications of the results of these tests.

The effects of parental smoking on anthropometric parameters, peak expiratory flow rate and physical condition in school children.

Passive smoking in children is a considerable health problem, mainly arising from parental smoking. The objectives of the present cross-sectional study were to assess the impact of passive smoking on 1) anthropometric parameters; 2) peak expiratory flow rate (PEFR); and 3) physical condition in school children. The target population included 177 children attending elementary school 5th to 8th grade. Study subjects were divided into two groups according to parental smoking habits. Body weight and height were determined using a digital weighing scale and digital stadiometer; PEFR was measured between 8 a.m. and 10 a.m. using a Peak Flow Meter; and physical condition was assessed by the 6-minute run test. Sixty-six percent of study children were exposed to passive smoking. The children of smoking parents had higher BMI [18.79 (17.50-21.13) kg/m2] than children of nonsmoking parents [17.90 (16.00-20.00) kg/m2; p = 0.036]. There was no statistically significant difference in body height and weight. The children of smoking parents had statistically lower values of PEFR [M(IQR) = 84 (78-88)%, M(IQR) = 94 (89-101)%, respectively; p < 0.0001] and 6-minute run test than children of nonsmoking parents [M(IQR) = 2(1-3), M(IQR) = 4(3-5); respectively; p < 0.0001]. The results of the present study showed that exposure of school children to passive smoking by their parents resulted in an increase of BMI, impairment of lung function, and impairment of physical condition, especially in children of both smoking parents.

GSTP1, GSTM1 and GSTT1 genetic polymorphisms and total serum GST concentration in stable male COPD.

The aim of this study was to test the hypothesis that glutathione- S-transferase (GST) genotypes were associated with COPD. GSTP1, GSTM1 and GSTT1 genotypes were determined by DNA methods and GST activity spectrophotometrically in older male Caucasian Croats (non- -smokers, ex-smokers, and smokers) with stable COPD (n = 30) and sex/age matched controls (n = 60). The distribution of GSTP1 genotypes and alleles in controls vs. COPD showed a statistical difference (p < 0.05). The odds ratio of CC/CT+TT (wild type GSTP1 exon 6 vs. joint heterozygous and mutant homozygous GSTP1 exon 6) was 10.000 and statistically different (p = 0.002). In this study, the GSTP1 mutant genotype of exon 5 (GG), as well as GSTP1 mutant and heterozygous genotypes of exon 6 (TT and CT), were suggested to be genetic contributors to COPD susceptibility. Null GSTM1, null GSTT1 and joint GSTM1/GSTT1 null genotypes were not disease associated. Serum GST was not associated with GST genotypes and COPD or smoking history in our study subjects. Conclusions drawn from the study should be further supported and clarified by studies with larger sample sizes.

Exhaled breath condensate--from an analytical point of view.

Over the past three decades, the goal of many researchers is analysis of exhaled breath condensate (EBC) as noninvasively obtained sample. A total quality in laboratory diagnostic processes in EBC analysis was investigated: pre-analytical (formation, collection, storage of EBC), analytical (sensitivity of applied methods, standardization) and post-analytical (interpretation of results) phases. EBC analysis is still used as a research tool. Limitations referred to pre-analytical, analytical, and post-analytical phases of EBC analysis are numerous, e.g. low concentrations of EBC constituents, single-analyte methods lack in sensitivity, and multi-analyte has not been fully explored, and reference values are not established. When all, pre-analytical, analytical and post-analytical requirements are met, EBC biomarkers as well as biomarker patterns can be selected and EBC analysis can hopefully be used in clinical practice, in both, the diagnosis and in the longitudinal follow-up of patients, resulting in better outcome of disease.

Hepatitis during respiratory syncytial virus infection--a case report.

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) occurred 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.

FABP2 gene polymorphism and metabolic syndrome in elderly people of croatian descent.

INTRODUCTION: Metabolic syndrome (MetS) is a multifactorial disorder in which dyslipidemia plays an important role. Fatty acid-binding protein 2 (FABP 2) is responsible for transport of free fatty acids in the intestinal endothelium cells. FABP2-genetic variants might affect plasma lipid concentrations and intracellular lipid transport. The aim of this study was to investigate the association between FABP2 Ala54Thr genetic polymorphism and metabolic syndrome and some biochemical and anthropological parameters in elderly subjects. MATERIALS AND METHODS: This cross-sectional study included 140 men and 176 women older than 70 years. Fasting serum concentration of glucose, lipid parameters, total proteins and C-reactive protein were determined by standardized methods. Presence (MetS(+)) or absence (MetS(-)) of MetS was determined according to criteria of the International Diabetes Federation. FABP2 genetic polymorphism Ala54Thr (rs1799883) was genotyped with PCR-RFPL. RESULTS: The genotype frequencies for Ala/Ala, Ala/Thr and Thr/Thr genotype were 60, 36 and 6 in MetS(-), and 131, 70 and 13 in MetS(+), respectively, without statistical significance (P = 0.567). Ala/Ala genotype was a subgroup of non-carriers, while Ala/Thr and Thr/Thr genotypes were Thr54-carriers. Median triglyceride concentration was significantly lower in carriers then in non-carriers for whole MetS(+) group (P = 0.050); there were no significant difference between men with MetS (P = 0.144), but there was a difference between women with MetS (P = 0.020). T-test showed that mean HDL cholesterol concentrations in MetS(+) group for Thr54-carriers was significantly higher in whole group (P = 0.001), and for both genders (men P = 0.039; women P = 0.004) as compared to non-carriers. CONCLUSIONS: FABP2 genetic polymorphism is associated with lower triglyceride and higher HDL-cholesterol concentrations in elderly subjects with MetS. This genetic variation might be a useful marker for understanding dyslipidemia in MetS.

Prostate cancer in elderly Croatian men: 5-HT genetic polymorphisms and the influence of androgen deprivation therapy on osteopenia--a pilot study.

BACKGROUND: The aim of this study was to determine the relationship between body mass index, biochemical parameters, and 5-hydroxytryptamine (5-HT) genetic polymorphisms and prostate dysfunction in an elderly general male population. RESULTS: One hundred and seventeen elderly male subjects [60 men without symptoms of prostate hyperplasia, 42 men with untreated benign prostatic hyperplasia (BPH), and 15 men with prostate cancer (PCa)] treated with finasteride or flutamide were included. Multiple comparisons showed significant difference in age, T-score, concentration of phosphorus, calcium, C-reactive protein, and prostate-specific antigen (PSA) between the groups. T-score was the lowest and phosphorus concentration was the highest in the PCa group. Highest PSA, proteins, calcium, and Hekal's formula score were found in the BPH group. Patients with PCa were more frequent GG+GA carriers of 5-HT1B 1997A/G gene polymorphism (p=0.035). Univariate regression analysis showed association of PCa-treated subjects with age (p=0.010) and 5-HT1B genetic polymorphism (p=0.018). Antiandrogen therapy affects T-score (p=0.017), serum phosphorus (p=0.008), glucose (p=0.036), and total proteins (p=0.050). Multivariate-stepwise logistic regression analysis showed the significant association of treated PCa with age (p=0.028) and inorganic phosphorus (p=0.005), and a marginal association with ultrasonographic T-score (p=0.052). CONCLUSIONS: Antiandrogen therapy might induce bone mineral loss in elderly PCa patients. Preliminary data imply that the genetic variants of the 5-HT1B receptor might be associated with PCa.

Serial interferon-γ release assay in children with latent tuberculosis infection and children with tuberculosis.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Eosinophil cationic protein--current concepts and controversies.

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.