Freezing of sputum as a way to improve the applicability of sputum studies

Rationale: Sputum examination is a valuable research tool to study airway diseases, but the requirement to process the samples within 2 hours of their collection poses limitations to its wider applicability. A way to bypass this hurdle would be to freeze the sample at the time of collection and to examine it at a later stage.Methods and Subjects: We developed a protocol for freezing of sputum upon collection by adding dimethylsulfoxide to it. We tested the reproducibility of cell counts in frozen samples and in fresh portions from the same sputum specimens. We took sputum from 41 asthmatics (18 males) with different levels of control of their disease: 19 of the samples were spontaneously produced and 22 were induced with hypertonic saline.Results: Significant correlations (p<0.05) were established between the total cell counts, the relative and absolute number of neutrophils, eosinophils and macrophages in the paired fresh and frozen sputum samples. Cell viability in frozen sputum was slightly but consistently lower. Only one frozen sample had viability < 50%. Outcomes in paired samples from induced sputum had better reproducibility than the spontaneous ones.Conclusion: Examination of frozen sputum samples does not change total cell counts and differential cell counts, despite consistently affecting cell viability compared with fresh sputum cellularity. Still cell viability in frozen sputum was above 50% in all but one examined specimens

Distribution of Killer Cell Immunoglobulin-Like Receptor Genes in Albanians from Republic of Macedonia

AIM: The aim of this study was to analyze Killer Ig-Like Receptor (KIR) gene polymorphisms in Albanians from Republic of Macedonia.MATERIAL NA METHODS: The studied sample consists of 104 healthy unrelated individuals, aged 20-45 years. All individuals are of Albanian nationality, residents of different geographical regions (Skopje, Gostivar, and Tetovo) in Republic of Macedonia. The population genetics analysis package, Arlequin, was used for analysis of the data.RESULTS: All 16 KIR genes known were observed in the Albanian individuals and framework genes (KIR3DL3, KIR3DP1, KIR2DL4, and KIR3DL2) were present in all individuals. The frequencies of other KIR genes were: KIR2DP1 (0.981), KIR2DL1 (1), KIR2DL2 (0.615), KIR2DL3 (0.865), KIR2DL5 (0.414), KIR3DL1 (0.933), KIR2DS1 (0.462), KIR2DS2 (0.606), KIR2DS3 (0.327), KIR2DS4 (0.875), KIR2DS5 (0.298), and KIR3DS1 (0.442). Tested linkage disequilibrium (LD) among KIR genes demonstrated that KIR genes present a wide range of linkage disequilibrium.CONCLUSION: This is the first study analyzing the polymorphism of KIR genes and genotype frequencies in Albanian individuals in the world. The results can be used for anthropological comparisons.

DETECTION OF A RARE MUTATION IN A NOONAN SYNDROME SUSPECTED PATIENT: A CASE REPORT

Noonan syndrome (NS) is a genetic autosomal dominant condition, caused by mutations in PTPN11 and other genes. The aim of this report is to highlight a finding of a rare mutation in the RAF1 gene in a six-year-old child evaluated for Noonan Syndrome. An Ampliseq Research Panel covering A2ML1, BRAF, CBL, HRAS, KRAS, MAP2K1, MAP2K2, NRAS, PTPN11, RAF1, RIT1, SHOC2, SOS1 and SPRED1 genes was used on the Ion Torrent platform. Out of 54 variants detected, a single nucleotide missense mutation c.483T>G in the RAF1 gene was classified as likely pathogenic, based on a single previous submission to Clinvar. Further investigations may shed light on the possible role of this variant in the pathogenesis of Noonan Syndrome and other RASopathies

COMPARISON OF ELISA AND CHEMILUMINESCENCE IMMUNOASSAY METHODS FOR QUANTIFICATION OF HUMAN PLACENTAL GROWTH FACTOR IN SERUM

Placental growth factor (PlGF) is crucial during placental development in early pregnancy. Several studies in pregnancies with complications such as preeclampsia or small for gestational age neonates find that PlGF levels are significantly lower in the first trimester, which implies that the concentration of PlGF could be used as an early screening biomarker for these conditions. This study aimed to compare the performance of chemiluminescence immunoassay (CLIA) and enzyme-linked immunosorbent assay (ELISA) for the quantification of human PlGF in serum. This is a comparative study on 88 pregnant women in the first trimester subjected to measurement of PlGF in serum using two commercially available kits: Human PlGF Quantikine HS ELISA (R&D Systems) and PlGF CLIA (Snibe). The overall coefficient of correlation between the tests was 0.93. When the cut-off value of 40 pg/mL was applied, it dropped significantly to 0.50 towards the lower values, while remaining an excellent 0.91 in the group with higher concentrations of PlGF. While R&D Systems’s ELISA seems to have better sensitivity, it is not very convenient to use for a small number of samples. Snibe’s CLIA automated method is user-friendly, fast and powerful. Both tests show excellent performance when indicating risk-free pregnancies

Food Allergy Based on Serum Specific IgE in Republic of Macedonia

ОСÐОВÐ:  Ðлергијата кон храна Ñе дефинира како неÑакан имун одговор кој Ñе јавува при повторувачки изложувања на било кој вид на храна и може да биде поÑредувана Ñо имуноглобулин Е (ИгЕ) противтела или не – ИгЕ поÑредувана. ÐлергиÑката реакција кон храна е доÑта чеÑта и Ñпоред поÑледните иÑтражувања Ñе јавува кај 6-8% од децата, а во поÑледните три декади забележан е пораÑÑ‚ во инциденцата на алергии кон храна. Доколку Ñе Ñомневаме за ИгЕ поÑредувана реакција на храната треба да Ñе направат лабораториÑки иÑпитувања и да Ñе одредат вкупните и Ñпецифичните ИгЕ противтела кон различните видови на храна.ЦЕЛ: Целта на овој труд е да Ñе преÑмета и прикаже процентот на алергии на храна во Република Македонија на оÑнова на позитивен наод на ИгЕ противтела во Ñерумот на пациенти упатени на ИнÑтитутот за имунобиологија и хумана генетика за алерголошки теÑтирања во деÑетгодишен период (2001-2011).ÐœÐТЕРИЈÐЛ И МЕТОДИ: Ðнализирани Ñе ретроÑпективно конÑекутивни пациенти во деÑет годишен период (од 01.01.2001 до 01.01.2011 година), упатени на ИнÑтитутот за имунобиологија и хумана генетика при МедицинÑкиот факултет во Скопје за иÑпитување на алергии. Ðнализите Ñе направени на 3312 иÑпитаника (пациенти) за кои имаме точни податоци за меÑтото од кое потекнуваат, за возраÑта и кај кои Ñе одредени вредноÑтите за вкупниот ИгЕ во Ñерумот, како и вредноÑтите за Ñпецифичните ИгЕ противтела наÑочени кон алергени од храната. Ðнализите за одредување на вкупен ИгЕ и Ñпецифичен ИгЕ во Ñерумот кај пациентите Ñе изработени на UniCAP100 System; Pharmacia, Uppsala, Sweden.РЕЗУЛТÐТИ: При анализата на диÑтрибуцијата на бројот на пациенти во Ñекоја група на концентрација на вкупниот ИгЕ покажуваат дека најголем дел од пациентите имале нормални вредноÑти за вкупниот ИгЕ во Ñерумот. Од вкупно 3312 пациенти Ñо Ñомневање за поÑтоење на ИгЕ поÑредувана алергија на храна кај 2367 Ñме добиле негативен наод за поÑтоење на зголемени нивоа на ИгЕ противтела, а Ñамо кај 945 иÑпитаници Ñме докажале поÑтоење на ИгЕ поÑредувана алергија на храна. ÐајчеÑтите позитивни алергени Ñе: белка од јајце, млеко, варено млеко, пченица, кикирики, протеини на млеко, лешник и јаболко, а од групно Ñпецифичните Ñе: Ñмешата од храна и Ñмешата од овошје.ЗÐКЛУЧОЦИ: Големиот број на негативни резултати ја покажува и потребата од зголемување на бројот на алерголошки лаборатории низ Република Македонија како и зголемување на едукацијата на матичните лекари преку Ñеминари, конференции и предавања. Ова е првата Ñтудија во Република Македонија за алергии кон храна во која Ñе прикажани резултати на оÑонова на добиените вредноÑти на Ñпецифични ИгЕ противтела кон различни алергени од храна во Ñерум.BACKGROUND: Food allergy is defined as an adverse immune response during repetitive exposure to any kind of food and may be mediated by immunoglobulin E (IgE) antibodies or not - IgE mediated. Food allergies are common and according to recent studies they occur in 6-8% of children, and in the last three decades there has been an increase in the incidence of food allergies. In IgE-mediated food reaction we should make laboratory tests to determine the total and specific IgE antibodies to various foods.AIM: The aim of this paper is to demonstrate the percentage of food allergies in the Republic of Macedonia based on positive IgE antibodies in the serum of patients referred to the Institute of Immunobiology and Human Genetics for allergies testing in ten year period (2001-2011).MATERIAL AND METHODS: Consecutive patients in ten year period (from 01.01.2001 to 01.01.2011), addressed to the Institute of Immunobiology and Human Genetics at the Medical Faculty tested for allergies were analyzed retrospectively. Three thousand and twelve (3312) patients with data for place of residence, age and serum values for  total IgE, specific IgE and group-specific IgE to food allergens were analyzed. The determination of total IgE and specific IgE for food allergens was performed with the UniCAP100 System (Pharmacia, Uppsala, Sweden).RESULTS: Analyzes of the distribution of the number of patients in each group of concentration of total IgE showed that most patients had normal serum total IgE levels. From a total of 3312 patients with suspected IgE-mediated food allergy, 2367 were found negative and only in 945 patients we determined the existence of IgE-mediated food allergy. The most common allergens in our study were: egg white, milk, boiled milk, wheat, peanuts, milk proteins, hazelnut and apple and from the group - specific allergens most common were food mixture and fruit mixture.CONCLUSIONS: The large number of negative results suggested the need for increasing the number of Allergologic Laboratories in Republic of Macedonia and increase of the need for better education of primary care physicians through seminars, conferences and lectures. This is the first study in the Republic of Macedonia for food allergies in which the presented results are based on the obtained specific IgE antibodies values in the sera of patients towards different food allergens

Distribution of CYP2C9 and VKORC1 Gene Polymorphisms in Healthy Macedonian Male Population

Background: Distribution of CYP2C9 and VKORC1 gene polymorphisms may vary significantly among different ethnic groups, and eventually influence the variation in drug metabolism or even failure.Objective: The aim of this study was to evaluate the prevalence of CYP2C9 and VKORC1 alleles in the healthy population of Republic of Macedonia compared to the global geographic data reported from different ethnic populations. Also, to genotype CYP2C9 and VKORC1 genes and eventually to divide individuals in poor, extensive, or intermediate metabolizer.Material and Methods: Blood samples were collected after signing written consent, DNA was isolated from peripheral blood, and CYP2C9 and VKORC1 genes were typed (n=124). Genotyping was performed by commercially available kits (GeneID GmbH, Strassberg, Germany, AID Diagnostica), based on the method of polymerase chain reaction with a subsequent hybridization. The population genetics analysis package, PyPop ver. 0.6.0, was used for analysis of the data.Results: The frequency of alleles varies from 0.931 for CYP2C9*3 to 0.109 for CYP2C9*2 indicating common “wild type†allele in those genes. The frequency ranges spanned ~50% for each allele of VKORC1 gene, indicating no common “wild type†allele in this gene. Test of neutrality showed significant negative value for VKORC1 polymorphism that indicates balancing selection operating on the alleles at that locus. All polymorphisms of CYP2C9*2, CYP2C9*3 and VKORC1 showed a good fit with Hardy-Weinberg expectations.Conclusion: The results of polymorphic alleles of CYP2C9 and VKORC1 genes in Macedonian population can be used for the variation in drug metabolism studies as well for adapting dosage regimes for oral anticoagulant therapies

Total IgE Distribution in Food Allergy Suspected Patients in Republic of Macedonia (2001-2011)

BACKGROUND: IgE may be considered the hallmark of allergic disorders. It is easily detected in serum and can be measured as total IgE and as allergen-specific IgE. In fact, the serum IgE assay is used to diagnose an allergy.AIM: The aim of this study is to evaluate, investigate and present the distribution of total serum IgE levels, determined with UniCap system, in food-allergy suspected patients in a Republic of Macedonia.MATERIAL AND METHODS: In this study we analyzed retrospectively 8898 consecutive patients that were admitted for allergy testing at the Institute of Immunobiology and Human Genetics during the ten year period between 01.01.2001 and 01.01.2011. Total IgE levels in patient sera were detected with the in vitro system UniCAP100 (Pharmacia, Uppsala, Sweden).RESULTS: When we analyzed the number of patients according to the total IgE groups, we noted that most of the patients have normal levels of total IgE in serum. However, we also discovered a group of patients with elevated levels of total IgE that are greater than 200 kU/L. The average concentration of total serum IgE is higher in women in the age group 6 (6-7 years), followed by a steep decrease in the age group 9 (9-10 years), and after that the average concentrations of total IgE were mostly constant with the exception of a partial increase in the age group 21 (65-69 years). For men, the average serum concentrations of total IgE were highest in the age group of 6 (6-7 years), which was significantly higher than the average concentrations of total IgE in all other age groups.CONCLUSION: The large number of enrolled patients, a particular strength of this study, revealed that average concentrations of total IgE in men are higher than in women and that total IgE did not decrease with age. On the contrary, increased total IgE levels were found in patients aged 65 and 69 of both genders. We continue our work with analyses of the specific IgE antibodies values toward food and the correlation with total IgE values

Immunonephelometry and Reverse Hybrydization Genotyping in Diagnosis of Alpha-1-Antitrypsin Deficiency in Macedonians

BACKGROUND: With a frequency of 1:1600, the alpha-1-antitrypsin deficiency is one of the most frequent hereditary diseases and can be recessively inherited. AAT deficiency is most often caused by inheritance of the so-called PiZ allele. Inheritance of this allele increases the risk of developing chronic obstructive pulmonary diseases (COPD) and liver disease.AIM: The aim of this study was to present immunonephelometry and reverse hybridization genotyping in diagnosis of alpha-1-antitrypsin deficiency in Republic of Macedonia.MATERIAL AND METHODS: At the Institute of Immunobiology and Human Genetics, part of the Faculty of Medicine in Skopje, in the previous 7 years, total of 361 patients with suspected alpha-1-antitrypsin (AAT) deficiency were referred for analysis of AAT concentration using nephelometry (Dade Behring) and subsequent AAT genotyping of individuals with alpha-1-antytripsin deficiency at protein level, based on reverse hybridization technique.RESULTS: Measurement of AAT concentration (g/l) by nephelometry have shown normal level in the range of 1.37-1.41 g/l (88%), lower than normal AAT levels in the range of 0.70-0.83 g/l (8.03%), and concentration above the normal levels in the range of 2.28-2.4 g/l (3.88%).CONCLUSION: Diagnosis in the case of a suspicion of AAT deficiency is carried out by measuring the alpha-1-antitrypsin level in blood and by genotyping of alpha-1-antytripsin allele

Association of Methylenetetrahydrofolate Reductase (MTHFR-677 and MTHFR-1298) Genetic Polymorphisms with Occlusive Artery Disease and Deep Venous Thrombosis in Macedonians

Cilj Ispitati moguću povezanost genetičkog polimorfizma metilen-tetrahidrofolatne reduktaze (MTHFR-677, MTHFR-1298) s okluzivnom arterijskom bolešću i dubokom venskom trombozom u Makedonaca. Postupci Radili smo s 83 zdrave osobe, 76 bolesnika s okluzivnom arterijskom bolešću i 67 bolesnika s dubokom venskom trombozom. Od njih su prikupljeni su uzorci krvi i iz leukocita je izolirana DNA. Mutacije gena za MTHFR identificirane su testom CVD StripAssay (ViennaLab, Labordiagnostika GmbH, Beč, Austrija), a za analizu je uporabljen sustav za genetičku analizu PyPop. Potom su izračunani Pearsonove P vrijednosti, grubi omjer izgleda (odds ratio, OR) i Waldovi 95% intervali pouzdanosti (confidence intervals, CI). Rezultati Frekvencija alela C lokusa za MTHFR-677 bila je 0,575 u bolesnika s dubokom venskom trombozom, 0,612 u onih a okluzivnom arterijskom bolešću i 0,645 u zdravih osoba. Frekvencija alela T lokusa za MTHFR-677 bila je niža u zdravih osoba (0,355) nego u bolesnika s okluzivnom arterijskom bolešću (0,388) i dubokom venskom trombozom (0,425). Frekvencija alela A u lokusu MTHFR-1298 bila je 0,729 u zdravih osoba, 0,770 u bolesnika s okluzivnom arterijskom bolešću i 0,746 u bolesnika s dubokom venskom trombozom. Frekvencija alela C lokusa za MTHFR-1298 bila je 0,271 u zdravih osoba, 0,230 u bolesnika s okluzivnom arterijskom bolešću i 0,425 u bolesnika s dubokom venskom trombozom. Nije opažena povezanost polimorfizma MTHFR-677 i MTHFR-1289 s okluzivnom arterijskom bolešću ili dubokom venskom trombozom, nego se samo pokazao protektivni učinak diplotipa MTHFR/CA:CC za okluzivnu arterijsku bolest. Zaključak Osim protektivnoga učinka diplotipa MTHFR/CA:CC za okluzivnu arterijsku bolest, nismo našli značajnu povezanost polimorfizma lokusa MTHFR-677 i MTHFR-1289 s okluzivnom arterijskom bolešću i dubokom venskom trombozom.Aim To analyze the association of methylenetetrahydrofolate reductase polymorphisms (MTHFR-677 and MTHFR-1298) with occlusive artery disease and deep venous thrombosis in Macedonians. Methods We examined 83 healthy respondents, 76 patients with occlusive artery disease, and 67 patients with deep venous thrombosis. Blood samples were collected and DNA was isolated from peripheral blood leukocytes. Identification of MTHFR mutations was done with CVD StripAssay (ViennaLab, Labordiagnostika GmbH, Vienna, Austria) and the population genetics analysis package, PyPop, was used for the analysis. Pearson P values, crude odds ratio, and Wald’s 95% confidence intervals were calculated. Results The frequency of C alleles of MTHFR-677 was 0.575 in patients with deep venous thrombosis, 0.612 in patients with occlusive artery disease, and 0.645 in healthy participants. The frequency of T allele of MTHFR-677 was lower in healthy participants (0.355) than in patients with occlusive artery disease (0.388) and deep venous thrombosis (0.425). The frequency of A allele for MTHFR-1298 was 0.729 in healthy participants, 0.770 in patients with occlusive artery disease, and 0.746 in patients with deep venous thrombosis. The frequency of C allele of MTHFR-1298 was 0.271 in healthy participants, 0.230 in patients with occlusive artery disease, and 0.425 in patients with deep venous thrombosis. No association of MTHFR-677 and MTHFR- 1289 polymorphisms with occlusive artery disease and deep venous thrombosis was found, except for the protective effect of MTHFR/CA: CC diplotype for occlusive artery disease. Conclusion We could not confirm a significant association of MTHFR- 677 and MTHFR-1289 polymorphisms with occlusive artery disease or deep venous thrombosis in Macedonians, except for the protective effect of MTHFR/CA:CC diplotype against occlusive artery disease

ПРАКТИКУМ ЗА ГЕНЕТИКА ВО СПОРТ: Учебник за практична настава по генетика во спорт

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