Search

Transfusion associated graft versus host disease (TA-GvHD) is a rare complication of transfusion treatment with blood products containing viable lymphocytes. The risk of developing TA-GvHD depends on number and sustainability of lymphocytes in blood product, host’s immune system reactivity and the difference in human leukocyte antigen (HLA) system between donor and recipient. Standard method of TA-GvHD prevention is irradiation of blood products with ionizing radiation and absorbed dose of 25-50 Gy. The aim of irradiation is inactivation of viable lymphocytes in blood product containing cells (red blood cells, platelets, granulocytes) and lymphocyte proliferation inhibition in blood product recipient. In the last two decades, gamma irradiation has been extensively abandoned and replaced with specially designed cabinet machines using X-ray irradiation which are suitable for hospital and blood bank use. This reduces the risk of accidental or deliberate misuse of radioactive source, solves radioactive waste disposal issues and reduces ionizing irradiation exposure of employees in blood establishments. In Blood Bank of Croatian Institute of Transfusion Medicine, X-ray irradiation of blood products has been carried out since 2014 with a year rate of 20,000 irradiated cell blood products used in treatment of patients at risk of developing TA-GvHD.

Objectives The objective of this study was to show experience of the Croatian Institute of Transfusion Medicine in monitoring and analysing collection failures caused by the venepuncture technique or occurred as a result of adverse reactions and complications experienced by donors during donation. Background Collection failures represent one of the leading nonconformities in blood establishments. Apart from being a negative motivating factor for blood donors, they also affect the blood components supply and have a negative financial impact. Methods Nonconformity records referring to collection failures were analysed retrospectively over a 6-year period (2013-2018) with regard to their frequency, causes, donor characteristics (age, gender, number of donations), place of occurrence (blood establishment, mobile sessions) and trends during the analysed period. Results A total of 5166 collection failures out of 618 251 donations (0.84%) were recorded during the analysed period. The leading cause was haematoma at puncture site (1676, i.e., 32.4%). Collection failures which are primarily attributed to the venepuncture technique or vein selection accounted for 91% of all cases, whereas collection failures which occurred as a result of discontinued punctures due to adverse reactions in donors accounted for 9% of all cases. A much higher frequency of all collection failure types was recorded in female donors, whereas younger donors experienced adverse reactions more frequently (median age of 24). Conclusion The analysis and monitoring frequency of collection failures play an important role in planning of staff training activities, work organisation and timely implementation of corrective actions.

COVID-19 presentations range from cold-like symptoms to severe symptoms with the development of acute respiratory distress syndrome (ARDS). We report on a severe COVID-19 patient who was mechanically ventilated and who developed ARDS and bacterial infection. Because of rapid clinical deterioration and the exhaustion of other treatment options, the family and attending physicians requested a compassionate use of adult allogeneic bone marrow-derived mesenchymal stem cells (MSC) in addition to commonly used immunosuppressive, antiviral, and supportive therapy. The clinical course is discussed thoroughly, with a special emphasis on the safety and effect of MSC therapy. Compassionate MSC treatment, given in three rounds, affected ARDS regression. The patient was discharged from the intensive care unit after 31 days and from hospital after 49 days in a good general condition. MSC treatment was not associated with any side effects and was well tolerated in a three-week period; therefore, it should be studied in larger trials and considered for compassionate use

ABO blood group is a risk factor for several cancers, but it is not clear yet whether the risk of breast cancer is greater in particular ABO blood type carriers. The aim of this casecontrol study was to examine the correlation between ABO blood group genotypes, estrogen receptor (ER), progesterone receptor (PR) and HER2 status as tumor grade markers (I-III), and the occurrence of breast cancer. The research included 59 patients with invasive breast cancer and 80 asymptomatic, healthy women, blood donors. Genomic DNA was isolated using QIAampDNA Blood Mini Kit (QIAGEN, Germany). Genotyping was performed using in-house polymerase chain reaction with sequence-specific primers (PCR-SSP) method. Comparison of genotypes and phenotypes of ABO blood groups between patients and control group yielded p>0.05. There was no statistical significance of correlation between ABO genotypes/phenotypes in either patient group or control group. Testing the significance of different tumor grade occurrence, and ER, PR and HER2/neu status showed no statistical significance in the occurrence of a particular tumor grade, or in ER, PR and HER2/neu status as tumor markers in O1A1 genotype compared to non-O1A1 genotypes. Our study results confirmed that there was no correlation between ABO blood type genotypes/phenotypes and breast cancer in study groups., ABO sustav krvnih grupa povezan je s rizikom od nekih tumorskih bolesti, ali još uvijek nije razjašnjeno je li rizik od tumora dojke veći kod nositeljica određene ABO krvne grupe. Cilj istraživanja bio je ispitati povezanost između genotipova ABO krvnih grupa, zastupljenosti gradusa tumora (I-III) te biljega ER, PR i HER2/neu i razvoja tumora dojke. Istraživanjem je obuhvaćeno 59 bolesnica s invazivnim tumorom dojke i 80 zdravih žena, dobrovoljnih darivateljica krvi. Genomska DNA izolirana je pomoću komercijalnog testnog paketa QIAampDNA Blood Mini Kit (QIAGEN, Njemačka). Genotipizacija uzoraka izvedena je pomoću in-house PCR-SSP metode. Usporedbom genotipova i fenotipova ABO krvne grupe između bolesnica i kontrolne skupine dobiven je p>0,05 te nije bilo statističke značajnosti za povezanost ABO genotipova/ fenotipova između skupine bolesnica s tumorom dojke i kontrolne skupine. Ispitivanjem značajnosti razlike zastupljenosti gradusa tumora, statusa ER, PR i HER2/neu nisu dobivene statistički značajne vrijednosti za pojavu određenog gradusa tumora te statusa biljega ER, PR i HER2/neu kod genotipa O1A1 u odnosu na genotipove ne-O1A1. Rezultati studije potvrdili su da ne postoji povezanost između genotipova/fenotipova ABO krvnih grupa i tumora dojke kod ispitivanih skupina.

Manufacture of lead-containing products has long been associated with various health risks. To get an insight into the related genotoxic risks, we conducted a biomonitoring study in 50 exposed workers and 48 matched controls using a battery of endpoints that sensitively detect the extent of genome instability in peripheral blood lymphocytes. The levels of primary DNA damage were estimated with the alkaline comet assay, while cytogenetic abnormalities were determined with the cytokinesis-block micronucleus (CBMN) cytome assay. Additionally, CBMN slides of 20 exposed and 16 control participants were subjected to fluorescence in situ hybridisation (FISH), coupled with pancentromeric probes to establish the incidence of centromere-positive micronuclei, nuclear buds, and nucleoplasmic bridges. Blood lead levels (B-Pb) were measured with atomic absorption spectrometry. To further characterise cumulative effects of occupational exposure, we measured erythrocyte protoporphyrin (EP) concentrations and delta-aminolevulinic acid dehydratase (ALAD) activity in blood. We also assessed the influence of serum folate (S-folate) and vitamin B12 (S-B12) on genome stability. Compared to controls, occupationally exposed workers demonstrated significantly higher B-Pb (298.36±162.07 vs 41.58±23.02), MN frequency (18.71±11.06 vs 8.98±7.50), centromere positive MN (C+ MN) (8.15±1.8 vs 3.69±0.47), and centromere negative MN (C- MN) (14.55±1.80 vs 4.56±0.89). Exposed women had significantly higher comet tail intensity (TI) and length (TL) than control women. Furthermore, workers showed a positive correlation between age and nuclear buds and MN, between MN and years of exposure, and between S-B12 levels and TI and ALAD activity, while a negative correlation was found between TI and B-Pb. These findings suggest that occupational settings in the manufacture of lead-containing products pose significant genotoxic risks, which calls for developing more effective work safety programmes, including periodical monitoring of B-Pb and genetic endpoints., Profesionalna izloženost u industriji olova štetno utječe na zdravlje radnika. Ovo je istraživanje provedeno na 50 radnika izloženih olovu te 48 ispitanika kontrolne skupine primjenom testova za procjenu genomske nestabilnosti u limfocitima periferne krvi. Razine primarnoga oštećenja DNA procijenjene su alkalnim komet-testom, a citogenetičke abnormalnosti utvrđene su citohalazin blokiranim mikronukleus-testom (CBMN). Na podskupini od 20 izloženih i 16 kontrolnih ispitanika dodatno je proveden mikronukleus-test u kombinaciji s fluorescencijskom in situ hibridizacijom (MN-FISH). Primjenom pancentromernih sonda istražili smo učestalost mikronukleusa pozitivnih na centromere, nuklearnih pupova I nukleoplazmatskih mostova. Razine olova u krvi (B-Pb) izmjerene su atomskom apsorpcijskom spektrometrijom. Kako bismo utvrdili kumulativne učinke profesionalne izloženosti, izmjerili smo koncentracije eritrocitnoga protoporfirina (EP) i aktivnost dehidrataze delta-aminolevulinske kiseline (ALAD) u krvi. Također smo procijenili utjecaj serumskoga folata (S-folata) i vitamina B12 (S-B12) na stabilnost genoma. U usporedbi s podudarnim kontrolama, profesionalno izloženi radnici imali su značajno višu razinu B-Pb (298,36±162,07 vs. 41,58±23,02), učestalost MN-a (18,71±11,06 vs. 8,98±7,50), mikronukleusa pozitivnih na centromere (C+ MN) (8,15±1,8 vs. 3,69±0,47) i mikronukleusa negativnih na centromere (C- MN) (14,55±1,80 vs. 4,56±0,89). Izložene radnice imale su značajno veći intenzitet (TI) i duljinu (TL) repa u komettestu od ženskih kontrola. Nadalje, u radnika je utvrđena pozitivna korelacija između učestalosti jezgrinih pupova i MN-a s dobi, između MN-a i godina izloženosti te između TI-ja i aktivnosti ALAD-a i razina S-B12. Negativna korelacija utvrđena je između TI-ja i B-Pb. Dobiveni rezultati upućuju na to da profesionalna izloženost olovu predstavlja značajan genotoksični rizik, što zahtijeva razvoj učinkovitijih programa zaštite na radu, uključujući povremeno praćenje B-Pb i genetičkih markera.

Red blood cell autoantibodies (RBC autoAbs) of IgG class are found in the majority of patients with warm autoimmune hemolytic anemia (wAIHA) but sometimes also during the pretransfusion testing of patients with different diagnoses but without hemolysis. The aim of the study was to identify the main differences between these two groups of patients according to age, gender, subclass and titer of IgG RBC autoAbs and diagnosis.In the 9-year retrospective study, data were collected from records of 291 patients with IgG RBC autoAbs detected by gel technique, from which 111 with wAIHA.More than 85% of patients in both groups were over 40 years old, with male to female ratio 1:1.9 in wAIHA vs 1:1.3 in patients without hemolysis (P=0.0916). The main characteristics of patients with wAIHA vs patients without hemolysis were: IgG only 38% vs 70%, IgG+Complement 62% vs 30%, total IgG1 79% vs 55%, IgG1+IgG3 35% vs 11%, titer of 100 for IgG1+IgG3 17% vs 3% (P0.0001), respectively, while titer of 100 for IgG1 18% vs 9% (P=0.0241). The underlying diagnosis in wAIHA vs patients without hemolysis: hematologic disorders 41% vs 22% (P=0.0006), autoimmune disorders 12% vs 13% (P=0.8033), solid tumors 5% vs 14% (P=0.0154) and surgery procedures 6% vs 26% (P0.0001).We observed more wAIHA patients with high titer of IgG1 and high prevalence of IgG1+IgG3 and consider that patients without hemolysis having identical results might be interesting to find out how they are protected from damage by RBC autoAbs.

SUMMARY – Recently an increase has been reported in the number of HBV transmissions from anti-HBc positive blood donors that were repeatedly negative in HBsAg and nucleic acid testing using the most sensitive tests available. The aim of the study was to show the effect of anti-HBc antibody testing performed in 2006 on permanent deferral of voluntary blood donors (VBDs), and to estimate occult hepatitis B infection (OBI) rate in this population after the introduction of mandatory molecular testing in the 2013-2016 period. More than 30, 000 blood donations collected during the 2005-2007 period and more than 14, 000 VBDs having donated blood during the 2013-2016 period after the introduction of molecular testing from eastern Croatia were included in the study. Serologic testing was performed with HBsAg assay throughout the study period, and anti-HBc assay was only performed in 2006. As part of the confirmatory algorithm testing, all HBsAg positive and unclear results were tested with molecular tests. Anti-HBc prevalence among VBDs in 2006 was 1.5%, with a rate of 1:197, whereas HBsAg prevalence was stable from 2005 to 2007 (0.04%, 0.1% and 0.1%, respectively). The calculated OBI rate from 2013 to 2016 was 1:30, 250. Ten of 161 (12.4%) VBDs had serologic anti-HBc-only pattern. Anti-HBc testing in 2006 resulted in statistically more deferrals of VBDs compared to 2005 and 2007, and to the rest of Republic of Croatia. The strategy of universal anti-HBc testing of VBDs in addition to the existing HBsAg and molecular screening could be an additional measure to prevent HBV transmission by blood and blood components.

BACKGROUND: The aim of this study was to determine the distribution of ABO and RhD blood group phenotypes in the general population in the Republic of Croatia and among hospitalized patients with severe COVID-19. MATERIALS AND METHODS: Data on ABO and RhD blood groups of all blood donors in Croatia (who donated blood during the period 2015–2020) and patients and pregnant women tested at the Croatian Institute of Transfusion Medicine during the 2-year period, 2019–2020, were obtained from the e-Delphyn blood bank information system. A total of 614,673 results were analyzed in this group. The other group consisted of 780 COVID-19 patients hospitalized with severe COVID-19. Data are presented as total number and percentages and a comparison of proportions test was performed. RESULTS: The most frequent ABO phenotype in the general population is A (38%), followed by O (37%), B (18%) and AB (7%). RhD positive individuals accounted for 81% of the general population and RhD negative for the other 19%. Among COVID-19 patients, phenotype A was the most frequent (42%), followed by phenotypes O (32%), B (17%) and AB (9%). Thus blood group A was significantly more common among COVID-19 patients than among the general population, whereas blood group O was significantly less frequent. DISCUSSION: This study provides the first official results of the distribution of ABO and RhD blood group phenotypes in the general population in Croatia. Moreover, this study confirms other researchers’ observations about the predominance of the A blood group phenotype among COVID-19 patients.

We would like to use the opportunity to discuss some of thesafety concerns of convalescent COVID- 19 plasma, raised by Mung-munpuntipantip and Wiwanitkit in their letter. We agree with the authors that risk- benefit analyses for the use of convalescentplasma are of great importance and contribute to the improvementof patient safety.

Objectives. – The anti-HBc prevalence over a 14- year period (2004-2017), trends, infectivity, residual risk, and need for testing in blood donors (BD) of the Croatian Institute of Transfusion Medicine were assessed. Material and Methods. – Anti-HBc was tested in 19, 969 BD serum samples collected in 2004 (n=7, 561), 2013 (n=7, 318) and 2017 (n=5, 090). All serums were initially screened for HBsAg, anti-HCV, HIV Ag/Ab, and anti-TP. 2013 and 2017 samples were also tested by ID-NAT. Results. – Over a 14-years period, the anti-HBc prevalence significantly decreased among Croatian BD (5.90% in 2004, 2.56% in 2013, and 1.32% in 2017). Similarly, the prevalence of anti-HBc-only profiles decreased from 0.62% in 2004, 0.25% in 2013, and 0.21% in 2017. The 4-time decreasing trend was observed in all age groups of BD from 2017 but mostly among repeat donors (5.90% to 1.38%). First-time donors showed no significant difference in anti-HBc prevalence probably due to their younger age and HBV vaccine status. However, similar anti-HBs carriage rates (86.4%, 87.6%, and 82.1%) were reported in anti-HBc positive donors over the study period. HBsAg and HBV DNA were not detected. No OBI infection was found in the study despite an OBI frequency of 1:10, 900 donations previously reported in Croatia. A HBV decreasing residual risks of 68, 88, and 12 per million donations were estimated for years 2004, 2013, and 2017, respectively. Conclusion. – Anti-HBc testing is an additional measure of preventing HBV infection by transfusion. Implementation of anti-HBc testing will result in the deferral of 1.3% BD and should be supported by cost-benefit analyses.

BACKGROUND: Passive immunotherapy is the century- old practice of administering antibodies from an exposed convalescent or vaccinated person to a patient susceptible to the disease in question. Experience from prior outbreaks with other coronaviruses (SARS-CoV-1) shows that such convalescent sera contain neutralizing antibodies (NAb) against relevant virus and that their use was beneficial in the treated patients. Collection of COVID-19 convalescent plasma (CCP) at Croatian Institute of Transfusion Medicine (CITM) started in July 2020 and first unit for clinical use was issued in December. Clinicians in Croatia started using CCP in second wave of pandemics, mostly for patients with haematological malignancies. Such patients are not able to fight the SARS-CoV-2 infection by producing their own antibodies. CCP with individually checked levels of NAb, as an external source of antibodies, showed promising results in improving condition of these patients during SARS-CoV-2 infection. AIMS: To present the establishment of relevant methodology to properly evaluate SARS-CoV-2 neutralization capacity testing and to assess the correlation of donor disease severity and demographic characteristics with antibody titre level. METHODS: Each donor had a documented history of laboratory-confirmed SARS- CoV-2 infection. All plasma was donated by recovered and healthy COVID- 19 patients and collected by apheresis. In the beginning of the collection, titre of NAb was measured with the SARS-CoV-2 neutralization assay for quantification of SARS-CoV-2 NAb on Vero E6 cell suspensions and with home working stocks of SARS-CoV-2 virus prepared from a clinical isolate of the Laboratory working stock label SARS-CoV-2 297/20 Zagreb virus. In the meantime, the regression analysis for Vidas SARS-CoV-2 IgG test was done. This resulted in high correlation coefficient with neutralization assay and the regression equation has been used for the estimation of cut-off NAb level. High titre was determined as ED50/ml ≥ 1200. Data were collected from national transfusion IT system (e-Delphyn) and CCP donor checklist. Chi-squared, Kruskal- Wallis and Mann- Whitney tests were performed in MedCalc software for statistical analysis. RESULTS: We collected data for donations during the eight months period (28th July 2020 to 28th March 2021). 256 CCP units have been collected from 162 apheresis procedures. The donors’ age median was 36 (18-62) years. 30.4 % procedures were collected from women. The NAb median level of CCP for clinical use was 2560 (274 - 145, 218). The most common blood type was A (43%), followed by O (33%), B (12%) and AB (12%). COVID-19 symptoms in donors varied: asymptomatic (2.5%), mild (75%), medium (19.4%) and severe (3.1%). 75.4% of all donations had high NAb titre. Titre level did not statistically differ between genders (P = 0.262, Z = 1.12), severity of symptoms (P = 0.072, χ2 = 6.99) nor blood types (P = 0.842, χ2 = 0.83). Severity of disease did not statistically differ between genders (P = 0.23, χ2 = 4.26) nor blood types (P = 0.803, χ2 = 5.34). SUMMARY/CONCLUSIONS: 3/4 of collected CCP had high NAb titre. In rare cases, when the CCP supplies were scarce, physicians required issuing of CCP with lower titres. NAb titre was not dependent on gender, symptom severity nor blood type. Application of serological test performed in CITM highly correlated with the neutralization assay and allowed quicker issuing of CCP for clinical use which is very important for due time therapy of COVID-19 patients.

Objectives: The patients with hematological malignancies are a vulnerable group to COVID-19, due to the immunodeficiency resulting from the underlying disease and oncological treatment that significantly impair cellular and humoral immunity. Here we report on a beneficial impact of a passive immunotherapy with convalescent plasma to treat a prolonged, active COVID-19 infection in a patient with a history of nasopharyngeal diffuse large B-cell lymphoma treated with the therapy inducing substantial impairment of particularly humoral arm of immune system. The specific aim was to quantify SARS-CoV2 neutralizing antibodies in a patient plasma during the course of therapy. Materials and methods: Besides the standard of care treatment and monitoring, neutralizing antibody titers in patient's serum samples, calibrated according to the First WHO International Standard for anti- SARS-CoV-2 immunoglobulin (human), were quantified in a time- dependent manner. During the immunotherapy period peripheral blood flow cytometry immunophenotyping was conducted to characterize lymphocyte subpopulations. Results: The waves of clinical improvements and worsening coincided with transfused neutralizing antibodies rises and drops in the patient's systemic circulation, proving their contribution in controlling the disease progress. Besides the patient's lack of own humoral immune system, immunophenotyping analysis revealed also the reduced level of helper T-lymphocytes and immune exhaustion of monocytes. Conclusion: Therapeutic approach based on convalescent plasma transfusion transformed a prolonged, active COVID-19 infection into a manageable chronic disease.

Objectives Croatian Institute of Transfusion Medicine (CITM) implemented non-invasive fetal RHD genotyping as a request for targeted antenatal anti-D prophylaxis. The diagnostic performance of in-house RT-PCR method for fetal RHD genotyping and preliminary results are analyzed. Materials and methods Evaluation included results of RHD genotyping for 205 RhD negative pregnant women, 12–36th week of gestation, whose samples were received in period between 2015 and 2020. QIAsymphony SP DSP Virus Midi Kit was used for cffDNA extraction on QIAsymphony SP platform (Qiagen, Germany). Fragments of RHD exons 7 and 10 and later exon 5 were RT-PCR amplified. As internal controls, amplification of SRY gene or RASSF1A fragment and β-actin genes digested with BsTUI were used. Results We identified 70.72% (145/205) positive and 28.78% (59/205) negative fetal RHD genotypes. We had one inconclusive result (0.50%) due to the interference of maternal DNA with variant genotype RHD*09.02.00/01/*01N.01. When compared to newborns RhD phenotypes, no false negative and three false positive results (3/199, 1.50%) were observed. The test yielded 100% sensitivity and 95.08% specificity, while diagnostic accuracy was 98.48%. We were able to determine one case of fetal variant genotype RHD*04.04/*01N.01 inherited from the father. The negative and positive predictive test values were 100% and 97.86%, respectively. Conclusion Automated cffDNA extraction and RT-PCR amplification of fetal RHD exons 5,7,10 and fragments of SRY, RASSF1A genes represents highly reliable system for determining fetal RHD status which enables targeted antenatal anti-D prophylaxis. To obtain high specificity of cffDNA extraction, strict and thoroughly cleaning procedures are required.

Pulmonary embolism (PE) is a potentially life-threatening condition that mainly affects the people of advanced age. While certain blood group phenotypes (non‑O blood group) are known risk factors for the development of venous thromboembolism (VTE), there is no research which investigated the association of blood group genotypes with severity of PE. The aim of this study was to investigate the frequency of ABO blood group genotypes among the population of patients with PE and to investigate the correlation of the pulmonary embolism severity index (PESI) score to specific ABO blood group genotypes. In this cross-sectional study 74 patients with PE diagnosed using CT pulmonary angiography were included and 303 blood donors without VTE or congenital thrombophilia participated as a control group. After isolation of genomic DNA ABO blood group genotype was determined using the polymerase chain reaction sequence-specific amplification (PCR-SSP) method. We observed a significantly higher frequency of A1B and BB genotypes in patients with PE compared to healthy individuals (A1B 14.9% vs. 4.3%, P

Brain-derived neurotrophic factor (BDNF) and its tropomyosin-related kinase B (TrkB) receptor might contribute to normal lung functioning and immune responses, however, their role in asthma remains unclear. Plasma BDNF concentration, as well as BDNF and NTRK2 (TrkB gene) polymorphisms, were investigated in 120 asthma patients and 120 healthy individuals using enzyme-linked immunosorbent assay and polymerase chain reaction, respectively. The genotype and allele frequencies of BDNF Val66Met (rs6265) and NTRK2 rs1439050 polymorphisms did not differ between healthy individuals and asthma patients, nor between patients grouped according to severity or different asthma phenotypes. Although plasma BDNF concentrations were higher among healthy subjects carrying the BDNF Val66Met GG genotype compared to the A allele carriers, such differences were not detected in asthma patients, suggesting the influences of other factors. Plasma BDNF concentration was not affected by NTRK2 rs1439050 polymorphism. Asthma patients had higher plasma BDNF concentrations than control subjects, however, no differences were found between patients subdivided according to asthma severity, or Type-2, allergic, and eosinophilic asthma. Higher plasma BDNF levels were observed in asthma patients with aspirin sensitivity and aspirin-exacerbated respiratory disease. These results suggest that plasma BDNF may serve as a potential peripheral biomarker for asthma, particularly asthma with aspirin sensitivity.

During a two-year period (2001-2003), 464 patients were treated for tuberculosis at Jordanovac Department for Lung Diseases in Croatia. Besides pulmonary tuberculosis in 97.7% of patients, patients were also treated for tuberculous pleurisy (0.9%), tuberculous laryngitis (0.6%), tuberculous meningitis (0.2%), tuberculous pericarditis (0.2%) and urogenital tuberculosis (0.4%). Out of the total number of patients, 57.3% declared themselves to be active smokers (men were predominant and made up to 80.8%) and 20.9% to be active alcohol consumers. Both risk factors, i.e. smoking and alcohol consumption, were present in 15.1% of all patients. The most common comorbidities were diabetes mellitus (30.4%), cardiac diseases (11.2%) and chronic obstructive pulmonary disease (8.0%). Lung carcinoma was the most common malignant disease (n=51), with Mycobacterium tuberculosis isolated in 33% of them. Seventy-two of 464 (15.5%) patients had recurrences of tuberculosis. Of these, 30.5% had one of the risk factors (20.8% were smokers and 9.7% consumed alcohol), while 32.5% of patients had both risk factors. In conclusion, cigarette smoking was proved to be the most significant risk factor for development of pulmonary tuberculosis and its recurrence., Tijekom dvije godine (2001.-2003.) u Klinici za plućne bolesti “Jordanovac”, Zagreb, Hrvatska, od tuberkuloze je liječeno 464 bolesnika. Osim najčešće plućne tuberkuloze u 97,7% bolesnika, oboljeli su liječeni i od eksudativnog tuberkuloznog pleuritisa (0,9%), laringealne tuberkuloze (0,6%), tuberkuloznog meningitisa (0,2%), perikardijalne tuberkuloze (0,2%) te tuberkuloze koja je zahvatila urogenitalni sustav (0,4%). Od ukupnog broja bolesnika 57,3% ih se izjasnilo kao aktivni pušači (muškarci 80,8%), dok je 20,9% deklarirano kao aktivni konzumenti alkohola. Ukupno je 15,1% bolesnika imalo oba rizična čimbenika u anamnezi, tj. i aktivno pušenje cigareta i konzumaciju alkohola. Od komorbiditeta najčešća je bila šećerna bolest u 30,4% bolesnika, od srčanih bolesti bolovalo je 11,2% bolesnika, dok je kronična opstruktivna plućna bolest bila prisutna u 8% bolesnika. Karcinom pluća bio je najčešće zastupljen među malignim bolestima. Od ukupnog broja oboljelih od karcinoma pluća (51 bolesnik), Mycobacterium tuberculosis izolirali smo u 33% bolesnika. Recidivi tuberkuloze su zabilježeni u 72 (15,5%) bolesnika. Jedan rizični čimbenik imalo je 30,5% bolesnika: pušača je bilo 20,8%, dok je alkohol konzumiralo 9,7% bolesnika, a 32,5% bolesnika imali su oba rizična čimbenika. Zaključno, pušenje cigareta pokazalo se kao najznačajniji rizični čimbenik za razvoj plućne tuberkuloze, kao i za pojavu recidiva tuberkuloze.

Asthma is one of the most common chronic diseases in the world. Obesity is the most common comorbidity of asthma and is connected to incidence and course of the disease. Obesity is associated with non-allergic asthma phenotype, but this relation could be influenced by gender. The aim of our study was to determine the relationship between BMI and asthma and to explore possible gender differences.Study included 149 patients with asthma (examined group) and 153 healthy blood donors (control group). Data from the medical records of patients with asthma were used, and all included subjects had their BMI calculated using standard formula. Data were analyzed using descriptive statistics methods. Data with non-parametric distribution were analysed with Mann-Whitney U test and showed through medians with corresponding interquartile ranges. Statistical significance of BMI differences between non-allergic asthma, allergic asthma and control groups were analyzed by one-way analysis of variance - ANOVA. The results were interpreted at a significance level of P0.05.The comparison between median BMI values of two groups shows that examined group of patients with astma has significantly higher median BMI value in comparison with control group (P=0.035). Correlation was stronger for women than men (P=0.002 vs P=0.898). Incresed BMI of the examined group of patients with asthma was not asociated with non-allergic asthma (P=0.085). However, when stratified according to gender, there was a strong association of increased BMI with non-allergic asthma in women (P0.001).Patients with asthma in our study have higher BMI in comparison to healthy individuals, which contributes to hypothesis that BMI is a risk factor for development of asthma. We found that possible effect that BMI has on asthma is stronger in women, since there was a strong association between increased BMI and non-allergic asthma only in women.

Objectives The aim of this study was to determine RHESUS D GENE (RHD) allelic variants among Croatian D-negative blood donors and compare our results with respective data from other European countries. Background Altered or reduced D antigen expression can result in D variants, which can be mistyped and can lead to the alloimmunisation of the blood recipient. RHD genotyping can distinguish D variants: weak D, partial D and DEL, thus preventing alloimmunisation. Material/methods A total of 6523 samples obtained from D-negative Croatian donors were screened for the presence of RHD using the real-time polymerase chain reaction (PCR) method. PCR-SSP was performed for D variant genotyping by using commercial genotyping kits (Inno-Train, Kronberg, Germany). Genomic DNA sequencing for all 10 exons of the RHD was performed when the genotyping kits failed to assign a D variant. Results RHD molecular screening revealed 23 (0.35%) RHD-PCR positive samples, all C/E positive, in decreasing frequency: 11 hybrid RHD-CE (2-9) D-CE variants, 4 weak partial D type 11 and 2 weak D type 2. Six samples remained unresolved and were sequenced. For 12 of 23 samples (excluding large hybrids), an adsorption/elution of anti-D serum was performed, confirming that all 12 were RhD+. The calculated frequency of clinically significant D alleles in RhD-negative blood donors was 1:543 (0.18%) or 1:53 (1.89%) in C/E blood donors. Conclusion Data on the significant frequency of D variants among serologically D-negative blood donors in the north-eastern region of Croatia could help in introducing RHD molecular screening of blood donors in a routine workflow.

Aim ABO blood group genotypes are established as a genetic factor in pathophysiology of various diseases, such as cardiovascular disorders, cancers, infectious diseases and there is rising evidence of their involvement in other conditions. The aim of this study was to determine if ventilatory changes of lung function in asthma, measured by biomarkers/parameters, are connected to certain ABO blood group genotypes in Croatia. Methods A case-control study included 149 patients with asthma and 153 healthy individuals (blood donors). ABO genotyping on five main alleles was performed using PCR-SSP method. All patients had spirometry performed and severity of asthma was estimated. Clinical parameters of spirometry (FEV1, FEV/FVC, PEF), biomarkers FeNO, IgE and pO2 were measured. The χ2 test, Fisher's test, Kruskal-Wallis test and Spearman's correlation coefficients with p˂0.05 were used as statistically significant. Results There was no determined statistically significant difference in both ABO genotypes and phenotypes between patient and control groups. Comparison of the lung function in different ABO phenotypes in asthmatic patients also did not show any statistically significant differences in FEV1 values, FEV/FVC ratio or PEF. Statistically significant differences in oxygenation between different ABO blood types have not been noticed (p=0.326). Differences in quantitative values of biomarkers (FeNO and IgE) between different ABO blood phenotypes in patients with asthma were not significant, except for IgE that had marginal values (p=0.074). Conclusion No correlation was found between certain ABO blood group genotypes and parameters/biomarkers of ventilatory dysfunction in patients with allergic and nonallergic asthma.

SUMMARY – Asthma is a chronic inflammatory disease that is characterized by reversible obstruction of airways, bronchial hyper-reactivity and airway remodeling. The etiology of asthma is multifactorial, with inheritance playing an important role. The aim of our study was to investigate the importance of biomarkers of asthma and the role of plasminogen activator inhibitor-1 (PAI-1) gene as a genetic factor that could be involved in the pathogenesis of asthma. The research was conducted at Jordanovac University Department for Lung Diseases and Croatian Institute of Transfusion Medicine. The research included 149 patients with asthma and 89 healthy individuals. We collected demographic data of both study groups, determined asthma severity using GINA guidelines, and the values of biomarkers and PAI-1 by using laboratory techniques. Based on the results, we concluded that patients with allergic phenotype of asthma were younger, had better lung function and higher levels of IgE. By observing FeNO values, we were not able to distinguish asthmatic patients that had been diagnosed with obstruction of airways from asthmatic patients with normal lung function because FeNO indicates the inflammatory component of disease. The 4G/5G polymorphism of PAI-1 gene did not show any statistically significant difference in the distribution of 4G/4G, 4G/5G and 5G/5G between the group of asthmatic patients and control group.

Plasminogen activator inhibitor-1 (PAI-1) is a glycoprotein which has a role in tissue remodelling after inflammatory processes. The objective is to investigate the frequency of PAI-1 gene polymorphism (4G/5G) in patients with a lung ventilation dysfunction in asthma and allergic rhinitis. Genomic DNA was isolated and genotypes of polymorphism of PAI-1 4G/5G and ABO were determined using the methods of RT-PCR and PCR-SSP. Study group includes 145 adult patients diagnosed with chronic asthma, with all clinically relevant parameters and the laboratory markers of pO2, IgE and eosinophils in sputum and nasal swab. In the processing of data, appropriate statistical tests (Kolmogorov–Smirnov test, median, interquartile ranges, χ 2 and Mann–Whitney U tests) were used. Patients with symptoms of allergic rhinitis were significantly younger and had an almost four time higher levels of IgE (P = 0.001), higher pO2 (P = 0.002) and PEF (P = 0.036), compared to those who do not have these symptoms. Genotype PAI 4G/4G is significantly more common in patients with allergic rhinitis (28.1% vs. 16.1%; P = 0.017) compared to the genotype 5G/5G. Carriers of the genotype 4G/5G also have a borderline statistical significance. There were no statistically significant difference in the incidence of allergic rhinitis in the carriers of any ABO genotypes. The frequency of PAI genotype 4G/4G is significantly more common in patients with allergic rhinitis. The results suggest that the carriers of at least one 4G allele are at a higher risk for developing symptoms of allergic rhinitis in asthma.

Chronic obstructive pulmonary disease (COPD) is a chronic disease characterized by a progressive decline in lung function due to airflow limitation, mainly related to IL-1β-induced inflammation. We have hypothesized that single nucleotide polymorphisms (SNPs) in NLRP genes, coding for key regulators of IL-1β, are associated with pathogenesis and clinical phenotypes of COPD. We recruited 704 COPD individuals and 1238 healthy controls for this study. Twenty non-synonymous SNPs in 10 different NLRP genes were genotyped. Genetic associations were estimated using logistic regression, adjusting for age, gender, and smoking history. The impact of genotypes on patients’ overall survival was analyzed with the Kaplan–Meier method with the log-rank test. Serum IL-1β concentration was determined by high sensitivity assay and expression analysis was done by RT-PCR. Decreased lung function, measured by a forced expiratory volume in 1 s (FEV1% predicted), was significantly associated with the minor allele genotypes (AT + TT) of NLRP1 rs12150220 (p = 0.0002). The same rs12150220 genotypes exhibited a higher level of serum IL-1β compared to the AA genotype (p = 0.027) in COPD patients. NLRP8 rs306481 minor allele genotypes (AG + AA) were more common in the Global Initiative for Chronic Obstructive Lung Disease (GOLD) definition of group A (p = 0.0083). Polymorphisms in NLRP1 (rs12150220; OR = 0.55, p = 0.03) and NLRP4 (rs12462372; OR = 0.36, p = 0.03) were only nominally associated with COPD risk. In conclusion, coding polymorphisms in NLRP1 rs12150220 show an association with COPD disease severity, indicating that the fine-tuning of the NLRP1 inflammasome could be important in maintaining lung tissue integrity and treating the chronic inflammation of airways.

Background. - Seroprevalence of hepatitis E virus (HEV) in blood donors presenting to the Croatian Institute of Transfusion Medicine was assessed with 4 available tests (3 ELISA tests and 1 immunoblot (IB) test). Materials and Methods. - In October and November 2014, a total of 1,036 serum samples of blood donors were collected for the study. Samples were primarily tested for total HEV antibodies by Dia.Pro HEV Ab test (a). All reactive samples were tested by ELISA tests: Dia.Pro HEV IgG (b) and IgM (c), Mikrogen recomWell HEV IgG_old (d) and IgM_old (e), recomWell HEV IgG_new (f) and IgM_new (g), and IB Mikrogen recomLine HEV IgG (h) and IgM (i). HEV IgM reactive samples also positive by the IB were further tested for HEV RNA. Results. - There were 21.5% of samples reactive for total HEV antibodies (a). Seroprevalence of HEV IgG according to the b, d, f and h tests was 20.2%, 9.6%, 18.1% and 17.8%, respectively. Seroprevalence of HEV IgM according to the c, e, g and i tests was 4.4%, 1.5%, 2.0% and 1.7%, respectively. Out of 46 HEV IgM (Dia.Pro HEV IgM) positive samples, 18 (39.1%) were also positive by IB. HEV RNA was not detected in any of those samples. There was a significant association between age and HEV seroprevalence (P Conclusion. - Different HEV antibody detection assays showed a high HEV IgG seroprevalence in Croatian blood donors. Among HEV IgG and HEV IgM positive samples HEV RNA was not detected.

Chronic obstructive pulmonary disease (COPD) is a chronic disease characterized by a progressive decline in lung function due to airflow limitation, mainly related to IL-1&beta, induced inflammation. We have hypothesized that single nucleotide polymorphisms (SNPs) in NLRP genes, coding for key regulators of IL-1&beta, are associated with pathogenesis and clinical phenotypes of COPD. We recruited 704 COPD individuals and 1238 healthy controls for this study. Twenty non-synonymous SNPs in 10 different NLRP genes were genotyped. Genetic associations were estimated using logistic regression, adjusting for age, gender, and smoking history. The impact of genotypes on patients&rsquo, overall survival was analyzed with the Kaplan&ndash, Meier method with the log-rank test. Serum IL-1&beta, concentration was determined by high sensitivity assay and expression analysis was done by RT-PCR. Decreased lung function, measured by a forced expiratory volume in 1 s (FEV1% predicted), was significantly associated with the minor allele genotypes (AT + TT) of NLRP1 rs12150220 (p = 0.0002). The same rs12150220 genotypes exhibited a higher level of serum IL-1&beta, compared to the AA genotype (p = 0.027) in COPD patients. NLRP8 rs306481 minor allele genotypes (AG + AA) were more common in the Global Initiative for Chronic Obstructive Lung Disease (GOLD) definition of group A (p = 0.0083). Polymorphisms in NLRP1 (rs12150220, OR = 0.55, p = 0.03) and NLRP4 (rs12462372, OR = 0.36, p = 0.03) were only nominally associated with COPD risk. In conclusion, coding polymorphisms in NLRP1 rs12150220 show an association with COPD disease severity, indicating that the fine-tuning of the NLRP1 inflammasome could be important in maintaining lung tissue integrity and treating the chronic inflammation of airways.

Flavonoids are natural polyphenolic compounds present in a wide spectrum of plants that have a beneficial effect on human health. In the context of cardiovascular diseases related to plaque and thrombus formation, flavonoids exhibit an anti-aggregatory effect. Previously, it has been reported that all tested flavonoids exhibit an antiaggregatory effect on platelet aggregation when measured by impedance aggregometry on whole blood, in the test of aggregation induced by adenosine diphosphate (ADP). As not all flavonoids have the same targets within signaling pathways, an assumption of a common non-specific mechanism related to lipophilicity is to be considered. To test this hypothesis, reverse-phase thin layer chromatography was used to assess the lipophilicity of flavonoids; impedance aggregometry was used for testing of platelet aggregation and flow cytometry to monitor the influence of flavonoids on platelet activation. Lipophilicity analysis showed a highly negative correlation of logP and MINaAC for groups of flavones and flavanones. As determined by flow cytometry, the exposition of receptors necessary for the promotion of platelet activation and primary clot formation was diminished, i.e., lowered expression of the activated form of integrin αIIbβ3 was observed in the presence of flavanone. Platelet membrane stabilization by flavonoids as a mechanism of antiaggregatory effect has been supported by impedance aggregometry experiments when specific inhibitors of platelet aggregation signaling pathways (U73122, indomethacin, verapamil) were used in the presence of a weak (ADP) and a strong (TRAP-6) agonist of aggregation. While individual flavonoids can have specific targets within aggregation signaling pathways, all flavonoids share a common non-specific mechanism of platelet aggregation inhibition related to their lipophilicity and membrane stabilization that, to some extent, contributes to their antiaggregatory effect.

SUMMARYObjectives The aim of this study was to assess the appropriateness of using combined cell index (CCI) in the assessment of iron stores in blood donors. This index is calculated by the formula: red blood cell distribution width (RDW) × 104 × mean corpuscular volume (MCV)−1 × mean corpuscular haemoglobin (MCH)−1. Background Ferritin measurement is a reliable method for estimating iron stores in blood donors. The sensitivity of red blood cell (RBC) parameters of complete blood count in detecting non-anaemic iron deficiency is significantly lower. Consequently, there were several attempts to increase the detection sensitivity by combining these parameters in different indices. Methods This study included 1084 male and 792 female whole blood donors accepted for blood donation. For six RBC parameters with the highest level of correlation relative to ferritin [Hgb, MCV, MCH, mean corpuscular haemoglobin concentration (MCHC), RDW and CCI], diagnostic efficacy in the detection of iron depletion (ferritin

Hepatitis E (HEV) has become the topic in transfusion medicine a few years ago since its occurence is increasing in developed countries. Therefore there is a risk of viral transmission by blood. Blood donors in Croatia are not routinely screened for HEV and there are no data regarding HEV seroprevalence in the general population. We also had no trace-back for HEV. Aims were to estimate the seroprevalence of HEV among voluntary blood donors (VBD) in the Croatian Institute of Transfusion Medicine (CITM). In October and November 2014 a total of 1036 serum samples of VBD were collected. All donors had previously completed the medical questionnaire to verify that they fulfilled the criteria for blood donation and had given informed consent. The study was approved by the Ethics Committee of CITM. There were 913 (88, 1%) males and 123 (11, 9%) females. All of the testing was done according to the producer’s instructions. Samples were primarily tested for total HEV antibodies using a commercial enzyme immunoassay, HEV Ab, Dia.Pro Srl, Milan, Italy (performed on Gemini analyzer). All reactive samples were tested for HEV IgG/IgM by comercial immunoassays, HEV IgG/IgM, Dia.Pro Srl, Milan, Italy (performed on Gemini analyzer). All HEV IgM reactive samples were confirmed by a comercial immunoblot assay, recomLine HEV IgM, Mikrogen, Neuried, Germany (performed on Dynablot Plus analyzer). IgM-positive samples were further tested for the presence of HEV RNA. Viral RNA extraction (QIAamp viral RNA Mini Kit®, Qiagen, Hilden, Germany) was performed from 140 µl of each sera sample. A real time RT-PCR protocol (Jothikumar et al., 2006) for detecting a highly conserved fragment within ORF3 was carried out. The amplification was done in a Rotor-Gene Q machine (Qiagen, Hilden, Germany) by the use of commercially available kits (Rotor-Gene Probe RT-PCR kit, Qiagen, Hilden, Germany) according to the producer’s instructions. 223/1036 (21, 5%) samples were reactive for total HEV antibodies. 210/223 were reactive for HEV IgG and overall anti-HEV IgG seroprevalence was 20, 3%. 45/223 samples were anti-HEV IgM reactive, 18/45 (1, 7%) were confirmed by immunoblot HEV IgM test and HEV RNA was not detected in any of those 18 samples. There is a significant association between age (less and more then 40) and a higher seroprevalence (P

Introduction: The investigation of biomarkers could have a predictive value for the degree of asthma severity, occurrence of exacerbations and response to therapy. Aims: The aim was to investigate the diagnostic value of biomarkers in allergic and non- allergic asthma phenotype. Methods 149 adult asthmatic patients without asthma exacerbation were investigated. We collected information about patient9s age, measured biomarkers: total serum IgE (sIgE), Fractional exhaled nitric oxide (FeNO) and determined their Forced Expiratory volume in 1 second FEV1(L). Distribution of biomarkers between allergic and non-allergic phenotype were analysed by Kolmogorov-Smirnov test. Results: In univariate model, age, sIgE and FEV1 (L) were significantly related to allergic asthma phenotype. These patients are younger (median 56 years) than patients with non-allergic phenotype (median 62.5 years). Among people with allergic phenotype, mean sIgE and mean FEV1 (L) values (300.2kIU/L, 2.3L) are higher than among non-allergic asthma phenotype (60.1kIU/L, 1.8L). The FeNO values do not significantly differ between allergic (24.3 ppb) and non-allergic (15.6 ppb) asthma phenotype. When adjusted for all variables included in model, only sIgE level was significantly associated to the allergic asthma phenotype. Conclusion: We have found that allergic asthmatic patients are younger, have milder degree of asthma severity, better lung function and higher values of total serum IgE and FEV1 than non-allergic. Better lung function could be related to their better respond to treatment with inhaled corticosteroids and cornerstone therapy while patients with non-allergic phenotype are older and could have airways remodelling.

SUMMARY Objectives Results are presented of the statistical quality control of red cell concentrate buffy coat removed in additive solution (RCC/BC/AS) and red cell concentrate buffy coat removed and leucoreduced in additive solution (RCC/BC/LR/AS) produced at the Croatian Institute of Transfusion Medicine during an 8-year period (2005–2012). The aim was to assess quality conformity of these products with specified requirements, as well as the suitability and justification of current regulations on the minimal quality requirements. Methods The measurements of all the study parameters of the products analysed are expressed using descriptive statistics and graphs showing the distributions of observed parameters. Results In RCC/BC/AS, the mean (±SD) volume was 279 ± 17 mL; haematocrit, 0·60 ± 0·03 L L−1; haemoglobin content, 55 ± 5 g; leucocyte count, 0·65 ± 0·41 × 109; and haemolysis at expiry date, 0·16 ± 0·13%. In RCC/BC/LR/AS (post-production filtration), the mean (±SD) volume was 255 ± 14 mL; haematocrit, 0·60 ± 0·02 L L-1; haemoglobin content, 51 ± 4 g; leucocyte count, 0·11 ± 0·16 × 106; and haemolysis at expiry date, 0·11 ± 0·07%. In RCC/BC/LR/AS (inline filtration), the mean (±SD) volume was 254 ± 15 mL; haematocrit, 0·61 ± 0·02 L L−1; haemoglobin content, 51 ± 5 g; leucocyte count, 0·04 ± 0·06 × 106; and haemolysis at expiry date, 0·16 ± 0·10%. The standards were just met for leucocyte count in RCC/BC/AS (90%), whereas for all other parameters satisfactory results were obtained in at least 99% of products analysed. Total incidence of bacterial contamination was 0·23% for all products. Conclusion Results of the RCC/BC/AS and RCC/BC/LR/AS quality control showed very high conformity with the specified requirements in the majority of study parameters, suggesting that the current requirements could be redefined and improved at the institutional level.

The results of our study and a brief overview of the literature aim to draw attention to the impact of smoking on blood donors and their donations in order to improve the safety of donors and transfusion treatment.