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Background: FVIII and D-dimer are included in different prediction models to risk stratification for thrombotic recurrence or anticoagulation cessation. However, the optimal timing of their measurement related to the last drug dose is not sufficiently examined. Aims: To determine FVIII and D-dimer levels at peak and trough plasma concentrations of DOACs in order to define optimal timing of blood drawing. Methods: Concentrations of rivaroxaban (n=32), apixaban (n=24) and dabigatran (n=28), D-dimer levels and FVIII activities were measured at trough (before the next drug dose) and peak (two hours after drug intake) DOAC levels in circulation of outpatients during their regular control clinical examination. Rivaroxaban and apixaban were determined using specific chromogenic anti-FXa assay, dabigatran with Innovance DTI assay, FVIII with APTT-based coagulometric method (Actin FS/FVIII deficient plasma) and D-dimer by quantitative immunoturbidimetric assay using monoclonal antibody (Innovance D-dimer), all from Siemens Healthineers, Germany on BCSXP analyzer. Statistical analysis was done with Wilcoxon and Friedman tests. The study was funded by the Croatian Science Foundation as part of the research project IP-2016-06-8208. Results: In contrast to D-dimer, FVIII values were significantly higher at trough in comparison with peak dabigatran and rivaroxaban concentrations (P = 0.013 and 0.024), whereas for apixaban FVIII also showed a trend of higher values at trough, but without significant difference (P=0.850) (Table 1). Significantly higher values of both D-dimer and FVIII were measured at trough and peak drug levels of apixaban compared to dabigatran and rivaroxaban (Table 1, P*). Conclusion(s): Plasma concentration of DOACs significantly affects FVIII values, unlike D-dimer. These findings are important in use of these tests in assissting clinical decisions related to risk stratification schemes for recurrent thrombotic event or anticoagulation cessation. For FVIII measurement blood drawing should be performed at trough DOAC levels exclusively whereas D-dimer may be measured at both trough or peak drug levels in circulation.

Uvod: Cilj ovog istraživanja je bilo utvrđivanje granice detekcije odnosno Ct vrijednosti i broja kopija virusne RNA pomoću komercijalnog kontrolnog uzorka EURM-019 s poznatim brojem kopija kako bi se jasno mogli razlikovati pozitivni od negativnih rezultata SARS-CoV-2 metodom RT-qPCR te pouzdano izdavati rezultati testiranja. Materijali i metode: Komercijalni uzorak EURM- 019 s poznatim brojem kopija RNA od 107 kopija/ μL do 100 kopija/μL je korišten za potvrdu granice detekcije. Ukupno 54 uzorka brisa nazofarinksa pacijenata ; 29 očekivano pozitivnih i 25 očekivano negativnih uzoraka je analizirano prema određenoj granici detekcije. Izolacija virusne RNA napravljena je MagMAX Viral/Pathogen II Nucleic Acid Isolation Kit-om (Applied Biosystems) na uređaju za automatsku izolaciju nukleinskih kiselina KingFisher Flex (Applied Biosystems). Umnažanje SARS-CoV-2 je napravljeno na tri različite RT-PCR platforme: ABI7500 i QS5 (Applied Biosystems) i AriaMx (Agilent) Berlinskim protokolom detekcijom E i RdRp gena i komercijalnim kitom LiliF COVID-19 Real-time RT- PCR Kit detekcijom E, RdRp i N gena. Rezultati: Za određivanje granice detekcije, uz stopu detekcije veću od 95%, kao kriterij za pozitivan rezultat, postavljeno je da svi geni koji se mogu detektirati u PCR kitovima budu iznad praga osnovne fluorescencije s prisutnom krivuljom umnažanja. Utvrđena granice detekcije je bila 15 kopija/reakciji odnosno 3 kopije/μL i po Berlinskom protokolu i Lilifom, što je odgovaralo vrijednosti Ct 32 tj. Ct 31. Od 54 uzorka očekivano pozitivnih je bilo 29. Po postavljanju Ct < 32 tj. Ct < 31 po Berlinskom protokolu tj. Lilifu, 21 uzorak (21/29) je označen kao pozitivan rezultat za oba PCR kita, dok je 8 (8/29) uzoraka za oba kita bilo iznad Ct > 32 i rezultat je očitan kao negativan. Svi očekivano negativni uzorci bili su negativni. Zaključak: Postavljanje granice detekcije važno je za kvalitativne metode kao što je korištena RT-qPCR za razlikovanje SARS-CoV-2 pozitivnih od negativnih pacijenata te pouzdano izdavanje rezultata koji mogu smanjiti potrebu za re-testiranjem pacijenata.

Uvod: Za izolaciju DNA preporuča se svježi uzorak pune krvi. Hipoteza istraživanja je da se prinos, čistoća i/ili integritet DNA mijenjaju u različitim uvjetima pohrane. Cilj je ispitati utjecaj različitih uvjeta pohrane (temperatura i duljina pohrane) krvi na količinu i kvalitetu DNA. Materijali i metode: U pilot istraživanje su uključena 2 ispitanika kojima je krv uzorkovana u 6 Vacuette® K 3 EDTA spremnika (Greiner Bio-One), a DNA izolira- na kako slijedi: (i) odmah, (ii) nakon 1, 3, 7 i 15 dana pohrane na sobnoj temperaturi (ST), (iii) nakon 1, 3, 7, 15, 30 i 60 dana na 2-8 °C, (iv) nakon 15, 30 i 60 dana na - 20 °C. DNA je izolirana na kolonicama uz High Pure PCR Template Preparation Kit reagens (Roche). Prinos i čistoća su određeni mjerenjem apsorbancije na 260, 280 i 230 nm na DS-11 FX+ spektrofotometru (DeNovix Inc). Brojčana vrijednost integriteta (DIN) i veličina vrpce određeni su elektroforetski Genomic DNA ScreenTape reagensom na 4200 TapeStation System (Agilent Technologies). Genotipizacija MTHFR C677T mutacije je određena analizom krivulje taljenja na LightCycler v1.5 (Roche) uz TibMolbiol® reagens. Kriteriji prihvatljivosti uključuju sve navedeno: (i) prinos 3, 00-6, 00 μg, (ii) čistoću A260/A230 = 1, 8-2, 4 i (iii) A260/A260 = 1, 7-2, 0, (iv) integritet DIN ≥ 7, 0 i (v) veličinu vrpce > 48500 bp, (vi) uspješnost PCR umnažanja. Rezultati: Prinos je bio prihvatljiv za sve uvjete, osim kod pohrane na – 20 °C (< 3, 00 μg). Čistoća na A260/A280 je zadovoljavajuća za sve, dok su na A260/A230 neprihvatljivi uzorci izolirani nakon 30 i 60 dana na –20 °C (> 2, 4). Integritet i veličina vrpce nisu postignuti za uzorke izolirane nakon 7 i 15 dana na ST te 30 i 60 dana na 2-8 °C (DIN < 7, 0 ; vrpca < 48500 bp). U svim izolatima dobiveni su očekivani genotipovi (TT i CC). Zaključak: Dulja pohrana na ST i 2-8 °C dovodi do fragmentiranosti, a zamrzavanje rezultira manjim prinosom i smanjenom čistoćom DNA. Odabir uvjeta pohrane ovisit će o vrsti i specifičnosti primijenjenih metoda.

Tekuća biopsija omogućava nov neinvazivan pristup u molekularnom profiliranju tumora uključujući kolorektalni karcinom (CRC). Cirkulirajuća tumorska DNA (ctDNA) jedan je od obećavajućih biomarkera za probir, praćenje odgovora na terapiju, procjenu progresije i ishoda bolesti. Kako je rukovanje uzorcima tekuće biopsije zahtjevno, a nije standardizirano, prikazana je optimizacija metode za genotipizaciju JAK2 V617F u uzorcima ctDNA dobivenih tekućom biopsijom pacijenata s CRC-om. U sklopu projekta HRZZ IP- 2019-04-4624 prikupljena je periferna venska krv u epruvete CellSave (Menarini Silicon Biosistems) pri +4 °C od 25 pacijenata s CRC te je isti dan odvojena plazma i pohranjena na –20 °C. ctDNA je izdvojena u duplikatu iz 1–1, 5 mL plazme pomoću kompleta QIAamp ccfDNA/RNA Kit (Qiagen). Koncentracije ctDNA određene su fluorimetrijski pomoću uređaja DS-11 FX (DeNovix) i reagensa Qubit dsDNA HS Assay (Thermo Fisher Scientific). Genotipizacija JAK2 V617F u uzorcima ctDNA provedena je na uređaju 7500 Real-Time PCR System (Applied Biosystems) metodom alelne diskriminacije nakon lančane reakcije polimerazom u stvarnom vremenu (qPCR) uz komplet početnica i sondi TaqMan Digital PCR Liquid Biopsy Assay (Applied Biosystems) i uz dva isprobana reagensa za umnažanje TaqMan Genotyping PCR Master Mix i TaqMan Universal PCR Master Mix (Applied Biosystems). Kao kontrolni uzorci korišteni su uzorci genomske DNA iz leukocita genotipizirani akreditiranom metodom. Utvrđene su koncentracije ctDNA u rasponu od 0, 002 do 0, 993 ng/μL. Početno ispitivanje umnažanja i uspješnosti genotipizacije dalo je zadovoljavajuće rezultate za kontrolne uzorke ; vidljivo je umnažanje do 30. ciklusa, a očekivana diskriminacija alela nakon 40 ciklusa umnažanja bez obzira na korišteni reagens. Za uzorke ctDNA nije dobiveno uspješno umnažanje pa je ono nastavljeno tijekom još 20 ciklusa. Nakon 60 ciklusa umnažanja uspješno je očitan rezultat genotipizacije u uzorcima ctDNA umnoženih pomoću TaqMan Universal PCR Master Mix-a, dok reagens TaqMan Genotyping PCR Master Mix i dalje nije dao zadovoljavajuće umnažanje. Opisanom metodom uspješno je provedena genotipizacija JAK2 V617F za 24 od 25 prikupljenih uzoraka ctDNA iz periferne krvi pacijenata s CRC- om, iako prisutnost mutacije nije utvrđena niti u jednom uzorku. Dobiveni rezultati usporedit će se s rezultatima genotipizacije DNA izdvojene iz tumorskog tkiva i tako pridonijeti daljnjem profiliranju CRC-a korištenjem tekuće biopsije.

Background: Coronavirus disease 2019 (COVID-19) can be associated with severe hemostatic disorders due to profound changes in coagulation and fibrinolytic systems. Aims: To investigate activities of coagulation factors II (FII), V (FV), VIII (FVIII), X (FX) and XIII (FXIII) in COVID-19 patients with mild and severe disease presentation. Methods: Activities of FII, FV and FX were measured using PT-based coagulometric method (Innovin/factor deficient plasmas), FVIII using APTT-based coagulometric method (Actin FS/FVIII deficient plasma) whereas FXIII was determined by chromogenic method (Berichrom FXIII Assay), all performed on BCSXP analyzer (Siemens Healthineers, Germany). The differences between groups were tested with Man-Whitney test (statistically significant P-values < 0.05). Results: The study included 80 consecutive patients: 40 with mild disease admitted at general wards and 40 patients with severe disease admitted at intensive care units. Clinical data of the groups are presented in Table1. In contrast to other coagulation factors studied, acitivities of XIII were significantly decreased in severe COVID-19 patients compared to patients with mild disease in which FXIII levels were within reference range (Table 2). Among patients with severe COVID-19 disease, lower acitivites of FXIII in non-survivors (n =25) compared to survivors (n=15) were observed, although without statistically significant difference (P=0.056, Table 2). Conclusion(s): Our results indicate an acquired FXIII deficiency in COVID-19 patients with severe disease. Significant differences of FXIII levels between patients with mild and severe disease suggest that FXIII levels are related to disease severity and mortality. Simultaneously measurable differences with slight decrease of FII and FX acitivites in severe ill patients compared to those with mild desease suggest that consumption, as the consequence of previous coagulation activation, could be the cause of marked decrease of FXIII in severely ill patients. Further studies are required for elucidating the cause(s) of FXIII deficiency and its association with profound hemostatic disorders in critically ill COVID-19 patients.

Exosomes, extracellular vesicles, and their cargo, such as microRNAs, are among the most promising emerging biomarkers of various diseases, including colorectal cancer (CRC). Since their handling and isolation are not standardised, in this study we aimed to evaluate the performance of two different methods for isolation of microRNA from CRC patients' exosomes. Exosomes and exosomal microRNA were isolated from peripheral blood of 11 CRC patients using miRCURY Exosome Serum/Plasma kit and miRNeasy Serum/Plasma Advanced kit (Qiagen) or Total Exosome isolation reagent and Total Exosome RNA and Protein isolation kit (Invitrogen). Concentration of microRNA was determined using DS- 11 spectrophotometer (Denovix), and quality of the samples was determined on Bioanalyzer 2100 (Agilent). cDNA for microRNA expression analysis was obtained using miRCURY RT LNA kit (Qiagen), while expressions of mir-103a-3p and mir-19a-3p were assessed with miRCURY LNA SYBR GREEN PCR system (Qiagen) on 7500 Real-Time PCR System (Applied Biosystems) with UniSp6 as internal control. Wilcoxon matched pairs test was used for statistical analysis in GraphPad Prism 6.01 software. There was no significant difference in RNA concentrations between the two methods (P=0.102), and both methods isolated RNAs less than 200 nt in size. ΔCt (corrected to UniSp6) for mir-103a-3p (P=0.123) and mir-19a-3p (P=0.413) was similar between the two methods. mir-19a-3p was also corrected to mir-103a-3p (usually used as reference gene) and we found no difference between the used methods (P=0.083). We concluded that both assessed methods showed similar performance and could be used for determination of microRNA expression from CRC patients' exosomes.

Background: Great contribution to the quality improvement of laboratory service is review of patient feedbacks, which is also one of the ISO 15189 requirements. Measurement of service quality is variable, intangible and heterogeneous, so it is necessary to use models for measuring quality of service, which establish dimensions that affect patients' satisfaction. The aim of the study was to evaluate appropriateness of SERVPERF (Service Performance) model for measuring patient satisfaction with laboratory service in outpatient unit. Materials and methods: Modified SERVPERF questionnaire included 26 questions covering demographic data, overall satisfaction and following dimensions: reliability, tangibility, empathy, assurance and responsiveness. Participants were asked to rank their satisfaction with the Likert 5-point scale from very dissatisfied (1) to very satisfied (5). One open- ended question were added for additional comments. The questionnaire was sent via Survey Monkey© platform to the patients who requested delivery of laboratory report by an e-mail. Data were analyzed using Microsoft Excel. Results: Totally, 843 patients have answered on the questionnaire, out of which 78 % were women. The majority of the respondents have university education (53 %) and already have used laboratory service in our hospital (73 %). Overall satisfaction of the respondents were 4.1. The respondents were dissatisfied with the tangible dimension such as appearance and size of laboratory space (2.6 and 2.9), while the most satisfied were with the responsiveness such as professionalism and courtesy (4.5) and reliability (4.5). Further, respondents were very satisfied with the laboratory reports delivery options (4.6) as a part of empathy dimension. Open-ended question have shown great dissatisfaction with tangible dimension. Conclusion: Although, response rate have not been known, the study have shown that the patient satisfaction with laboratory service mainly depends on the reliability and responsiveness dimension. In our case, further improvement of laboratory service should be focused on arranging and extension of waiting room and blood collection spaces.

Uvod: Humana serumska paraoksonaza 1 (PON1) je kalcij ovisan enzim koji se sintetizira u jetri, a u krvi je vezan za HDL. Zbog antiaterogenog i antioksidacijskog djelovanja sve se više istražuje u kliničkim i epidemiološkim ispitivanjima. Arilesterazna aktivnost PON1 (ARE) određuje se pomoću supstrata fenilacetata i to najčešće iz uzorka seruma. Cilj ispitivanja bio je odrediti može li neki drugi uzorak osim seruma biti prikladan za određivanje ARE. Materijali i metode: ARE je određena kontinuiranim spektofotometrijskim mjerenjem oslobođenog fenola iz fenilacetata na 270 nm. Enzimska aktivnost mjerena je u uzorcima plazme (uzete na antikoagulanse EDTA, citrat i heparin) i u uzorku seruma 10 zdravih dobrovoljaca. Statistička obrada podataka provedena je statističkim programskim paketom SigmaStat 3.0 za Windows. Rezultati: Kruskall- Wallisov test pokazao je statistički značajnu razliku (P

Liquid biopsy offers a promising non-invasive approach for determination of the molecular profile of various tumours, including colorectal cancer (CRC). Exosomal microRNA represents one of most promising biomarkers for diagnostic screening, as well as for determining a response to therapy and evaluating disease progression and outcome. Since handling of liquid biopsy specimens is demanding and nonstandardized, in this study we aim to evaluate and optimise a method for determining selected microRNAs expression in exosome-derived microRNA samples from CRC patients. Exosomal microRNA was isolated from peripheral blood liquid biopsy samples obtained from 18 CRC patients using miRCURY Exosome Serum/Plasma kit and miRNeasy Serum/Plasma Advanced kit (Qiagen). cDNA was obtained using miRCury RT LNA kit (Qiagen) while expression of eight selected microRNAs was assessed with miRCURY LNA SYBR GREEN PCR system (Qiagen) on 7500 Real- Time PCR System (Applied Biosystems) with UniSp6 as internal control. Initial examination of expression of three microRNAs with four different starting sample volumes showed satisfactory results with the starting microRNA volume of 2 µl. miR-103a-3p was shown to be the best reference microRNA candidate, with Ct values ranging from 27.40 to 36.05 and ΔCt (corrected to UniSp6) from 13.36 to 22.31, since miR-1228-3p and miR-520d-3p were not detected. miR-125-3p was detected in three samples with Ct ranging from 33.58 to 35.86, and ΔCt from 20.01 to 22.40 ; miR-193a-3p was detected in seven samples with Ct ranging from 36.13 to 42.17, and ΔCt from 21.15 to 27.70 ; miR- 210-3p was detected in 14 samples with Ct ranging from 31.15 to 42.91 and ΔCt from 17.71 to 29.18, whereas miR-19a-3p and miR-92a-3p were detected in all samples with Ct ranging from 32.20 to 32.67 and 23.87 to 31.96, and ΔCt ranging from 9.90 to 16.74 and 10.35 to 16.97, respectively. The described optimisation of the method for the detection of selected exosomal microRNAs in samples obtained by the peripheral blood biopsy will contribute to the further profiling of CRC utilizing liquid biopsy through comparison with expression in tumour tissue samples.

Background: Rapid and accurate determination of direct oral anticoagulants (DOACs) is still a major medical need in urgent clinical situations. Aims: The aim of this study was to identify the feasibility of algorithm for rapid exclusion of clinically significant concentration of DOACs (>30 ng/mL) by data from in patients from cardiology and neurology departments. Methods: The study included 128 paired plasma and urine samples from patients treated for non-valvular atrial fibrillation of venous thromboembolism with an oral direct factor Xa inhibitor (DXI) (apixaban, n=31, rivaroxaban, n=53) and direct thrombin inhibitor (DTI) (dabigatran, n=44) at trough drug levels. Plasma DOACs concentrations were determined by quantitative chromogenic substrate assays (Innovance anti-FXa assay for rivaroxaban and apixaban and Innovance DTI assay for dabigatran) on BCSXP analyzer (Siemens Healthineers, Germany). Urine samples were evaluated by DOAC Dipstick test (DOASENSE GmbH, Heidelberg, Germany) to determine the presence or absence of DOACs. Parameters of diagnostic accuracy were determined by receiver operating curve (ROC) analysis. Comparison between DOAC Dipstick test results and plasma concentrations of DOACs were obtained using kappa statistics. The study was funded as a part of Croatian Science Foundation research project IP-2016-06-8208. Results: ROC analysis revealed threshold values of plasma concentrations for DXI ≥14 ng/mL and ≥19 ng/mL for DTI for detection of DXI and DTI by DOAC Dipstick, respectively. At cut-off value ≥19 ng/mL for DTI there was no false negative or false positive results (kappa value=1.0). For DXI at cut-off ≥14 ng/mL one false positive result was obtained, κ=0.92 (95 % CI 0.74-1.00). Conclusion(s): Application of an algorithm for exclusion of relevant plasma concentrations by DOAC Dipstick can be proposed by testing first DOACs in urine and if positive followed by quantitative DOAC assays if clinically required.

Uvod: U pandemiji COVID-19 pokazalo se da je jedan od mehanizama ulaska SARS-CoV-2 u stanice posredovan angiotenzin-konvertirajućim enzimima ACE i ACE2, čije varijante gena mogu utjecati na vezanje i ulazak virusa u stanice ili pojačati oštećenje zahvaćenih tkiva. Materijali i metode: U svrhu testiranja hipoteze o utjecaju varijanti ACE i ACE2 gena na težinu i tijek COVID-19 bolesti, ovim istraživanjem ispitali smo najčešće polimorfizme ACE i ACE2 gena (rs1799752, rs2285666, rs1978124 i rs3827466) u 231 nehospitalizirana bolesnika s blažim simptomima (djelatnici bolnice, medijan dobi 46 godina, 83% žena) i u 52 hospitalizirana bolesnika (sa teškim simptomima, medijan dobi 74 godine, 44% žena, od kojih je 22 preminulo). Pacijenti su podijeljeni u 2 skupine prema težini tijeka bolesti: A (asimptomatski, blagi i srednje teški tijek bolesti) nasuprot skupine B (teški i kritični simptomi). Između listopada i prosinca 2021., tijekom primitka na bolničko liječenje pacijentima je uzet bris nazofarinksa u svrhu testiranja na COVID-19. Alikvoti briseva čuvani su na + 4°C, i iz njih je izolirana stanična DNA (iz prisutnog epitela) te je učinjena genotipizacija (metodom real time PCR, LightCycler 1.5, Roche, Basel, Švicarska). Rezultati: Genotipizacijom ACE rs1799752, ACE2 rs1978124 i rs2285666 polimorfizma između ispitiva- nih skupina nisu utvrđene razlike, dok je za polimor- fizam ACE2 rs3827466 utvrđena značajna razlika iz- među kliničke slike bolesti za skupinu A (genotip AA: N = 40, AG: N = 84 i GG: N = 107) i skupinu B (genotip AA: N = 13, AG: N = 4 i GG: N = 35) ; chi-kvadrat, P < 0, 001. Razlika kliničke slike bolesti osobito je izražena u bolesnika mlađih od 60 godina A skupine (genotip AA: N = 35, AG: N = 76 i GG: N = 94) i B skupine (ge- notip AA: N = 0, AG: N = 0 i GG: N = 7) ; chi-kvadrat, P = 0, 019. Zaključak: Kako utjecaj ACE2 rs3827466 polimorfiz- ma na teži klinički tijek bolesti do sada nije eviden- tiran u sličnim istraživanjima, a u našem istraživanju veličina ispitivanih skupina nije ujednačena, ispitiva- nje je potrebno proširiti na veću skupinu bolesnika s težim kliničkim tijekom bolesti.

Uvod: Egzosomi su izvanstanične vezikule promjera 30-150 nm, bogate proteinima, RNA molekulama i dijelovima genomske DNA. Egzosomi imaju važnu ulogu u dijagnostici tumorskih bolesti primjenom tekuće biopsije, ali nanometarska veličina otežava njihovu izolaciju i karakterizaciju. Cilj ovoga rada bio je uspostaviti metodu za izolaciju egzosoma iz plazme bolesnika s kolorektalnim karcinomom (CRC). Materijali i metode: Plazma je dobivena centrifugiranjem pune krvi 11 bolesnika s CRC-om. Izolacija egzosoma provedena je korištenjem dva komercijalno dostupna kompleta, miRCURY Exosome Serum/Plasma Kit (Qiagen, Njemačka) ili Invitrogen Total Exosome Isolation Kit (from plasma) (ThermoFisher Scientific, SAD). Egzosomi su lizirani RIPA puferom i sonikacijom. Koncentracija proteina utvrđena je kolorimetrijskom metodom s bicinkoniničnom kiselinom ili mjerenjem apsorbancije na 280 nm korištenjem DS-11 spektrofotometra (DeNovix, SAD). Uzorci egzosoma pripremljeni su za SDS-PAGE miješanjem s puferom za pripremu uzoraka (s ili bez β-merkaptoetanola). Karakterizacija je provedena Western blot metodom uz upotrebu mišjih monoklonskih antitijela za CD9, HSC70, aktin i kalneksin (Invitrogen, SAD). Rezultati: Određivanjem koncentracije proteina kolorimetrijskom metodom dobivena je zadovoljavajuća preciznost u seriji (7, 91 %), točnost (96, 53 %) i linearnost (r = 0, 9964), dok rezultati dobiveni mjerenjem apsorbancije na 280 nm nisu bili prihvatljivi zbog nemogućnosti otklanjanja interferencije RIPA pufera. Sveukupni rezultati volumena i broja egzosoma te određivanja koncentracije proteina u egzosomima i njihovim vizualnim očitanjem na gelu za SDS-PAGE razvidno je da Qiagenov komplet daje bolji prinos egzosoma od ThermoFisherovog kompleta. U svim je uzorcima egzosoma Western blotom dokazana prisutnost njihovog biljega CD9, uz nereducirajuće uvjete (bez β-merkaptoetanola). U pojedinim je uzorcima dobiven signal za HSC70, a niti u jednom uzroku nije dokazan aktin. Odsutnost endoplazmatskog retikula u uzorcima dokazana je izostankom vrpce karakteristične za kalneksin. Zaključak: Pokazano je da je za izolaciju egzosoma primjeren Qiagenov komplet, a za određivanje koncentracije proteina u uzorcima egzosoma s RIPA puferom kolorimetrijska metoda s bicinkoniničnom kiselinom.

Background: Knowledge related to expected peak concentrations of apixaban in children and adolescent populations is very scarce, since it has only recently been approved for treatment in pediatric population. Aims: To compare peak plasma concentrations of apixaban in adolescents and adults. Methods: Innovance heparin (Siemens Healthineers, Germany) calibrated with apixaban calibrators (Hyphen BioMed, France) was used for quantitative determination of apixaban concentration. The study included plasma samples from adult patients (N=80 ; 47-89 yrs) treated with standard dose (2×5 mg/day) and from adolescents (N=59 ; 13-22 yrs) treated with two dosing regimens: 2×2.5 mg/day (N=38) and 2×5 mg/day (N=21). Blood samples were taken two to four hours after drug administration in order to obtain peak apixaban concentrations. The study was funded by the Croatian Science Foundation as part of the research project IP-2016-06-8208. Statistical analysis was done using Wilcoxon test by MedCalc Statistical Software version 11.5.1. Results: Concentrations of apixaban showed significantly lower peak values in adolescents treated with 2×2.5 mg/day compared with those treated with 2×5 mg/day (P=0.003), as well as compared with adults treated 2×5 mg/day (P

Background: In special clinical conditions quantitative measurement of direct oral anticoagulants (DOACs) in plasma, should be performed to help clinical decision making and treatment. Aims: To present a case report of a patient treated with dabigatran in whom low peak drug concentrations in plasma suggested inadequate anticoagulation. Methods: A 88 years old male was hospitalized due to recurrent symptoms of cerebral ischemia for last few days and brain ischemia was confirmed on CT scan. Before hospitalization, the patient was taking dabigatran (110 mg twice daily) due to persistent atrial fibrillation. Dabigatran was measured by Innovance DTI assay and rivaroxaban was measured by anti-FXa assay on BCSXP analyzer (Siemens Healthineers, Germany). The study was funded by the Croatian Science Foundation as a part of the research project IP-2016-06-8208. Results: Peak (two hours after the last dose) concentrations of dabigatran measured at three consecutive days were 35, 16 and 19 ng/mL, whereas trough (before the next dose) concentrations were and 34

Drug-specific therapeutic approaches for colorectal cancer (CRC) have contributed to a significant improvement in the health status of patients. However, a great need to improve personalization of treatments based on genetic and epigenetic tumor profiles to maximize quality and efficacy while limiting cytotoxicity remains. Currently, CEA and CA 19- 9 are the only validated blood biomarkers in clinical practice. For this reason, laboratories are trying to identify new specific prognostic and, more importantly, predictive biomarkers for CRC patient profiles. Thus, the unique landscape of personalized biomarker data should have a clinical impact on CRC treatment strategies and molecular genetic screening tests should become the standard method for CRC diagnosis, as well as detection of disease progression.

Background: As Other Coagulation Thrombophilia Assays, Testing of Protein S (PS) Activity Can also Be Influenced and Misinterpreted in Patients on Direct Oral Anticoagulants (DOACs) Aims: To investigate the effect of DOACs on PS activity results and to evaluate the use of our own optimized method with activated charcoal (AC) in removing potential interference. Methods: The study included 45 plasma samples from patients treated with three DOACs (Table 1). DOACs concentrations were determined in native plasma. Then, using our method earlier optimized for other thrombophilia assays, 100 mg of medical AC powder (Jadran Galenski laboratorij, Rijeka, Croatia) was added to 500µL plasma allowing DOACs adsorption for 10 minutes. Treated samples were centrifuged 20 minutes at 1800xg and DOACs concentrations and PS activity were measured in supernatant. Rivaroxaban and apixaban were determined using commercial anti-FXa assay (Siemens Healthineers, Germany) with drug specific calibrators (Hyphen Biomed, France). Dabigatran was measured using Innovance DTI assay (Siemens Healthineers, Germany). Protein S activity was determined using coagulometric method (Protein S Ac, Siemens Healthineers, Germany). All measurements were performed on BCSXP analyzer (Siemens Healthineers, Germany). Statistical analysis was done using Wilcoxon test by MedCalc Statistical Software version 11.5.1 (MedCalc Software, Belgium). The study was funded as part of Croatian Science Foundation research project IP-2016-06-8208. Results:All three DOACs have shown significant interfering effect on PS activity results in terms of false increased values. In 37/45 of native plasma samples, results of PS activity were above upper limit of measuring range (>130%) with significantly higher values in native than in AC treated samples (P=0.0001) (Figures 1 and 2). Conclusions: All DOACs have strong interfering effect on protein S activity in terms of possible false negative result. Our optimized method using medical AC has been found effective in overcoming interference of DOACs on PS activity testing.

Background: Direct oral anticoagulants (DOACs), i.e. dabigatran, rivaroxaban and apixaban, have strong effect on dilute Russell viper venom screen (dRVVTs) and confirm (dRVVTc) test results. Therefore, it is possible to assume potential application of these assays in exclusion of DOACs presence in circulation. Aims: To investigate whether normal dRVVT test result expressed as calculated lupus anticoagulant (LA) ratio i.e., dRVVTs/dRVVTc, can exclude clinically relevant concentration (>30 ng/mL) of DOACs. Methods: DOACs concentrations were measured using commercial assays on BCS XP analyzer (Siemens Healthineers, Germany): Innovance heparin (Siemens Healthineers, Germany) calibrated with specific calibrators (Hyphen BioMed, France) for rivaroxaban and apixaban ; Innovance DTI assay (Siemens Healthineers, Germany) for dabigatran. LA ratio was calculated from measured dRVVTs and dRVVTc tests with commercial LA1 Screening and LA2 Confirmation reagents using original protocols (Siemens Healthineers, Germany). Differences between groups were tested using Kruskal-Wallis test. Receiver operating characteristics (ROC) analysis was performed for diagnostic accuracy estimation at cut-off value of 30 ng/mL for each DOACs. The study was funded by the Croatian Science Foundation as part of the research project IP- 2016-06-8208. Results: LA ratios showed significant differences between false positive (FP) and true negative (TN) LA results (P< 0.001) for all three DOACs (Table 1). Among patients with FP and TN results of LA, concentrations of DOACs were significantly different for rivaroxaban only (P< 0.001). ROC analysis confirmed that normal LA ratio result can´t exclude concentration below 30 ng/mL for dabigatran and apixaban (area under curve (AUC=0.625, P>0.05), whereas normal LA ratio could exclude rivaroxaban concentration below 30 ng/mL, AUC=0.967, P< 0.001 (Table 1). Conclusions: Based on the normal dRVVT result (LA ratio), it is not possible to exclude low concentration of dabigatran and apixaban. On the contrary, normal dRVVT (LA ratio) could be potentially useful for excluding low concentration of rivaroxaban.

Background: Clinical application of dabigatran does not require routine therapeutic monitoring. However, there are special clinical situations in which measurement of dabigatran concentration in circulation should be performed using specific quantitative methods. Aims: The aim of this study was to compare two quantitative methods for dabigatran concentration i.e. commercial chromogenic Innovance Dabigatran assay (DTI) and in house optimized coagulometric diluted thrombin time (dTT) test. Methods: Dabigatran concentrations were determined in 38 patients using two methods on BCS XP coagulation analyzer (Siemens Healthineers, Germany): Innovance Dabigatran assay (Siemens Healthineers, Germany) with drug specific calibrators (Siemens Healthineers, Germany) and dTT test as an in house applied protocol with thrombin time reagent (BC Thrombin, Siemens Healthineers, Germany) calibrated with commercial dabigatran calibrators (Diagnostica Stago, France). Passing-Bablok regression and Bland-Altman methods were used for method comparison and Wilcoxon test was used to test differences between pairs of samples.The study was funded by the Croatian Science Foundation as part of the research project IP- 2016-06-8208. Results: Passing and Bablok regression analysis (y=-7.653 (-15.085 to -2.782) +0.986x (0.939 to1.033) revealed constant difference between two methods (Figure 1). Bland-Altman analysis showed statistically significant mean difference of 7.7% (P< 0.001) between dTT assay compared with DTI. Further, comparison of the results for dabigatran concentrations using DTI and dTT assays showed statistically significant difference (P=0.0002), but without clinically significant difference (Table 1). Conclusions: Although two evaluated methods for quantitative determination of dabigatran concentration showed significant constant difference, that difference is not clinically significant, so both can be used interchangeably. However, commercial DTI assay could be recommended as a method of choice for quantitative measurement of dabigatran concentration, since it has much wider measurement range without sample dilution (up to 500 ng/mL) compared to dTT assay (up to 256 ng/mL), uses chromogenic principle and time of testing is much shorter.

Background: Although clinical application of apixaban does not require coagulation monitoring, there are clinical situations in which measurement of drug concentration should be performed. Anti-activated factor Xa (anti- FXa) inhibition methods for all anti-FXa drugs are based on the same principle, so there are attempts to evaluate potential clinical application of heparin-calibrated anti-FXa assay as an alternative method for direct FXa inhibitors. Aims: To assess whether commercial low molecular weight heparin (LMWH)-calibrated anti-FXa assay can be used for assessing anticoagulant effect of apixaban in adolescents. Methods: LMWH-calibrated anti-FXa method (Innovance heparin, Siemens Healthineers, Germany) was used for measurement of LMWH anti- FXa activity. Innovance heparin (Siemens Healthineers, Germany) calibrated with apixaban calibrators (Hyphen BioMed, France) was used for quantitative determination of apixaban concentration. Agreement between LMWH and apixaban calibrated anti-FXa assays was tested using kappa statistics whereas receiver operating characteristics (ROC) analysis was performed for LMWH therapeutic range. The study was funded by the Croatian Science Foundation as part of the research project IP-2016-06- 8208. Results: In 36 plasma samples of 11 adolescents (13-22 years), concentrations of apixaban ranged from 20 ng/mL to 267 ng/mL (median 94 ng/mL). LMWH anti-FXa activity ranged from 0.11 to 1.50 (upper LMWH measuring range) (median 0.68 IU/mL) (Table 1). Inter-rater aggrement analysis showed poor agreement (κ=0.16 ; 95%CI – 0.0327 to 0.3386) between therapeutic range of LMWH-calibrated anti-FXa activity and clinically relevant apixaban concentrations (>30 ng/mL) (Table 2). ROC analysis showed that only apixaban concentrations between 57 and 121 ng/mL could be expected with certainty within therapeutic range for LMWH anti-FXa activity. Cut-off value for LMWH anti-FXa activity for apixaban concentrations < 30 ng/mL is 0.12 IU/mL. Conclusions: LMWH anti-FXa activity assay is not appropriate method for quantitative estimation of apixaban concentration and should not be used as an interchangeable method. Quantitative measurement of apixaban concentration should be performed in all clinical situations.

Ishemijski moždani udar je multifaktorijalna bolest koja se razvija međudjelovanjem genske osnove i okolišnih čimbenika rizika. U nastanak i razvoj moždanog udara uključeni su sustavi zgrušavanja i fibrinolize, upalnog odgovora, metabolizma lipida, regulacije krvnog pritiska. Do sada je znanstvenoj literaturi opisano više desetaka potencijalnih genskih čimbenika rizika za nastanak moždanog udara, ali je njihova pojedinačna povezanost s moždanim udarom slaba. Ciljevi ovog istraživanja bili su ispitati: učestalost varijanti F5 Leiden, G20210A gena F2, V34L gena F13A1, -445G/A gena FGB, 5G/4G gena SERPINE1, I/D gena ACE i -1131T/C gena APOA5 u zdravih ispitanika i ispitanika s ishemijskim moždanim udarom; razliku u koncentraciji lipidnih parametara (ukupni kolesterol, HDL- i LDL-kolestrol i trigliceridi) i parametara upale (CRP i broj leukocita); povezanost genskih čimbenika, lipidnih parametara i parametara upale s nastankom moždanog udara. U istraživanje je uključeno 250 ispitanika kojima je dijagnosticiran ishemijski moždani udar na osnovu kliničkog pregleda i kompjuterizirane tomografije i 100 zdravih ispitanika regrutiranih tijekom sistematskog pregleda bez podatka o cerebrovaskularnom i/ili kardiovaskularnom incidentu u osobnoj anamnezi. Svim ispitanicima su natašte uzeti uzorci krvi za genotipizaciju i određivanje lipidnog statusa, CRP-a i broja leukocita. Rezultati su pokazali da ispitanici s moždanim udarom imaju značajno niže koncentracije ukupnog kolesterola i LDL-kolesterola i značajno više koncentracije glukoze i CRP-a te značajno veći broj leukocita u odnosu na kontrolne ispitanike. Varijanta I/D gena ACE povezana je s težinom moždanog udara, a varijanta -1131 T/C gena APOA5 je uz dob i pretilost neovisan čimbenik nastanka teškog ishemijskog moždanog udara. Nije pronađena razlika u učestalosti ispitivanih genskih čimbenika između ispitanika s moždanim udarom i kontrolnih ispitanika. Stroke is a multifactorial disease developing through the complex interaction of both genetic and environmental risk factors. Initiation and progression of stroke involves disorders in coagulation cascade, fibrinolytic system, inflammatory response, lipid metabolism and blood pressure homeostasis. Until today a large number of potential genetic risk markers have been studied but their individual association with stroke is weak. We aimed to investigate: the prevalence of F5 Leiden, F2 G20210A, F13A1 V34L, FGB -455G/A, SERPINE1 5G/4G, ACE I/D and APOA5 -1131T/C variants in healthy subjects and subjects with acute ishemic stroke; differences in blood lipid concentrations (total, HDL- and LDL-cholesterol and triglycerides) and inflammatory markers (CRP and white blood cell count); association of genetic variations, blood lipids and inflammatory markers with the ischemic stroke risk. The study included 250 subjects with acute ishemic stroke which was diagnosed by clinical examination and computerized tomography and 100 healthy subjects without known cerebrovascular and/or cardiovascular incidents in anamnesis regruted during general medical examination. Blood samples for genotyping and determination of blood lipids, CRP and WBC count were drawn to all subjects in fasting state. Results have shown that subjects with acute ishemic stroke have significantly lower concentrations of total and LDL-cholestrol whereas concentrations of glucose, CRP and WBC are significantly higher than in control subjects. ACE variant I/D has shown association with the stroke severity. APOA5 variant -1131T/C along with age and obesity is independent predictor of severe stroke. There are no differences in the frequencies of the investigated genetic variants between studied groups of subjects.

Background: Treatment with direct oral anticoagulants (DOACs) has significant interfering effect on results of many specialized coagulation tests, such as thrombophilia assays, due to possible false negative (FN) or false positive (FP) results. Aims: To investigate the impact of DOACs (dabigatran, rivaroxaban and apixaban) on thrombophilia assays: resistance to activated protein C (APCR), coagulation factor VIII (FVIII) and antithrombin (AT) activities and to evaluate the efficiency of our own optimized method using activated charcoal (AC) in removing interference. Methods: DOACs concentrations and thrombophilia assays were firstly determined in native patient plasma samples (total n=65). Secondly, 100 mg of medical AC powder (Jadran Galenski laboratorij, Rijeka, Croatia) was added to 500 µL plasma allowing DOACs adsorption for 10 minutes. Treated samples were centrifuged 20 minutes at 1800xg and all measurements were repeated in supernatant. All assays were performed using commercial methods on BCSXP analyzer (Siemens Healthineers, Germany): Innovance anti-FXa assay with drug specific calibrators for rivaroxaban and apixabann (Hyphen BioMed, France) ; Innovance DTI assay for dabigatran ; ProC Global for APCR ; one-stage coagulation assay for FVIII and Innovance Antithrombin for AT activities. Wilcoxon test was used to test differences between pairs of samples. The study was funded by the Croatian Science Foundation as part of the research project IP- 2016-06-8208. Results: All three DOACs have shown significant and different interfering effect on particular thrombophilia assays evaluated in this study (Table 1), indicating that most of these assays could not be performed in patients on DOACs, and suggesting the effective solution in removing interference by application of procedure with AC (Figure 1). In AC treated samples, concentrations of DOACs were below limit of detection. Conclusions: AC has been found an effective in vitro agent to overcome effect of dabigatran, rivaroxaban and apixaban on particular thrombophilia assays, confirming potential of AC application before testing in patients treated with DOACs.

TA was associated with higher PAI-1 activity rise in acute STEMI patients treated with primary PCI. The clinical implications of this association should be tested in larger studies.

Background: Although clinical application of dabigatran does not require routine monitoring, there are special clinical situations in which laboratory measurement of drug concentration in plasma should be performed. In literature, there are increasing evidence of case reports of serious bleeding on dabigatran in the elderly, especially in those who develop acute renal failure. Aims: To present three patients treated with dabigatran in which quantitative measurement of dabigatran concentrations in plasma have confirmed as very effective approach in clinical decision making and treatment. Methods: Case 1: 85 years old female presented at consultant neurologist office with multiple skin hematomas. Case 2: 85 years old female presented at Emergency Department (ED), referred from general practitioner with severe normocytic anemia and clinical symptoms of melena/hematochezia and chronic renal insufficiency in anamnesis. Case 3: 89 years old male presented at ED in soporous state, with severe hypotension as a result of afterwards diagnosed aortic dissection. Dabigatran was prescribed by cardiologist to all patients, in 110 mg twice daily regimen. Concentrations of dabigatran were measured by Innovance DTI assay on BCSXP analyzer (Siemens Healthineers, Germany). The study was funded by the Croatian Science Foundation as a part of the research project IP- 2016-06-8208. Results: Case 1: Peak concentrations of dabigatran were 473, 373 and 446 ng/mL, while trough concentrations were 257, 237 and 292 ng/mL, respectively. Case 2: Dabigatran concentration in random sample was 1269 ng/mL. Case 3: Dabigatran concentration in random sample was 968 ng/mL. Results of screening coagulation assays and dabigatran concentrations are presented in Table 1. Conclusions: In all three cases, measurement of dabigatran concentration directed further clinical decision making and treatment. In all three cases dabigatran treatment was immediately discontinued. In Cases 1 and 2 treatment was continued with apixaban, while in Case 3 surgical treatment was not done due to high risk of bleeding.

Background: It has already been shown that some antiepileptic drugs, especially valproic acid (VA) and its derivatives, alter platelet function. However, little is known about effect of dual antiepileptic therapy and/or combination of antiepileptic drugs with antiplatelet therapy such as acetylsalicylic acid (ASA). Aims: To investigate an effect of mono and dual antiepileptic therapy and in combination with ASA applied as an antiplatelet drug. Methods: The study included 102 patients using antiepileptic therapy. Testing was done on Sysmex CS2500 analyzer (method: light transmission aggregometry (LTA)) using ADP (2 µmol/L), epinephrine (5 µmol/L) and collagen (2 µg/mL) as agonists (all HYPHEN BioMed, France). Platelet rich plasma and platelet poor plasma were obtained from 3.2% citrate plasma centrifuged at 500 xg/10 minutes and 2000 xg/10 minutes, respectively. The differences between subgroups were tested using Kruskal-Wallis test. Results: LTA results were: ADP 50% (44-74) ; epinephrine 54% (41-78) and collagen 71% (43- 79). One third (31.3%) of patients were using combination of drugs (other antiepileptic or ASA) and 65 (63.7%) were taking VA. There was no difference between ADP (P=0.144), epinephrine (P=0.246) and collagen (P=0.431) results depending on antiepileptic used in all patients. Results differed between patients using monotherapy vs dual antiepileptic therapy for epinephrine 65% (44-79) vs 44% (37-51) ; P=0.001] and collagen [73% (47-79)] vs 43% (29- 70) ; P=0.005] (Table 1). Difference is obtained in patients using only VA vs VA combined with ASA for epinephrine [73% (44-80) vs 42% (23-74) ; P=0.020] and in patients using only VA vs dual antiepileptic therapy [65% (41-78) vs 84% (53-85) ; P=0.020] (Table 2). Conclusions: Dual antiepileptic drug therapy inhibit platelet aggregation induced by epinephrine and collagen. Platelet aggregation is inhibited if induced by epinephrine in patients treated with valproic acid as monotherapy and if it is combined with ASA.

Scientific research question: Inter-individual variability of both peak and trough plasma concentrations of direct oral anticoagulants (DOACs), dabigatran, rivaroxaban and apixaban, is still insufficiently investigated. Therefore, the aim of our study was to assess inter-individual variability of peak and trough plasma levels of all three DOACs in patients with non valvular atrial fibrillation (NVAF). Methodology: The study included plasma samples from patients treated with dabigatran (N = 106, 150 mg twice-daily), rivaroxaban (N = 123, 20 mg once-daily) and apixaban (N = 69 ; 5 mg twice-daily). Blood samples were taken on the same day to obtain both trough (immediately prior the next drug dose) and peak (two hours after drug administration) DOACs concentrations. Both rivaroxaban and apixaban were measured using chromogenic anti-FXa assay (Innovance anti-FXa, Siemens Healthineers, Germany) calibrated with drug specific calibrators (Hyphen BioMed, France). Dabigatran was measured using commercial chromogenic method (Innovance DTI assay, Siemens Healthineers, Germany). All coagulation assays were performed on Behring Coagulation System XP (BCSXP) analyzer (Siemens Healthineers, Germany). Statistical analysis was done using Mann-Whitney test by MedCalc Statistical Software version 11.5.1. The inter-individual variability for trough and peak concentrations was assessed by calculating mean values and standard deviation (SD) for each DOAC concentration measured for all samples. The study was funded as an integral part of the Croatian Science Foundation research project IP-2016-06-8208, entitled New oral anticoagulants: relationship between drug concentration and anticoagulant effect. Findings: Concentrations for all three DOACs ranged as follows: dabigatran (peak 3 - 473 ng/mL ; trough 0 - 292 ng/mL) ; rivaroxaban (peak 13 - 468 ng/mL ; trough 1 - 311 ng/mL) and apixaban (peak 56 - 396 ng/mL ; trough 10 - 259 ng/mL) with significant differences between peak and trough concentrations (P

Background: Recently, platelet function testing is increasingly included in assessment of the effectiveness of antiplatelet therapy in research and clinical settings. Literature data suggest high residual platelet reactivity measured by light transmission aggregometry (LTA) as >20% when induced with arachidonic acid (AA) and >70% when induced with adenosine diphosphate (ADP). Aims: To evaluate platelet reactivity by LTA using drug specific agonists in patients on long-term mono and dual antiplatelet therapy. Methods: Study included 191 consecutive patients on dual antiplatelet therapy, 158 patients on monotherapy with acetylsalicylic acid (ASA) and 37 on clopidogrel referred to our laboratory for platelet aggregation testing. Platelet rich plasma was obtained from 3.2% citrate plasma centrifuged at 500xg/10 minutes. LTA was done on Sysmex CS2500 (Siemens Healthineers, Germany) using AA (1 mmol/L) and ADP (2 µmol/L) as agonists (Hyphen Biomed, France). Parametric and nonparametric analysis of variance was used to test the differences between studied groups. Results: Among 191 patients with median age 64 yrs (range 26-90) on dual therapy, 170 were using clopidogrel (75 mg) and ASA (100 mg) whereas 21 were using ticagrelor (90 mg) and ASA (100 mg). Results for AA were 7% (interquartile range (IQR) 3-10)) and for ADP 35%±17%, respectively. Group of patients using ticagrelor and ASA has significantly lower ADP results than those using clopidogrel and ASA (median 20% ; IQR 16-27) vs median 37% ; IQR 23- 49 ; P< 0.001). Patients on ASA monotherapy (N=158) had AA 9% (IQR 4-16) while in those on clopidogrel therapy (N=37), aggregation with ADP agonist was 68%±10%. Only 8 (4%) patients on dual therapy did not reach suggested cut-off for AA in contrary to 20 (13%) patients on monotherapy with ASA (Table 1). Conclusions: Our study has shown that a higher proportion of patients on long term ASA monotherapy has high residual platelet reactivity compared to patients on dual antiplatelet therapy.

Aims. To investigate whether plasminogen activator inhibitor 1 (PAI­1) activity detected during first 24 hours of treatment of ST elevation myocardial infarction (STEMI) differs in patients with and without acute onset heart failure (HF). Methods. A total of 87 STEMI patients treated with primary percutaneous coronary intervention (PCI) were enrolled in the prospective observational single center cohort study. PAI­1 activity was determined on admission (prior to PCI) and after exactly 24 hours by using commercially available test Berichrom PAI (Siemens, Marburg, Germany). PAI­1 activity rise was defined as activity in second sample subtracted by activity at admission. Primary end­point was defined as an episode of HF requiring intravenous therapy within index hospital treatment, regardless of ejection fraction. Secondary end­point was defined as death at 5­ year follow­up. Results. Primary end­point occured in 9 patients (10.3%). In this group of patients, cardiogenic shock was more common (22.2% vs. 1.3%, chi square, test P=0.001), and ejection fraction was lower (median 45% vs. 55%, Mann­Whitney U test, P=0.001), while differences in other variables did not reach statistical significance level. Median PAI­1 activity rise in patients with HF was 4.10 U/mL (interquartile range (IQR) 1.75­7.65 U/mL), and in patients without HF 1.18 U/mL (IQR ­0.04­2.22 U/mL). In linear regression model, PAI­1 rise was independently related to HF (odds ratio (OR) 4.4), use of thrombus aspiration (OR 3.8) and body weight (OR ­2.2). Secondary endpoint occured in 2 patients during hospital treatment, and in 11 during follow up. Higher mortality was found in patients older than 65 (chi square test, P=0.034), females (chi square test, P=0.030), patients with occurrence of HF (Fisher exact test, P=0.026), worse final Thrombolysis in myocardial infarction (TIMI) flow (Fisher­Halton­Freeman test, P=0.001), higher PAI­1 activity rise (Mann­Whitney U test, P=0.004), lower left ventricular ejection fraction (Mann­Whitney U test, P=0.014) and lower body mass index (Mann­Whitney U test, P=0.024). In multivariate Cox regression analysis, with dichotomised PAI­1 activity rise (expressed as >3.7 U/mL; cut­off point found by receiver operating characteristic curve analysis), independent predictors of death were final TIMI flow and PAI­1 activity rise >3.7 U/mL, but not HF. Conclusion. Higher PAI­1 activity rise was observed in STEMI patients treated with primary PCI in whom acute HF occured during index hospitalisation. Whether the detected association is clinically significant, and contributes to long term outcome of the patients with HF in coronary artery disease, is to be determined in further studies.