ࡱ> [bjbj  ΐΐQB,B,99a;a;a;4;;;h;=;DT?TG(9G9GGZ^/A1A1A1A1A1A1A$FvIFUAa;>ZUA999GGO^:D^09V9Ga;G/A/A@3 :#?Gr;X2<&APD0D=IIL#?#?Ia;7?UAUADIB, K8:Marta KmetPovezanost stresa i aktivacije koagulacijskog sustavaDIPLOMSKI RADPredan Sveu iliatu u Zagrebu Farmaceutsko-biokemijskom fakultetu  Zagreb, 2017. Sadr~aj  TOC \h \z \t "N 1.;1;N 1.1.;2;N 1.1.1.;3" HYPERLINK \l "_Toc483773251"1. UVOD  PAGEREF _Toc483773251 \h 1 HYPERLINK \l "_Toc483773252"1.1. Stres  PAGEREF _Toc483773252 \h 1 HYPERLINK \l "_Toc483773253"1.1.1. Akutni stres  PAGEREF _Toc483773253 \h 2 HYPERLINK \l "_Toc483773254"1.1.2. Kroni ni stres  PAGEREF _Toc483773254 \h 2 HYPERLINK \l "_Toc483773255"1.1.3. Stres na poslu  PAGEREF _Toc483773255 \h 3 HYPERLINK \l "_Toc483773256"1.1.4. Povezanost stresa i kardiovaskularnih oboljenja  PAGEREF _Toc483773256 \h 4 HYPERLINK \l "_Toc483773257"1.2. Sustav hemostaze  PAGEREF _Toc483773257 \h 5 HYPERLINK \l "_Toc483773258"1.2.1. Komponente hemostaze  PAGEREF _Toc483773258 \h 5 HYPERLINK \l "_Toc483773259"1.2.2. Koagulacijske pretrage  PAGEREF _Toc483773259 \h 9 HYPERLINK \l "_Toc483773260"1.3. Laboratorijski pokazatelji stresa  PAGEREF _Toc483773260 \h 11 HYPERLINK \l "_Toc483773261"1.3.1. Biokemijski pokazatelji stresa  PAGEREF _Toc483773261 \h 11 HYPERLINK \l "_Toc483773262"1.3.2. Koagulacijski pokazatelji stresa  PAGEREF _Toc483773262 \h 11 HYPERLINK \l "_Toc483773263"2. OBRAZLO}ENJE TEME  PAGEREF _Toc483773263 \h 14 HYPERLINK \l "_Toc483773264"3. MATERIJALI I METODE  PAGEREF _Toc483773264 \h 15 HYPERLINK \l "_Toc483773265"3.1. Ispitanici  PAGEREF _Toc483773265 \h 15 HYPERLINK \l "_Toc483773266"3.2. Uzorkovanje  PAGEREF _Toc483773266 \h 15 HYPERLINK \l "_Toc483773267"3.3. Priprema uzorka  PAGEREF _Toc483773267 \h 15 HYPERLINK \l "_Toc483773268"3.4. Automatizirane metode  PAGEREF _Toc483773268 \h 16 HYPERLINK \l "_Toc483773269"3.4.1. Kalibracija i kontrola kvalitete  PAGEREF _Toc483773269 \h 16 HYPERLINK \l "_Toc483773270"3.4.2. Odreivanje PV-a  PAGEREF _Toc483773270 \h 16 HYPERLINK \l "_Toc483773271"3.4.3. Odreivanje APTV-a  PAGEREF _Toc483773271 \h 17 HYPERLINK \l "_Toc483773272"3.4.4. Odreivanje fibrinogena  PAGEREF _Toc483773272 \h 18 HYPERLINK \l "_Toc483773273"3.4.5. Odreivanje TV-a  PAGEREF _Toc483773273 \h 18 HYPERLINK \l "_Toc483773274"3.5. Neautomatizirana metoda: euglobulisnki test  PAGEREF _Toc483773274 \h 19 HYPERLINK \l "_Toc483773275"3.5.1. Reagensi  PAGEREF _Toc483773275 \h 19 HYPERLINK \l "_Toc483773276"3.5.2. Na elo i postupak  PAGEREF _Toc483773276 \h 19 HYPERLINK \l "_Toc483773277"3.6. Statisti ke metode  PAGEREF _Toc483773277 \h 19 HYPERLINK \l "_Toc483773278"4. REZULTATI I RASPRAVA  PAGEREF _Toc483773278 \h 21 HYPERLINK \l "_Toc483773279"4.1. Deskriptivna statistika rezultata mjerenja  PAGEREF _Toc483773279 \h 21 HYPERLINK \l "_Toc483773280"4.2. Usporedba rezultata s obzirom na vrijeme uzorkovanja  PAGEREF _Toc483773280 \h 23 HYPERLINK \l "_Toc483773281"4.3. Ispitivanje korelacije  PAGEREF _Toc483773281 \h 25 HYPERLINK \l "_Toc483773282"4.4. Rasprava  PAGEREF _Toc483773282 \h 26 HYPERLINK \l "_Toc483773283"5. ZAKLJU CI  PAGEREF _Toc483773283 \h 31 HYPERLINK \l "_Toc483773284"6. LITERATURA  PAGEREF _Toc483773284 \h 32 HYPERLINK \l "_Toc483773285"7. SA}ETAK / SUMMARY  PAGEREF _Toc483773285 \h 35 HYPERLINK \l "_Toc483773286"7.1. Sa~etak  PAGEREF _Toc483773286 \h 35 HYPERLINK \l "_Toc483773287"7.2. Summary  PAGEREF _Toc483773287 \h 36 HYPERLINK \l "_Toc483773288"8. PRILOZI  PAGEREF _Toc483773288 \h 37 HYPERLINK \l "_Toc483773289"8.1. Popis kratica  PAGEREF _Toc483773289 \h 37 Temeljna dokumentacijska kartica/Basic documentation card UVOD STRES Svatko se od nas povremeno nae u nekoj stresnoj situaciji te su nam dobro poznati neki osnovni simptomi stresa, kao ato su poja ano znojenje ili ubrzani sr ani ritam. O stresu se puno govori u svakodnevnom ~ivotu, naj eae u negativnom kontekstu, kao o ne emu ato trebamo izbjegavati ili protiv ega se trebamo boriti. No, kratkoro no, stres mo~e biti koristan obrambeni mehanizam koji u prirodi jedinkama omoguuje pre~ivljavanje. Pojam stresa mo~e se definirati na razli ite na ine. Mo~emo rei da je stres proces u kojem neki stresor, odnosno neato ato percipiramo kao opasnost, pokree odreeni psiho-bioloaki odgovor koji jedinki poma~e u upravljanju situacijom(McVicar i sur., 2014). U stanju stresa remeti se unutraanja ravnote~a organizma, zbog ega je potrebno ulo~iti dodatne napore kako bi se on prilagodio. U kojoj e mjeri, i hoe li uope, neki stresor izazvati stres ovisi o subjektivnom zna enju tog podra~aja za pojedinca. To zna i da e razli ite osobe razli ito reagirati u nekoj stresnoj situaciji(Havelka, 1995). Dakle, osim o stresoru, reakcija pojedinca na stres ovisi i o njegovoj individualnoj osjetljivosti, koja je odreena osobnoau, dobi i stilom ~ivota, te vanjskim okolnostima poput obitelji, prijatelja i radne atmosfere(Kne~evi i sur., 2009). Psiholoaka komponenta odgovora na stres poja ava svjesnost, poti e br~u kogniciju i reflekse. Bioloaka komponenta se sastoji iz aktivacije simpatikusa, koji priprema organizam na neposrednu fizi ku aktivnost, te od lu enja kortizola, koji je odgovoran za metaboli ku stimulaciju i imunosne promjene(McVicar i sur., 2014). Stresna reakcija zapo inje impulsima koji kreu iz korteksa, prolaze kroz limbi ki sustav do hipotalamusa. U hipotalamusu se stvara kortikotropin-otpuatajui hormon (CRH) koji preko portalne cirkulacije dolazi do hipofize koju stimulira na lu enje adrenokortikotropnog hormona (ACTH). ACTH poti e koru nadbubre~ne ~lijezde na sintezu i lu enje glukokortikoida. Ti glukokortikoidi zatim inhibiraju aktivnost osi hipotalamus-hipofiza-nadbubre~na ~lijezda mehanizmom negativne povratne sprege tako da se ve~u na receptore u hipofizi, hipotalamusu i srednjem prefrontalnom korteksu uzrokujui smanjenje sekrecije CRH, a time i otpuatanje ACTH(Gu i sur., 2012). Hipotalamus je i ishodiate vlakana autonomnog ~iv anog sustava. Za vrijeme stresa dolazi do aktivacije simpatikusa ato dovodi do lu enja adrenalina i noradrenalina iz sr~i nadbubre~ne ~lijezde te do ~iv ane stimulacije unutarnjih organa(Havelka, 1995). Reakcije na stres mogu biti psiholoake, ponaaajne i fizioloake. Psiholoake reakcije na stres uklju uju porast tjeskobe, negativne emocije, probleme u koncentraciji, depresiju, sindrom izgaranja (eng. burnout syndrome) ili porast samoubojstava. Primjeri ponaaajnih reakcija na stres su povla enje i izolacija na poslu ili kod kue, razdra~ljivost ili pak vea konzumacija cigareta, alkohola ili kave. Fizioloake reakcije na stres su poviaenje krvnog tlaka, palpitacije, bolovi u prsima, nesanica, pojava nekih vrsta karcinoma, probavne smetnje, glavobolje, koatano-miaine tegobe te oslabljeni imunitet(Kne~evi i sur., 2009). Akutni stres Akutni stres je stres koji traje u periodu od nekoliko minuta do nekoliko sati, a takav stresni odgovor ima pozitivne u inke za organizam. Naime, on ga priprema na borbu ili bijeg, djelujui na kardiovaskularni, miainoskeletni i neuroendokrini sustav. Pretpostavlja se da akutni stres djeluje na imunoloaki sustav tako da ga priprema na izazove poput ranjavanja ili infekcija koje mogu nastati u reakciji na stresor(Dhabhar, 2009). Zbog otpuatanja katekolamina dolazi do poveanja minutnog volumena srca, poveanog protoka krvi kroz miaie, retencije natrija, smanjenog motiliteta probavnog sustava i kutane vazokonstrikcije, poveava se koncentracija glukoze u krvi te se dilatiraju bronhiole(Ranabir i Reetu, 2011). Puno du~i u inak imaju kortikosteroidi. Mineralokortikoidi, poput aldosterona, reguliraju reapsorpciju vode i natrija, te aktivnu sekreciju kalija u bubrezima, ime se poveava krvni tlak i volumen krvi. Glukokortikoidi, u koje spada kortizol, poti u metabolizam masti i proteina i sintezu glukoze. Kortizol regulira mnoatvo kardiovaskularnih, metaboli kih, imunoloakih i homeostatskih funkcija(Lee i sur., 2015). Kroni ni stres Kroni ni se stres javlja kada izmeu stresnih podra~aja ne postoji potrebno razdoblje oporavka,ve se stresni dogaaji ni~u jedan za drugim. Takvo stanje dovodi do poja anog lu enja kortikosteroida, hipertrofije kore nadbubre~ne ~lijezde te atrofije timusa i drugih limfati kih struktura(Kne~evi i sur., 2009). Mehanizam negativne povratne sprege ovisan o glukokortikoidima prestaje funkcionirati jer glukokortikoidni receptori razvijaju rezistenciju. Razine molekula medijatora stresa u organizmu ostaju visoke, ime se kompromitira imunosni sustav te se dugoro no oateuju organi i tkiva. Naime, u kroni nom se stresu uspostavlja stanje blage, tinjajue upale koja mo~e biti pokreta  razli itih bolesti, kao ato su kardiovaskularni poremeaji, dijabetes, rak, autoimuni sindromi i mentalna oboljenja kao ato su depresija i anksioznost(Mariotti, 2015). Stres na poslu Stres na poslu definira se kao neravnote~a izmeu zahtjeva i sposobnosti da se tom zahtjevu udovolji, a javlja se u situaciji kada bi, po prosudbi radnika, neuspjeh u zadovoljavanju zahtjeva imao zna ajne posljedice. Umjereni stres mo~e djelovati motivirajue, no prevelika koli ina stresa dovodi do niskog radnog u inka(Kne~evi i sur., 2009). Kada se broj izvora stresa poveava, zaposlenicima je sve te~e nositi se s njima ato dovodi do loaijih rezultata rada i sindroma izgaranja na poslu(Strapajevi, 2015). To je stanje emocionalnog i psihi kog iscrpljenja na poslu uzrokovano nerazmjerno visokim trudom, u vidu ulo~enog vremena, emocionalnog anga~mana i empatije, i niskom razinom zadovoljstva u kombinaciji sa stresnim uvjetima na radnom mjestu(Varja i i sur., 2013). Taj se sindrom javlja kod ljudi koji obavljaju zahtjevne poslove i poslove koji uklju uju brigu za druge, kao ato su socijalni radnici, nastavnici i zdravstveni radnici. Zdravstveni radnici, posebice lije nici, izlo~eni su visokim razinama stresa. Istra~ivanje provedeno na 500 lije nika iz Ujedinjenog Kraljevstva pokazalo je da barem jedna treina njih ima neke karakteristike izgaranja na poslu. To uklju uje emocionalnu iscrpljenost, depersonalizaciju koja dovodi do indiferentnosti zdravstvenog djelatnika prema pacijentu te neefikasnost i nezadovoljstvo vlastitim postignuima(Romani i Ashkar, 2014). U istra~ivanju provedenom 2004. godine u Irskoj 56% lije nika svoj je posao ocijenilo kao izrazito stresan, a nezadovoljstvo na radu navelo je 79% lije nika. Naj eai navedeni stresori bili su hitna stanja, de~urstva, donoaenje odluka nakon nonog de~urstva, rutinski medicinski rad i mala mogunost napredovanja(Strapajevi, 2015). Noni rad i rad u smjenama interferiraju s druatvenim ~ivotom radnika, oni su eae emocionalno iscrpljeni, nezadovoljni te iskazuju viae znakova izgaranja na poslu. Sna~an utjecaj na psihi ko i fizi ko zdravlje ima naruaavanje cirkadijarnih ritmova, bioloakih endogenih ritmova koji kontroliraju budnost, temperaturu tijela, krvni tlak, vrijeme reakcije itd. To dovodi do jakog umora i smanjene funkcionalne sposobnosti razli itih tjelesnih sustava(Pti ar, 2013). U epidemioloakim studijama pronaena je poveznica izmeu stresa na poslu i kardiovaskularnih i metaboli kih bolesti, i to tako da stresan i zahtjevan posao udvostru uje rizik od smrti uzrokovane tim bolestima. Posljedice kumulativne izlo~enosti takvom poslu nisu do kraja poznate, no zna se da uklju uju autonomnu disfunkciju i neuroendokrinu aktivaciju. Takoer, postoje dokazi da rad u smjenama poveava rizik od koronarne bolesti srca te da ak i kod kratkih epizoda mentalnog stresa dolazi do tranzitorne disfunkcije endotela i aktivacije koagulacije kod zdravih pojedinaca(Matejovic i sur., 2011). Povezanost stresa i kardiovaskularnih oboljenja Kardiovaskularne bolesti (KVB) su bolesti srca i krvo~ilnog sustava za iji je nastanak naj eae odgovorna ateroskleroza. One su vodei uzrok smrti u suvremenom svijetu, sa zna ajnim udjelom u prijevremenom umiranju, morbiditetu i dizabilitetu stanovniatva. U Hrvatskoj su 2010. godine bile vodei uzrok smrti s udjelom od 49,2%(Kralj, 2011). Ateroskleroti ne krvne ~ile karakterizirane su disfunkcijom endotela sa smanjenom proizvodnjom duaikovog oksida, ime se smanjuju antikoagulantna i profibrinoliti ka svojstva endotelnih stanica(von Knel, 2015). Intenzivni mentalni stres mo~e biti okida  ishemije ili infarkta miokarda, ventrikularne aritmije i iznenadne sr ane smrti kod pacijenata s prethodnim oateenjem krvnih ~ila aterosklerozom. Glavni mehanizam koji povezuje psihi ki stres i KVB je brzo poveanje arterijskog tlaka i sr anog ritma zbog poveane aktivnosti simpatikusa u kombinaciji s disfunkcijom endotela i aterotrombotskom aktivacijom. Kod kroni nog psihosocijalnog stresa, koji uzrokuju primjerice obiteljski problemi ili nezaposlenost, mozak pokree obrambene mehanizme iako ne postoji stvarna ozljeda tkiva, a KVB se pojavljuju kao posljedica toga. Takav stres dovodi do kognitivnih i emocionalnih promjena koje pokreu molekularne kaskade koje rezultiraju otpuatanjem glukokortikoida i katekolamina(Vale, 2005). U istra~ivanju koje se bavilo utjecajem stresa na poslu na razli ite metaboli ke i hemostatske rizi ne imbenike pokazano je da zaposlenici pod visokim stresom na radnom mjestu imaju oslabljenu fibrinolizu. To su potvrdili visoki PAI-1 i niska t-PA aktivnost, to zajedno doprinosi formaciji ugruka(Vrijkotte i sur., 1999). SUSTAV HEMOSTAZE Hemostaza je odgovor organizma na ozljedu krvne ~ile i krvarenje u kojem sudjeluju stjenke krvnih ~ila, trombociti i faktori zgruaavanja(Labar, 2007). Time se omoguava cijeljenje oateenih krvnih ~ila, krv se odr~ava u tekuem obliku i uklanjaju se krvni ugruaci nakon uspostavljanja vaskularnog integriteta(Versteeg i sur., 2013). Kod ozljede krvne ~ile dolazi do vazokonstrikcije malih arterija i arteriola, ime se usporava protok krvi u podru ju ozljede ato smanjuje krvarenje, ali i omoguava kontaktna aktivacija trombocita i faktora zgruaavanja. Trombociti oslobaaju sadr~aj svojih granula i agregiraju na mjestu ozljede, stvarajui primarni ugruaak koji se dodatno stabilizira fibrinom nastalim pomou sustava zgruaavanja. Ubrzo zapo inje i proces uklanjanja ne~eljenih depozita fibrina enzimskim cijepanjem, ime nastaju topljivi fibrinski fragmenti(Labar, 2007). esto se koriste i pojmovi primarne i sekundarne hemostaze, pri emu su glavni sudionici primarne hemostaze krvne ~ile i trombociti, dok sekundarna hemostaza obuhvaa faktore zgruaavanja ija aktivacija dovodi do stvaranja fibrina. Treu fazu hemostaze ini fibrinoliza. Iako se pojmovi hemostaza i koagulacija esto koriste kao sinonimi, bitno je naglasiti da je koagulacija samo jedan dio hemostaze(Lutze, 2000). Komponente hemostaze Vaskularni sustav Ranije spomenuta vazokonstrikcija nije jedina uloga vaskularnog sustava u hemostazi. Naime, taj je sustav klju an u prokoagulantnoj i antikoagulantnoj regulaciji funkcije trombocita i faktora zgruaavanja. Endotel izlu uje von Willebrandov faktor, na njegovoj se povraini odvija vezanje trombina na trombomodulin, aktivacija proteina C i uklanjanje faktora zgruaavanja pomou antitrombina. Takoer, endotel doprinosi aktivaciji i regulaciji fibrinolize izlu ivanjem tkivnog aktivatora plazminogena. U vaskularnom glatkom miaiju i pericitima koji okru~uju ~ile nalazi se tkivni faktor koji sudjeluje u koagulacijskoj kaskadi(Burtis i sur., 2012). Trombociti Trombociti nastaju u koatanoj sr~i procesom megakariocitopoeze, odnosno diferencijacijom nezrelih hematopoetskih stanica u megakariocite te fragmentacijom citoplazme zrelih megakariocita. Jedan megakariocit stvara 2000 do 3000 trombocita, normalno ih ima 250"109/L te u perifernoj krvi ~ive 9 do 11 dana. Diskoidnog su oblika, bez jezgre, endoplazmatskog retikuluma ili Golgijevog aparata, a na njihovoj se povraini nalaze brojni glikoproteini koji su bitni za njihovu adheziju i agregaciju(Labar, 2007). Endotelne stanice odr~avaju trombocite u diskoidnom obliku izlu ivanjem tvari koje ih inhibiraju, kao ato su prostaglandin I2 i NO, ali i metaboliziranjem trombocitnih agonista kao ato su ADP i trombin(Chapin i Hajjar, 2015). Nakon oateenja krvne ~ile, trombociti dolaze u doticaj s bazalnom membranom i kolagenom te adheriraju za subendotel vezanjem za von Willebrandov faktor na njegovoj povraini, no u tom procesu sudjeluju i brojni drugi adhezijski proteini. Adhezija izaziva aktivaciju trombocita, odnosno promjenu njihovog oblika, oslobaanje granula i njihovu meusobnu agregaciju(Labar, 2007). Alfa granule sadr~e proteine kao ato su fibrinogen ili von Willebrandov faktor, dok guste granule sadr~e neproteinske komponente kao ato su adrenalin, serotonin i ADP. Takoer, na njihovo se povraini nalaze makronuklearni kompleksi uklju eni u koagulacijsku kaskadu. Trombociti sudjeluju i u retrakciji ugruaka pomou kontraktilnih proteina i time su~avaju ranu(Burtis i sur., 2012). Sustav zgruaavanja Zgruaavanje krvi je kompleksan proces koji dovodi do konverzije topljivog fibrinogena u netopljivi fibrin. Kaskada enzimskih reakcija, u kojoj sudjeluju faktori zgruaavanja i koja dovodi do formacije fibrina, omoguava amplifikaciju po etnog signala nakon ozljede i regulaciju samog procesa(Burtis i sur., 2012). Faktori zgruaavanja su plazmatski glikoproteini koji se veinom sintetiziraju u jetri. Protrombin (FII), prekalikrein, FXII, FXI, FX, FIX i FVII se u cirkulaciji nalaze u obliku proenzima, a tijekom procesa zgruavanja prelaze u aktivne serinske proteaze, dok su kininogen visoke molekularne mase, tkivni faktor i FV i FVIII kofaktori(Labar, 2007). Prema stani nom modelu proces zgruaavanja se mo~e podijeliti u tri faze. U inicijaciji se stvaraju male koli ine aktiviranih faktora zgruaavanja, u amplifikaciji njihove koncentracije rastu, a u fazi propagacije se ve~u za prokoagulantne membrane aktiviranih trombocita te se stvara fibrinski ugruaak. Danas je i dalje u airokoj upotrebi kaskadni model koji zgruaavanje dijeli na vanjski, unutarnji i zajedni ki put(Versteeg i sur., 2013). Nakon ozljede naj eae se aktiviraju oba puta(Lutze, 2000), te se spajaju zajedni ki put koji obuhvaa zavrane korake u nastajanju ugruaka(Burtis i sur., 2012). Unutarnji put zapo inju komponente koje su ve prisutne u krvi kada dou u kontakt s nekom tvari s kojom ina e nisu u kontaktu(Lutze, 2000). To su prekalikrein, kininogen velike molekularne mase, FXII i faktor XI koji se aktiviraju u kontaktu s negativno nabijenom povrainom, primjerice kolagenom i endotoksinima in vivo ili staklom in vitro. Iako ovi faktori imaju ulogu u stvaranju kinina, fibrinolizi i upalnom procesu, njihova je va~nost u aktivaciji sustav zgruaavanja in vivo zanemariva(Labar, 2007).  Vanjski put je glavni put aktivacije zgruaavanja (Labar, 2007), ija je klju na komponenta tkivni faktor, poznat i pod nazivom tromboplastin, transmembranski glikoprotein koji je eksprimiran u ekstravaskularnom tkivu, osobito na fibroblastima i stanicama glatkih miaia te slu~i kao tzv. hemostatska ovojnica(Versteeg i sur., 2013). On se u prisutnosti kalcija ve~e za FVII ime zapo inje koagulacijska kaskada. FVIIa aktivira FX koji svojom enzimskom aktivnoau prevodi protrombin u trombin. Trombin tada katalizira pretvorbu fibrinogena u fibrin, ali i aktivaciju FXIII, (Labar, 2007) koji posreduje u unakrsnom povezivanju fibrina, ato dodatno u vrauje strukturu ugruaka(Chapin i Hajjar, 2015). Nekontrolirano stvaranje ugruaaka sprje ava sustav fizioloakih inhibitora sustava zgruaavanja. Cirkulirajui inhibitori proteaza, kao ato su antitrombin ili inhibitor faktora tkivnog puta, ve~u se za aktivna mjesta aktiviranih faktora zgruaavanja (Versteeg i sur., 2013). Pored toga, kompleks trombina i trombomodulina, koji nastaje na povraini endotelnih stanica, aktivira protein C. On, u kompleksu s proteinom S, inaktivira FVa i FXIIIa ime se sprje ava daljnje stvaranje trombina(Labar, 2007). Fibrinoliti ki sustav Fibrinoliza je mehanizam enzimske razgradnje unakrsno povezanog fibrina, ato katalizira enzim plazmin, pri emu nastaju topljivi fragmenti fibrina razli itog sastava i molarne mase(Burtis i sur., 2012). Dvije su bitne uloge fibrinolize u hemostazi: razgradnja fibrinskog ugruaka nakon ato je ispunio svoju fizioloaku ulogu, ali i ograni avanje stvaranja ugruaka ime se odr~ava prohodnost krvnih ~ila(Thomas, 1998). Do aktivacije fibrinolize, odnosno pretvorbe plazminogena u plazmin, mo~e doi unutarnjim putem, u emu sudjeluju kontaktni faktori FXIIa, prekalikrein i kininogen visoke molekularne mase, te vanjskim putem, preko aktivatora plazminogena: tkivnog aktivatora plazminogena (t-PA) i urokinaze (u-PA). t-PA uglavnom nastaje u endotelnim stanicama, a u krvi se nalazi u kompleksu s inhibitorom tkivnog aktivatora plazminogena (PAI-1). Urokinaza se sintetizira u epitelnim stanicama bubre~nih tubula te se u urinu nalazi u visokim koncentracijama(Thomas, 1998). Monomeri fibrina se umre~uju u fibrinsku nit na na in da se dva monomera spojena preko D-domena spajaju s treim preko njegove E domene. Plazmin kida fibrinsku molekulu izmeu D i E domena te nastaju topljivi fragmenti fibrina koji sadr~e razli iti broj D-D domena, odnosno D-dimera(Burtis i sur., 2012). D-dimeri imaju prognosti ko zna enje u mnogim bolestima kao ato su diseminirana intravaskularna koagulacija ili duboka venska tromboza(Chapin i Hajjar, 2015). Koagulacijske pretrage Za koagulacijska mjerenja koristi se mnogo metoda, naj eae su to globalne pretrage bazirane na ugruaku i kromogene metode(Bates i Weitz, 2005). Globalni testovi ne mjere neku specifi nu komponentu ve cijelu fazu hemostatskog procesa, na primjer, aktivaciju viae faktora zajedno. Oni ukazuju na abnormalnost u hemostazi, ali malo govore o uzroku te abnormalnosti te je njihova osjetljivost niska(Lutze, 2000). Kromogene metode mjere razinu ili funkciju specifi nog faktora(Bates i Weitz, 2005). Princip metoda baziranih na ugruaku je detekcija vremena od inicijacije zgruaavanja do formiranja ugruaka i to mjerenjem promjena u mehani kim svojstvima ili propuatanjem svjetla kroz uzorak. Primjerice, u jednoj od mehani kih metoda magnet pokree metalnu kuglicu u kiveti s uzorkom sve dok se ne stvori ugruaak koji to kretanje prekida. Od opti kih se metoda koriste nefelometrija i turbidimetrija. Kromogene metode koriste svojstva serinskih proteaza koagulacijske kaskade da kidaju oligopeptidne supstrate otpuatajui kromogene koji se opti ki mjere(Burtis i sur., 2012). Primarni uzorak za koagulacijske pretrage je venska krv uzeta u spremnik s antikoagulansom natrijevim citratom. Od nje se naj eae dobiva plazma bogata trombocitima (PRP) za ispitivanje funkcije trombocita i plazma siromaana trombocitima (PPP) za veinu ostalih pretraga(Lutze, 2000). Omjer krvi i antikoagulansa treba biti 10:1, a preporu uje se da natrij citrat bude 3,2 postotni. Krv i antikoagulans se trebaju odmah promijeaati kako bi se sprije ilo in vitro zgruaavanje, a uzorci koji sadr~e vidljive ugruake se ne analiziraju. Uzorci se do laboratorija trebaju prenositi na sobnoj temperaturi jer hlaenjem mo~e doi do aktivacije trombocita i FVII te do gubitka dijela FVIII i von Willebrandovog faktora. Stabilnost uzoraka ovisi o pojedinoj metodi te o temperaturi i uvjetima skladiatenja uzoraka. Primjerice, uzorci za analizu funkcije trombocita na sobnoj su temperaturi stabilni 3 do 4 sata, a za analizu PV-a 24 sata(Bonar i sur., 2010). Pored uobi ajenih analiti kih interferencija hemolize, ikterije i lipemije, u koagulacijskim je mjerenjima vrlo bitno uzeti u obzir joa neke predanaliti ke imbenike, a to su zgruaani uzorak, neprimjereni omjer krvi i antikoagulansa te kontaminacija uzorka heparinom ili nekim drugim antikoagulansom. Zgruaavanje e potroaiti faktore zgruaavanja i u initi testove nepouzdanima. Ako se uzorkuje manji volumen od preporu enog, produ~it e se vrijeme zgruaavanja zbog suviaka antikoagulansa. Takoer, uzorci s poviaenim hematokritom (>55%) trebat e smanjeni volumen antikoagulansa zbog smanjenog volumena plazme(Burtis i sur., 2012). LABORATORIJSKI POKAZATELJI STRESA Biokemijski pokazatelji stresa Kako je ve re eno, u akutnom i kroni nom stresu dolazi do aktivacije osi hipotalamus-hipofiza-nadbubre~na ~lijezda, ato dobro odra~ava biokemijski biljeg kortizol. On se pouzdano mo~e mjeriti u razli itim bioloakim materijalima, pri emu se za odreivanje njegove trenutne sistemske koncentracije koriste slina ili krv, dok se kumulativna sekrecija kortizola tijekom du~eg vremenskog perioda mo~e mjeriti u urinu i kosi. Kao biomarkeri aktivnosti autonomnog ~iv anog sustava koriste se adrenalin i noradrenalin, no njihova je loaa strana ato je za mjerenje potrebno pacijentu oduzeti krv. Kako bi se izbjegla invazivnost ovih metoda, posljednjih se godina kao biomarker u istra~ivanjima sve viae koristi salivarna alfa-amilaza. Naime, njezino stvaranje i izlu ivanje uklju uje kontrolu autonomnog ~iv anog sustava, odnosno raste s njegovom aktivacijom. Poznato je da su proupalni citokini, kao ato su IL-6, IL-1 i TNF-, te C-reaktivni protein uklju eni odgovor organizma na akutni i kroni ni stres. Njihova bi sekrecija u stresu mogla rasvijetliti patofizioloake procese utjecaja stresa na razvoj kroni nih bolesti, odnosno mogli bi poslu~iti kao biomarkeri imunosne aktivacije u stresu(Nater i sur., 2013). Koagulacijski pokazatelji stresa Veliki broj istra~ivanja hemostaze ukazuje na to da akutni psihosocijalni stres dovodi do istovremene aktivacije i koagulacije i fibrinolize, s time da se koagulacija aktivira u veoj mjeri, ato rezultira hiperkoagulabilnim stanjem. U tablici 1 prikazane su promjene laboratorijskih biljega koagulacije, antikoagulacije i fibrinolize u hiperkoagulabilnom stanju(von Knel i sur., 2001). Tablica SEQ Tabela \* ARABIC 1.Promjene laboratorijskih biljega u hiperkoagulabilnom stanju (preuzeta iz von Knel i sur., 2001) KoagulacijaAntikoagulacijaFibrinoliza! vrijeme zgruaavanja krvi ! aktivnost/antigeni faktora zgruaavanja ! fibrinogen ! vWF ! pretrombotski markeri! antitrombin III! fibrinoliti ka aktivnost ! euglobulinski test ! aktivnost t-PA ! t-PA antigen ! aktivnost/antigen PAI-1 U daljnjem tekstu biti e detaljnije objaanjene neke osnovne koagulacijske pretrage koje slu~e za procjenu stanja hemostaze, te mogu ukazati na eventualnu zna ajniju aktivaciju sustava zgruaavanja i fibrinolize za vrijeme psihi kog stresa.Ipak, takozvani pretrombotski markeri, poput D-dimera ili fibrinopeptida A, mnogo su osjetljiviji u detekciji hiperkoagulabilnog stanja od uobi ajenih koagulacijskih metoda kao ato su PV, euglobulinski test i aktivnosti faktora zgruaavanja. Njihove poviaene koncentracije ukazuju na poja anu koagulacijsku aktivnost, a onda i fibrinoliti ku aktivaciju, i prije same pojave tromboze(von Knel i sur., 2001). Globalne pretrage: PV, APTV i TV Protrombinsko vrijeme (PV) je metoda bazirana na ugruaku koja odra~ava aktivnost vanjskog i zajedni kog puta zgruaavanja (Burtis i sur., 2012) odnosno ukazuje na postojanje poremeaja u jednom od faktora protrombinskog kompleksa (FII, FVII, FX), FV ili fibrinogenu. Odreuje se zbog praenja terapije s antagonistima vitamina K te kod praenja deficijencije vitamina K i bolesti jetre, te predoperativno (Thomas, 1998).Svaki pripravak tromboplastina ima razli itu osjetljivost na nedostatak pojedinih faktora zgruaavanja pa je potrebno da se svaki pripravak ba~dari prema internacionalnom referentnom tromboplastinu i da ima ISI vrijednost (Internacionalni indeks osjetljivosti)(Labar, 2007). Aktivirano parcijalno tromboplastinsko vrijeme (APTV) je metoda bazirana na ugruaku koja odra~ava aktivnost unutarnjeg i zajedni kog puta zgruaavanja. Aktivacija kontaktnih faktora posti~e se dodatkom aktivatora, naj eae s negativno nabijenom povrainom, poput kaolina. APTV se odreuje zbog praenja terapije s heparinom te za detekciju deficijencija faktora unutarnjeg i zajedni kog puta te njihovih inhibitora(Burtis i sur., 2012). Takoer, APTV mo~e biti produ~eno i zbog drugih stanja, poput bolesti jetre(HYPERLINK "http://klinkemija.kbcsm.hr/test.php?recordID=226"http://klinkemija.kbcsm.hr/test.php?recordID=226). Trombinsko vrijeme (TV) je metoda bazirana na ugruaku koja odra~ava pretvorbu fibrinogena u fibrinski monomer uz pomo trombina te polimerizaciju fibrinskih monomera, a odreuje se tako da se u citratnu plazmu dodaje mala koli ina trombina te se mjeri vrijeme nastajanja ugruaka(Burtis i sur., 2012). Patoloake vrijednosti trombinskog vremena prisutne su kod razli itih deficijencija (hipofibrinogenemija) ili abnormalnosti (disfibrinogenemija) fibrinogenske molekule. Nadalje, prisutnost heparina ili hirudina, degradacijskih produkata fibrina ili fibrinogena te patoloakih inhibitora takoer e dovesti do patoloakih vrijednosti TV-a(Lutze, 2000). Fibrinogen Fibrinogen je protein koji se u krvi nalazi u koncentaciji od 2 do 4 mg/mL. Sastoji se od dva seta od po tri razli ita polipeptidna lanca povezana disulfidnim vezama (A, B i ). Trombin uklanja N-terminalne fibrinopeptide A i B, pri emu nastaju monomeri fibrina koji se dalje umre~uju preko lizinskih ostataka tvorei fibrinski ugruaak(Chapin i Hajjar, 2015). Fibrinogen je pozitivni reaktant akutne faze ato zna i da njegova koncentracija raste u upalnim stanjima, te se tradicionalno smatra rizi nim faktorom za razvoj KVB jer poti e formiranje ugruaaka. Studije su pokazale povezanost stresa s poviaenom koncentracijom fibrinogena u plazmi(Lazzarino i sur., 2013). Uz to, mjerenje fibrinogena je indicirano za detekciju uroenih deficijencija fibrinogena, poput afibrinogenemije, te za praene tromboliti ke terapije(Thomas, 1998). Osim u upalama i infekcijama, vrijednosti fibrinogena poviaene su i nakon operativnog zahvata, traume ili opeklina, akutnog infarkta miokarda, nefrotskog sindroma ili zloudnih bolesti. Fibrinogen mo~e, primjerice, biti sni~en u bolestima jetre, u akutnim krvarenjima te kod nasljednog nedostatka ili defekata fibrinogena(HYPERLINK "http://klinkemija.kbcsm.hr/test.php?recordID=752"http://klinkemija.kbcsm.hr/test.php?recordID=752). Fibrinoliza Globalna fibrinoliti ka aktivnost plazme ispituje se euglobulinskim testom. Pokazano je da hipofibrinoliza izmjerena ovim testom ima prediktivnu vrijednost za razvoj venske tromboze te da je koristan u predvianju infarkta miokarda kod muakih osoba ispod 50 godina, dok se hiperfibrinoliza nije pokazala korisnom u predvianju bolesti krvarenja(Chapin i Hajjar, 2015). Smanjene vrijednosti eulobulinskog testa nalazimo kod razli itih stanja hiperfibrinolize poput ciroze jetre, ali i kod malignih bolesti, tromboliti ke terapije, sepse i drugih stanja. Usporenu fibrinolizu, odnosno poveane vrijednosti euglobulinskog testa nalazimo kod poremeaja fibrinoliti kog sustava zbog poveane aktivnosti t-PA, te u tromboembolijskim i drugim stanjima(HYPERLINK "http://klinkemija.kbcsm.hr/test.php?recordID=58"http://klinkemija.kbcsm.hr/test.php?recordID=58). Obrazlo~enje teme Rad u zdravstvu sa sobom donosi brojne odgovornosti i posebne obrasce rada. Dvadeset etverosatna de~urstva predstavljaju est izvor stresa za lije nike te mogu utjecati na njihovo zdravlje.Dosadaanja istra~ivanja povezuju izlo~enost stresu na radnom mjestu s poremeajem hemostaze u vidu nastanka hiperkoagulabilnog stanja. Fibrinogen, kao bitna komponenta hemostaze, igra zna ajnu ulogu i u formiranju aterosklerostskog plaka. Sve to ukazuje na poveani rizik od razvoja kardiovaskularnih bolesti kod lije nika. U ovom smo radu ~eljeli ispitati u kojoj mjeri pojedina no dvadeset etirisatno de~urstvo utje e na aktivaciju sustava koagulacije i fibrinolize, odnosno dolazi li, barem kratkotrajno, do dovoljno jakog dizbalansa da to mo~emo detektirati najosnovnijim koagulacijskim pretragama. Cilj diplomskog rada: Ispitati utjecaj akutnog stresa kao posljedice dvadeset etirisatnog de~urstva na koagulabilnost krvi kod lije nika na specijalisti kom usavraavanju. Specifi ni ciljevi: 1. Odrediti APTV, PV, TV, fibrinogen i vrijeme fibrinolize u uzorcima ispitanika prije, tijekom i nakon dvadeset etirisatnog de~urstva. 2. Utvrditi je li doalo do promjene u vrijednostima navedenih parametara tijekom i nakon dvadeset etirisatnog de~urstva u odnosu na vrijednosti prije de~urstva. 3. Dobivene rezultate staviti u kontekst dosadaanjih znanja o utjecaju stresa na hiperkoagulabilnost krvi. Materijali i metode Ispitanici Ispitanici su lije nici na specijalisti kom usavraavanju kojima je rad povremeno organiziran u obliku dvadeset etverosatnog de~urstva. Ukupni broj ispitanika je 29, od toga je 15 ~ena i 14 muakaraca. Dob ispitanika je u rasponu od 28 do 41 godine, s medijanom od 31 godine. Iz istra~ivanja su isklju ene trudnice, korisnice hormonske terapije, osobe s pozitivnom anamnezom tromboembolijskih incidenata i nikotinizma, osobe s akutnom infektivnom bolesti i osobe koje imaju indeks tjelesne mase manji od 18.5 i vei od 25 kg/m2. Takoer, isklju eni su ispitanici kojima je vrijeme od zadnjeg de~urstva krae od 5 dana kako bi dobiveni rezultati bili posljedica samo akutne izlo~enosti stresu. Svi su ispitanici potpisali informirani pristanak i suglasnost na tri venepunkcije u asepti nim uvjetima, izvedene od stru nog medicinskog osoblja bolni kog centra. Jedini osobni podaci uzeti od ispitanika bili su dob i spol. Uzorkovanje Uzorci venske krvi ispitanika dobiveni su venepunkcijom na radnom mjestu u tri navrata. Prvo uzorkovanje provedeno je 15 minuta prije po etka dvadeset etirisatnog de~urstva. Drugo uzorkovanje provedeno je 12 sati nakon po etka de~urstva. Tree uzorkovanje provedeno je 24 sata nakon po etka de~urstva. Krv je izvaena iz kubitalne vene u epruvetu s plavom bojom epa koja sadr~i 3,2%-tni Na-citrat. Mjesto uboda trebalo je prethodno dezinficirati alkoholom i pustiti da se osuai, dok je podvezu, koja se koristi za poveanje tlaka u venama kako bi bile vidljivije, trebalo dr~ati najviae minutu, kako ne bi doalo do hemokoncentracije.Koriatene epruvete s vakumom osigurale su odgovarajui volumen krvi kako bi omjer krvi i antikoagulansa bio 10:1 (9 dijelova krvi i 1 dio antikoagulansa). Dozvoljeno je odstupanje volumena krvi za maksimalno 10% od oznake na epruveti. Nakon ato se epruveta napunila krvlju morala se 3 do 4 puta okrenuti kako bi doalo do mijeaanja krvi i antikoagulansa. Priprema uzorka Nakon uzorkovanja potrebno je uzorak upisati u laboratorijski informacijski sustav (LIS) kako bi mu se dodijelio jedinstveni barkod kojim se ozna ava epruveta. Na taj se na in smanjuje mogunost zamjene uzorka, te omoguava lakae praenje uzorka tijekom analize.Analiti ki uzorak za sve pretrage je plazma siromaana trombocitima (PPP), pa je u svrhu njezinog dobivanja potrebno centrifugirati primarni uzorak pune krvi 10 minuta na 4000 okretaja/min. Automatizirane metode Pretrage PV, APTV, TV i fibrinogen izvode se na automatskom koagulometru Behring Coagulation System XP (BCS XP) tvrtke Siemens. Kalibracija i kontrola kvalitete APTV i TV se ne kalibriraju jer ih analizator mjeri izravno u sekundama. PV se izra~ava kao postotak aktivnosti pa je za njega potrebna kalibracijska krivulja, a isto vrijedi i za fibrinogen koji se izra~ava u g/L. Kalibrator za odreivanje PV-a je PT Multi Calibrator, a za odreivanje fibrinogena Fibrinogen Calibrator kit. Postupak kalibracije se provodi nakon svake promjene serijskog broja reagensa za tu pretragu te u slu aju da postupak redovne unutarnje analiti ke kontrole ne zadovoljava kriterije prihvatljivosti kontrolnog postupka. Svako pakiranje navedenih reagensa sadr~i 6 komercijalnih kalibratora s nazna enim vrijednostima koji slu~e za izradu kalibracijske krivulje. Neposredno nakon postupka kalibracije izvodi se postupak unutarnje analiti ke kontrole s normalnim i patoloakim komercijalnim kontrolnim plazmama, ime se provjerava i potvruje provedena kalibracija. U slu aju da su rezultati kontrolnog postupka neprihvatljivi, potrebno je ponoviti kalibraciju, a onda i postupak unutarnje analiti ke kontrole. Uz to, postupak unutarnje analiti ke kontrole se provodi svakog dana prije po etka rada na analizatoru, zatim kada se stavlja novi reagens istog serijskog broja, kada se sumnja na ispravnost analizatora i nakon servisnog postupka. Odreivanje PV-a Reagensi Reagens za odreivanje PV-a je jednokomponentan: Dade Innovin Reagent je liofilizirani reagens koji se sastoji od rekombinantnog humanog tkivnog faktora i sinteti kih fosfolipida (tromboplastina), kalcijevih iona, komponente koja neutralizira heparin, pufera i stabilizatora. Reagens je potrebno pripremiti otapanjem u 10 mL destilirane vode i ostaviti 15 min na sobnoj temperaturi prije uporabe. Na elo i postupak Plazma siromaana trombocitima najprije se inkubira s optimalnom koli inom tkivnog tromboplastina i kalcijevih iona pri emu dolazi do aktivacije vanjskog i zajedni kog puta zgruaavanja te se mjeri vrijeme potrebno za stvaranje fibrinskog ugruaka. Izmjereno vrijeme u sekundama se automatski prera unava u postotak aktivnosti od normale na temelju kalibracijske krivulje dobivene komercijalnim kalibracijskim plazmama. Tromboplastinski reagens sadr~i tkivni faktor koji zapo inje vanjski put, kalcij koji je potreban za aktivaciju faktora zgruaavanja te fosfolipide koji slu~e kao podloga za odvijanje reakcija koagulacijske kaskade. Rezultati se izra~avaju kao omjer srednjih vrijednosti protrombinskog vremena bolesnikove plazme i normalne kontrolne plazme. Odreivanje APTV-a Reagensi Reagens za odreivanje APTV-a je dvokomponentan: Dade Actin FS sadr~i pro iaene fosfolipide soje u elaginskoj kiselini s dodanim puferom, stabilizatorima i konzervansima. CaCL2 Solution 0,025 mol/L Oba reagensa su spremna za upotrebu. 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Nakon inkubacije dodaju se kalcijevi ioni zbog ega zapo inje proces zgruaavanja koji rezultira pretvorbom fibrinogena u fibrin. Vrijeme potrebno za stvaranje fibrinskog ugruaka mjeri se u sekundama. Analizator automatski prera unava izmjereno vrijeme u omjer tako da APTV izmjeren u sekundama u plazmi ispitanika podijeli sa srednjom vrijednosti referentnog intervala APTV-a za koriateni reagens ato je zadano u kompjuterskom programu analizatora. Odreivanje fibrinogena Reagensi Reagens je jednokomponentan: Multifibren U sadr~i govei trombin, peptid koji usporava agregaciju fibrina, kalcij klorid, heksadimetrin bromid, polietilen glikol, natrijev klorid, Tris, govei albumin i natrijev azid kao konzervans. Reagens je potrebno otopiti u 5 mL destilirane vode i ostaviti na sobnoj temperaturi 15 do 30 min prije upotrebe. Na elo i postupak Metoda odreivanja fibrinogena je modifikacija Claussove metode. Uzorku plazme dodaje se trombinski reagens u suviaku ato dovodi do pretvorbe fibrinogena u fibrin. Mjeri se vrijeme zgruaavanja u sekundama pri emu je brzina stvaranja ugruaka razmjerna koncentraciji fibrinogena u plazmi. Analizator automatski prera unava izmjereno vrijeme u koncentraciju (g/L) na temelju kalibracijske krivulje dobivene komercijalnim kalibracijskim plazmama. Odreivanje TV-a Reagensi Reagens prireen za upotrebu je jednokomponentan, a kit sadr~i: BC Thrombin Reagent koji sadr~i liofilizirani govei trombin i govei albumin Buffer Solution for BC Thrombin Reagent koji sadr~i HEPES, pH 7,4, i konzervanse Reagens se priprema otapanjem liofilizata reagensa u 5 mL puferske otopine koja se nalazi u originalnom pakiranju Na elo i postupak Nakon dodatka trombinskog reagensa odreuje se vrijeme zgruaavanja citratne plazme. Trombin iz reagensa prevodi fibrinogen u fibrin te se turbidimetrijski mjeri vrijeme u sekundama potrebno za stvaranje fibrinskog ugruaka. neautomatizirana metoda: euglobulisnki test Reagensi Octena kiselina (1%) Boratno pufer, pH 7,6% Otopina kalcijevog klorida 0,025 mol/L Na elo i postupak U epruvetu od 10 mL otpipetira se 9 mL destilirane vode, 100 L 1%-tne octene kiseline i 500 L uzorka, epruveta se lagano promijeaa i ostavi 30 min u hladnjaku, pri emu nastaje precipitat, tzv. euglobulinska frakcija koja sadr~i aktivator plazminogena, plazminogen, PAI-1 i fibrinogen. Nakon stajanja i centrifugiranja 10 minuta na 4000 okretaja, supernatant se odlije, a epruveta s preostalim sadr~ajem ostavi 5 minuta u obrnutom polo~aju na stalku. Epruveta se premjesti u vodenu kupelj na 37(C te joj se dodaje 500 L boratnog pufera kako bi se nastali precipitat bogat fibrinoliti kim sastavnicamaotopio, a dodatkom 500 L CaCl2 istalo~i se fibrinogen te nastaje ugruaak. Vrijeme nastanka ugruaka zabilje~i se te se svakih 15 minuta makroskopski prati vrijeme liziranja tog ugruaka. Rezultat se izra~ava kao vrijeme u minutama u kojem se makroskopskim pregledom utvrdi potpuna liza precitipata, odnosno kada on iz mlije no bijelog prijee u proziran. Statisti ke metode Za statisti ku obradu podataka koriateni su ra unalni programi Microsoft Excel 2010 ( Microsoft Corporation) i MedCalc v.17.4 ( 1993-2017 MedCalc Software). Koriatenjem deskriptivne statistike dobiveni su podaci o aritmeti koj sredini, njezinom 95%-tnom intervalu pouzdanosti, standardnoj devijaciji, medijanu i njegovom 95%-tnom intervalu pouzdanosti, minimumu i maksimumu te interkvartilnom rasponu (25-75 P). Normalna distribucija podataka ispitivana je D'Agostino-Pearsonovim testom, na temelju kojeg se opredjeljujemo za parametrijske ili neparametrijske testove za usporeivanje skupova podataka. Svi su rezultati interpretirani s razinom zna ajnosti (( 0,05. Analiza varijance za normalno distribuirane uzorke u injena je jednosmjernim ANOVA testom kojim se usporeuju rezultati jedne zavisne varijable s jednom nezavisnom varijablom koja ima viae razli itih skupina ili kategorija. Za varijable koje nisu distribuirane po Gaussovoj razdiobi koriaten je Kruskal-Wallisov neparametrijski test, koji je za razliku od ANOVA-e baziran na medijanu, a ne aritmeti koj sredini. Za grafi ki prikaz rezultata koristio se box and whisker dijagram (slike x do y) u kojem centralni zeleni kvadrat predstavlja vrijednosti izmeu 1. i 3. kvartila, a sredianja zelena linija u kvadratu predstavlja medijan. Vertikalna zelena linija prote~e se od minimlne do maksimalne vrijednosti s time da su isklju ene outside vrijednosti, koje su 1,5 puta vee od interkvartilnog raspona, te far out vrijednosti koje su 3 puta vee od interkvartilnog raspona. Naran asti kru~i ozna ava srednju vrijednost, a crvena linija povezuje srednje vrijednosti 3 mjerenja. Plava linija predstavlja 95%-tni interval pouzdanosti za medijan. Za ispitivanje korelacije izmeu varijabli koje su normalno distribuirane ra unao se Pearsonov koeficijent korelacije i njegova neparametrijska verzija, Spearmanov koeficijent korealcije ranga s pridru~enom statisti kom zna ajnosti P. Koeficijent korelacije mo~e poprimiti vrijednost izmeu -1 i 1, a ato je ona bli~a nuli, to je korelacija slabija. Ako se jedna varijabla poveava poveavanjem druge, korelacija e biti pozitivna, a ako se jedna varijabla smanjuje dok se druga poveava, korelacija e biti negativna. Koeficijent korelacije tuma en je prema Coltonu (Tablica 2). Tablica SEQ Tabela \* ARABIC 2. Tuma enje koeficijenta korelacije prema Coltonu Koeficijent korelacijePovezanost0 do 0,25Nema povezanosti 0,26 do 0,50Slaba povezanost0,51 do 0,75Umjerena do dobra povezanost 076 do 1Dobra do izvrsna povezanostRezultati I RASPRAVA Deskriptivna statistika rezultata mjerenja U tablici 2 prikazani su statisti ki obraeni rezultati mjerenja 5 koagulacijskih parametara u skupini od N=29 ispitanika, u sve tri to ke uzorkovanja krvi.Uz podatke deskriptivne statistike u tablici su prikazane P-vrijednosti dobivene D'Agostino-Pearsonovim testom za svaku to ku mjerenja zasebno. One ukazuju da varijable fibrinogen, PV i TV slijede Gaussovu normalnu distribuciju jer im je P-vrijednost vea od nivoa signifikantnosti 0,05. Vrijednosti fibrinolize nisu normalno distribuirane te jekod nje maksimalna vrijednost ujedno i mod, odnosno vrijednost s najveom frekvencijom pojavljivanja. Iako se P-vrijednosti za Tablica  SEQ Tabela \* ARABIC 3. Deskriptivna statistika Varijable1. uzorkovanje95% CISDM95% CIMinMax25-75 PPAPTV(omjer) 0,940,9 -1,00,0990,90,9 - 1,00,71,20,9  1,00,685APTV QUOTE  26,025,0 - 27,02,722624,9 - 27,3213224,2 - 27,50,691Fibrinogen  QUOTE  2,602,4 - 2,80,4782,42,3 - 2,81,93,62,2  3,00,215Fibrinoliza  QUOTE  166,9149,6-184,245,40180120,0 - 210,090210120,0 - 210,0<0,0001PV  QUOTE  99,393,8 - 104,714,3310091,7 - 107,36912690,8 - 108,10,764Starost(godine)32,230,8 - 33,63,743130,0 - 33,0284130,0 - 34,30,028TV  QUOTE  18,918,5 - 19,31,041918,4  19,0172218,2 - 19,40,087Varijable2. uzorkovanje95% CISDM95% CIMinMax25-75 PPAPTV(omjer) 0,960,9 - 1,00,1041,00,9 - 1,00,71,20,9 - 1,00,045APTV QUOTE  26,525,4 - 27,62,872625,3 - 27,6213524,2 - 27,90,046Fibrinogen  QUOTE  2,512,3 - 2,70,4692,42,2 - 2,71,83,62,2 - 2,80,248Fibrinoliza  QUOTE  128,5110,9-146,046,20120103,8 - 140,06021090,0 - 157,50,217PV  QUOTE  96,690,9 - 102,415,159788,2 - 108,26612386,2 - 109,70,431Starost(godine)32,230,8 - 33,63,743130,0 - 33,0284130,0 - 34,30,028TV  QUOTE  18,618,2 - 19,11,281917,9 - 19,1172117,5 - 19,20,086Varijable3. uzorkovanje95% CISDM95% CIMinMax25-75 PPAPTV(omjer) 0,960,9 - 1,00,0981,00,9  1,00,71,20,9 - 1,00,049APTV QUOTE  26,525,4 - 27,52,692625,5 - 27,5213425,1 - 27,90,050Fibrinogen  QUOTE  2,582,4 - 2,80,48922,3 - 2,81,93,72,2 - 3,00,184Fibrinoliza  QUOTE  166,7147,9-185,649,55190140,3 - 210,080210120,0 - 210,00,032PV  QUOTE  97,291,6 - 102,814,699988,9 - 104,96812387,7 - 107,70,652Starost(godine)32,230,8 - 33,63,733130,0 - 33,0284130,0 - 34,30,028TV  QUOTE  18,818,4 - 19,31,281918,2 - 19,5152118,1 - 19,60,118 QUOTE  -aritmeti ka sredina, 95% CI -95%-tni interval pouzdanosti za aritmeti ku sredinu, odnosno medijan, SD-standardna devijacija, M-medijan, Min-minimum, Max-maksimum, 25-75 P-raspon izmeu 25. i 75. percentila, P-vjerojatnost normalne distribucije APTV (izra~eno kao omjer i u sekundama) u 2. i 3. to ki uzorkovanja nalaze u grani nom podru ju, dalje smo ih podvrgli parametrijskom ANOVA testu. Dob ispitanika ne slijedi normalnu raspodjelu jer D'Agostino-Pearsonovim testom dobivena P-vrijednost iznosi 0,0005 (slika 3). Raspon godina je od 28 do 41, s time da je medijan 31, zbog ega nismo donosili zaklju ke o njihovom utjecaju na do~ivljaj stresa niti samu analizu.  Slika  SEQ Slika \* ARABIC 3. Raspodjela ispitanika po dobi USPOREDBA REZULTATA S OBZIROM NA VRIJEME UZORKOVANJA Jednosmjernim ANOVA testom ispitali smo razlikuju li se izmjerene, normalno distribuirane vrijednosti (zavisne varijable) s obzirom na vrijeme uzorkovanja (nezavisne varijable). Dobivene P-vrijednost za PV, TV i fibrinogen vee su od nivoa signifikantnosti 0,05, ato zna i da za izmjerene vrijednosti nema statisti ki zna ajne razlike s obzirom na vrijeme uzorkovanja, ato je grafi ki prikazano na slikama 4, 5 i 6.     Na temelju aritmeti ke sredine i medijana za APTV moglo bi se zaklju iti da tijekom dvadeset etverosatnog de~urstva dolazi do poveanja vrijednosti. Meutim, rezultati Kruskal-Wallisovog neparametrijskog testa pokazali su da nema statisti ki zna ajne razlike izmeu mjerenja u razli itim vremenima uzorkovanja kao ato je prikazano na slici (slika 7). Naime, P-vrijednost za APTV izra~en kao omjer je 0,725, a za APTV u sekundama P=0,714.  Kruskal-Wallisovim testom pokazalo se da postoji statisti ki zna ajna razlika u izmjerenim vrijednostima fibrinolize izmeu barem dvije to ke uzorkovanja (P=0,0043). Daljnjom post hoc analizom utvreno je da ta razlika proizlazi iz razlike izmeu prvog i drugog mjerenja, te drugog i treeg mjerenja, odnosno da je vrijeme fibrinolize krae nave er nego ujutro prije i nakon de~urstva, ato se mo~e vidjeti na slici 8.  Sve navedene testove napravili smo i posebno za muaku i ~ensku populaciju, iako male razlike postoje, one nisu statisti ki zna ajne. Ispitivanje korelacije Ra unanjem Pearsonovog i Spearmanovog koeficijenta korelacije za svaki par varijabli utvreno je da kod veine ispitivanih varijabli nema meusobne zna ajne korelacije. Primijeena je umjerena povezanost vrijednosti PV-a i APTV-a (koeficijent korelacije=-0,558, P=<0.0001), te PV-a i fibrinogena (koeficijent korelacije=0,551, P=<0,0001) u uzorku ispitanika N=29. Kada smo usporeivali vrijednosti kod muake populacije (N=14) naena je dobra povezanost vrijednosti PV-a i fibrinogena (koeficijent korelacije=0,757, P=<0,0001). Kod ~enske populacije (N=15) dobro koreliraju vrijednosti PV-a s APTV-om, s koeficijentom korelacije -0,715 za omjer i -0,714 za APTV u sekundama (P=<0,0001). Rasprava Brojni radovi dokazuju povezanost stresa na radnom mjestu s poveanim rizikom za razvoj kardiovaskularnih bolesti. Rad lije nika u dvadeset etverosatnom de~urstvu ne samo da je fizi ki naporan zbog nedostatka odmora i sna, ve predstavlja i psihosocijalno optereenje zbog odgovornosti za pacijente, a esto i vremenske ograni enosti za donoaenje odluka. U studiji koju su proveli Tr i suradnici dokazano je da se lije nici razli itih specijalnostikoji rade u nonim smjenama esto suo avaju sa stresom(Tr i sur., 2016). Uz to, mladi lije nici na specijalisti kom usavraavanju mogu biti nesigurniji zbog nedostatka iskustva. U gore navedenoj studiji dokazano je da su takvi lije nici pod veim stresom od starijih kolega te da imaju manju kontrolu nad poslom. Za naae ispitivanje odabrali smo upravo mlade lije nike na specijalisti kom usavraavanju. Za potrebe ovog rada nismo proveli upitnik o dvadeset etirisatnom de~urstvu kao stresoru kod lije nika, no iz dosadaanje literature pretpostavili smo da je on prisutan i meu naaim ispitanicima koji svi rade u bolnicama u Zagrebu. Naime, istra~ivanje provedeno u Zagrebu 2009. godine na 1900 zdravstvenih djelatnika u bolnicama ispitivalo je njihov do~ivljaj odreenih stresora na radu te razlikuje li se taj do~ivljaj stresa izmeulije nika i medicinskih sestara, odnosno tehni ara. Pokazalo se da su glavni stresori kod lije nika vezani uz financijske mogunosti, organizaciju i neadekvatan prostor, no dvadeset etirisatna de~urstva su takoer svrstana meu naj eae stresore meu lije nicima, kako je prikazano na slici 9(Kne~evi i sur., 2009). Naae istra~ivanje obuhvatilo je gotovo podjednako muakarce (N=14) i ~ene (N=15), no zna ajne razlike izmeu spolova nisu uo ene te ne mo~emo donositi zaklju ke o razli itom utjecaju dvadeset etirisatnog de~urstva na muakarce i ~ene. Ipak, Santhi i sur. u svom radu pokazuju da ~ene te~e podnose none smjene nego muakarci te su pod veim rizikom od ozljeda na radu tijekom produ~enog radnog vremena ili nestandardnih obrazaca rada(Santhi i sur., 2016). Populacija obuhvaena naaim istra~ivanjem su mladi lije nici (medijan za godine je 31), te stoga nismo zaklju ivali o povezanosti stresa i dobi. S druge strane, odabirom tako uskog raspona u godinama mogli smo zanemariti utjecaj kardiovaskularnih oateenja koja se gomilaju s godinama na rezultate analize ato nam je omoguilo promatranje samo akutnih promjena u hemostazi. Dobiveni rezultati pokazali su da nema statisti ki zna ajne promjene u koagulacijskim parametrima: PV-u, APTV-u, TV-u i koncentraciji fibrinogena 12 i 24 sata nakon po etka de~urstva u odnosu na vrijednosti 15 minuta prije po etka dvadeset etirisatnog de~urstva, dok je vrijeme fibrinolize pokazalo zna ajne promjene vrijednosti. Meutim, to ne zna i da tijekom de~urstva nije doalo do aktivacije koagulacijskog sustava, ve da provedene analize koje odra~avaju aktivaciju koagulacijske kaskade nisu dovoljno osjetljive na njezine male promjene. Hemostaza je vrlo dobro regulirani sustav te aktivacijom koagulacije dolazi i do aktivacije sustava fibrinolize upravo kako ne bi doalo do hiperkoagulabilnosti i nastanka lokalnih ugruaaka. Zbog toga se eventualne promjene nisu odrazile na rezultate mjerenja. Na to da i kratke epizode stresa na radnom mjestu mogu dovesti do aktivacije koagulacije ukazuje studija provedena 2011. godine u eakoj na skupini 13 zdravih lije nika s odjela intenzivne skrbi. U studiji se prou avao upalni i mikrovaskularni odgovor te koagulacijske promjene koje nastaju zbog dvadeset etirisatnog de~urstva. Praene su promjene u upalnim biljezima, adhezijskim molekulama i sublingvalnoj mikrocirkulaciji prije i nakon de~urstva, no primijeen je jedino zna ajan porast plazmatskih razina TF-a. Znamo da TF igra klju ne uloge u hemostazi te je glavni fizioloaki inicijator koagulacije, a njegove poveane razine induciraju blago hiperkoagulabilno stanje(Matejovic i sur., 2011). Rezultati naaih mjerenja pokazali su da je vrijeme fibrinolize kod lije nika krae nave er (M=120 min) nego ujutro prije (M=180 min) i nakon dvadeset etirisatnog de~urstva (M=190 min). Produ~eno vrijeme fibrinolize (hipofibrinolizu) u jutarnjim satima mo~emo objasniti fizioloakim cirkadijarnim promjenama u aktivnosti fibrinoliti kog sustava. Naime, rezultati jedne studije na mladim, zdravim ispitanicima oba spola upuuju na smanjenu bazalnu sistemsku aktivaciju plazminogena u jutarnjim satima. Mjerenja u 8 ujutro i 8 nave er pokazala su smanjenje vrijednosti inhibitora fibrinolize PAI-1 tijekom dana, ato je praeno porastom plazmatske aktivnosti t-PA (Kapiotis i sur., 1997). Mogue je da dnevne varijacije u fibrinoliti koj aktivnosti diktira upravo cirkadijarni ritam PAI-1, na koji ne utje u doba dana i budnost(Irokawa i sur., 1998). Pored fizioloakog cirkadijarnog ritma, naai su rezultati pokazali blago poviaenje vrijednosti fibrinolize ujutro nakon dvadeset etirisatnog de~urstva u odnosu na jutro prije po etka de~urstva. To zna i da je potrebno viae vremena za razgradnju ugruaka, ato bismo mogli povezati s do~ivljenim stresom na radnom mjestu. Ta se razlika, iako ne zna ajna, poklapa sa rezultatima studije koju su proveli von Diest i suradnici(van Diest, 2002). Oni su ispitivali povezanost stanja nazvanog vitalna iscrpljenost s izra~enijim dnevnim promjenama u hemostatskim parametrima. Kod ispitanika s ~ivotnom iscrpljenoau izmjerili su viae razine t-PA antigena, PAI-1 i tPA-PAI antigena u 7 sati ujutro u odnosu na kontrolnu skupinu. Te se razine nisu puno mijenjale u 6 sati popodne u odnosu na kontrolu skupinu. Njihova koagulacijska mjerenja nisu pokazala zna ajnu razliku izmeu pojedinaca sa ~ivotnom iscrpljenoau i kontrolne skupine, ato se takoer poklapa s naaim mjerenjima prema kojima nismo imali mjerljivu aktivaciju koagulacijskog sustava. Nedostatak naaeg istra~ivanja zasigurno je to ato nismo imali kontrolnu skupinu koja bi se sastojala od lije nika na specijalisti kom usavraavanju, iste dobne skupine, koji bi na dan uzorkovanja imali slobodan dan. To je bilo teako organizirati jer bi se vaenje krvi trebalo provesti van bolnice, idealno u domu ispitanika, i to u tri navrata. Pomou kontrolne skupine mogli bismo zaklju iti u kojoj mjeri su promjene fibrinolize uzrokovane stresom u de~urstvu, a u kojoj mjeri su posljedica fizioloakog cirkadijarnog ritma. Uz to bismo mogli zaklju iti i o promjenama u vrijednostima ostalih mjerenih koagulacijskih parametara. Takoer, vei broj ispitanika omoguio bi jasnije razlikovanje promjena koagulacijskih parametara izmeu muakaraca i ~ena. U istra~ivanju smo promatrali utjecaj samo jednog dvadeset etirisatnog de~urstva na koagulacijske parametre ato o ito nije dovoljno velik psihosocijalni stres za pokretanje veih promjena u hemostazi. Stoga je trebalo odabrati pretrage na koje e se odraziti i vrlo male promjene u hemostazi, kao ato su ve spomenuti pretrombotski markeri u koje spadaju D-dimeri i fibrinopeptid A. Ostali koagulacijski parametri ije poviaene vrijednosti ukazuju na mogui razvoj KVB su aktivnosti FVII i FVIII, vWF antigen, t-PA antigen, PAI-1 antigen i kompleks plazmin-2-antiplazmin. Slabljenje fibrinoliti ke aktivnosti odra~avaju niske razine antitrombina III potroaenog u antikoagulantnom procesu (von Knel i sur., 2001). Predla~emo i odreivanje tkivnog faktora, ije smo zna enje objasnili ranije u ovoj raspravi. ak i kada bismo utvrdili male promjene u hemostazi zbog jednog dvadeset etirisatnog de~urstva, to ne bi bilo klini ki zna ajno jer je mogue da se odmorom nakon de~urstva ti parametri uravnote~e. Zbog toga bi ispitanike trebalo pratiti kroz du~i vremenski period kako bi se ispitale kumulativne posljedice rada u de~urstvima na zdravlje lije nika. Tada bismo vidjeli postoje li promjene izmeu mladih lije nika iji je rad povremeno organiziran u dvadeset etirisatna de~urstva i kontrolne skupine lije nika koji ne de~uraju nou, kao ato su primjerice lije nici obiteljske medicine. Saznanja o utjecaju nonog rada na razvoj kardiovaskularnih bolesti bitna su radi praenja zdravlja radnika preko preventivnih pregleda. Preporu uje se da se zdravstveni pregledi radnika koji rade nou provode redovito svake 3 godine, uz mogunost obavljanja i ranijeg pregleda prema indikaciji nadle~nog specijalista medicine rada, lije nika ope medicine ili poslodavca. Uz zdravstveni pregled se preporu uje i pregled kod psihologa. 2013. godine stupio je na snagu Pravilnik o sadr~aju, na inu i rokovima zdravstvenih pregleda nonih radnika (http://narodne-novine.nn.hr/clanci/sluzbeni/2013_10_122_2613.html) koji utvruje sadr~aj, na in i rokove zdravstvenih pregleda nonih radnika, odreuje na in utvrivanja zdravstvene sposobnosti, rokove u kojima se mora ponoviti utvrivanje zdravstvene sposobnosti te sadr~aj i na in izdavanja svjedod~be o zdravstvenoj sposobnosti nonog radnika(Pti ar, 2013). U ovom smo se radu fokusirali isklju ivo na lije nike koji rade u bolnicama, jer za razliku od drugih zanimanja, uz manjak sna i poremeaj cirkadijarnog ritma, lije nici moraju donositi odluke koje izravno utje u na zdravlje, a mo~da i ~ivot njihovih pacijenata, a uz to su pod vremenskim pritiskom, ato zbog hitnosti stanja, ato zbog veeg broja pacijenata po jednom lije niku. No, ne smijemo zaboraviti i koliko zamorno mo~e biti neprestano raditi s ljudima koji nisu uvijek suradljivi. Iako u ovom radu nismo dokazali vee promjene u koagulacijskim parametrima, smatramo da su potrebna dodatna istra~ivanja zbog vrlo stresnog posla koji lije nici obavljaju, kako bi se ato viae saznalo o njihovom riziku za razvoj kardiovaskularnih i drugih bolesti. Ti bi rezultati mogli utjecati na eventualnu reorganizaciju rada u de~urstvima i uvoenje eaih preventivnih pregleda te odreivanja korisnijih biokemijskih pokazatelja promjena uzrokovanih stresom, a sve to s ciljem prevencije razvoja KVB u populaciji lije nika. Zaklju ci Nisu dokazane statisti ki zna ajne promjene u vrijednostima PV-a, APTV-a, TV-a i koncentracije fibrinogena tijekom i nakon dvadeset etirisatnog de~urstva u odnosu na vrijednosti prije de~urstva. Navedene globalne pretrage i koncentracija fibrinogena nisu dovoljno osjetljive na eventualne male promjene u koagulacijskom sustavu. 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Soc Psihijatr, 2013, 41, 118 122. Versteeg HH, Heemskerk JWM, Levi M, Reitsma PH. New fundamentals in hemostasis. Physiol Rev, 2013, 93, 327 358. von Knel R, Mills PJ, Fainman C, Dimsdale JE. Effects of psychological stress and psychiatric disorders on blood coagulation and fibrinolysis: a biobehavioral pathway to coronary artery disease? Psychosom Med, 2001, 63, 531 544. von Knel RM. Acute mental stress and hemostasis: when physiology becomes vascular harm.Thromb Res, 2015, 135(1), 52-55. Vrijkotte TGM, van Doornen LJP, de Geus EJC. Work Stress and Metabolic and Hemostatic Risk Factors Psychosom Med, 1999, 61, 796 805. Sa~etak / Summary Sa~etak Dvadeset etirisatna de~urstva predstavljaju jedan od veih stresora za lije nike jer osim umora i poremeaja cirkadijarnog ritma uklju uju odgovornost u donoaenju odluka i rad s pacijentima koji nisu uvijek suradljivi. Poznato je da stres na radnom mjestu dovodi do aktivacije koagulacijskog sustava, a nastalo hiperkoagulabilno stanje pogoduje razvoju kardiovaskularnih oateenja. Cilj ovog rada bio je ispitati utjecaj dvadeset etirisatnog de~urstva na globalne koagulacijske pretrage: PV, APTV i TV te na koncentraciju fibrinogena i vrijeme fibrinolize. U istra~ivanju je sudjelovalo 29 lije nika na specijalisti kom usavraavanju. Lije nicima je krv uzeta tri puta: prije, tijekom i nakon de~urstva. Rezultati su pokazali da nije doalo do zna ajne aktivacije koagulacijskog sustava koja bi se odrazila na globalne pretrage i koncentraciju fibrinogena. Meutim, pokazalo se da je vrijeme fibrinolize dobiveno euglobulinskim testom produ~eno u dva jutarnja mjerenja u odnosu na mijerenje tijekom de~urstva u ve ernjim satima. Nije jasno u kojoj je mjeri navedena promjena uzrokovana stresnim de~urstvom, a u kojoj fizioloakim oscilacijama u aktivaciji fibrinolize. SUMMARY 24-h work shifts are known to be one of the major stressors for physicians because of the circadian rhythm disruption and tiredness. Besides that, they include responsibility in making decisions and working with patients who are not always cooperative.Studies have shown that work stress leads to the activation of the coagulation system, and that the resulting hypercoagulable state favors the development of cardiovascular damage. The aim of this study was to examinethe effects of a 24-h work shift on global coagulation parameters: prothrombin time (PT), activated partial thromboplastin time (APTT) and thrombin time (TT), but also the fibrinogen concentration and fibrinolysistime. The study group included 29 physicians on education. The blood was sampled three times: before, during and after the work shift. Our results have shown that there was no coagulation system activation that would affect global tests and fibrinogen concentration. However, fibrinolysis timeobtained with the euglobulin test was extended in two morning measurements compared to the evening measurement. It is not clear if this is caused by work stress or the physiologic oscillations in fibrinolysis. Prilozi Popis kratica ACTH -adrenokortikotropni hormon APTV -aktivirano parcijalno tromboplastinsko vrijeme CRH -kortikotropin-otpuatajui hormon F  faktor zgruaavanja IL -interleukin KVB -kardiovaskularne bolesti PAI-1 -inhibitor tkivnog aktivatora plazminogena PV -protrombinsko vrijeme TNF- - faktora tumorske nekroze alfa t-PA -tkivni aktivator plazminogena TV -trombinsko vrijeme u-PA -urokinaza Temeljna dokumentacijska kartica Sveu iliate u Zagrebu Farmaceutsko-biokemijski fakultet Studij: Medicinska biokemija Zavod za medicinsku biokemiju i hematologiju Domagojeva 2, 10000 Zagreb, Hrvatska Diplomski radPOVEZANOST STRESA I AKTIVACIJE KOAGULACIJSKOG SUSTAVAMarta KmetSA}ETAKDvadeset etirisatna de~urstva predstavljaju jedan od veih stresora za lije nike jer osim umora i poremeaja cirkadijarnog ritma uklju uju odgovornost u donoaenju odluka i rad s pacijentima koji nisu uvijek suradljivi. Poznato je da stres na radnom mjestu dovodi do aktivacije koagulacijskog sustava, a nastalo hiperkoagulabilno stanje pogoduje razvoju kardiovaskularnih oateenja. Cilj ovog rada bio je ispitati utjecaj dvadeset etirisatnog de~urstva na globalne koagulacijske pretrage: PV, APTV i TV te na koncentraciju fibrinogena i vrijeme fibrinolize. U istra~ivanju je sudjelovalo 29 lije nika na specijalisti kom usavraavanju. Lije nicima je krv uzeta tri puta: prije, tijekom i nakon de~urstva. Rezultati su pokazali da nije doalo do zna ajne aktivacije koagulacijskog sustava koja bi se odrazila na globalne pretrage i koncentraciju fibrinogena. Meutim, pokazalo se da je vrijeme fibrinolize dobiveno euglobulinskim testom produ~eno u dva jutarnja mjerenja u odnosu na mijerenje tijekom de~urstva u ve ernjim satima. Nije jasno u kojoj je mjeri navedena promjena uzrokovana stresnim de~urstvom, a u kojoj fizioloakim oscilacijama u aktivaciji fibrinolize.Rad je pohranjen u Sredianjoj knji~nici Sveu iliata u Zagrebu Farmaceutsko-biokemijskog fakulteta.Rad sadr~i:37 stranica, 9 grafi kih prikaza, 3 tablice i 37 literaturnih navoda. Izvornik je na hrvatskom jeziku.Klju ne rije i:Stres na poslu, smjenski rad, hiperkoagulabilnost, fibrinoliza, koagulacijske pretrageMentor:Dr. sc. Nada Vrki, izvanredni profesor Sveu iliata u Zagrebu Farmaceutsko-biokemijskog fakulteta.Ocjenjiva i:Dr. sc. Nada Vrki, izvanredni profesor Sveu iliata u Zagrebu Farmaceutsko-biokemijskog fakulteta.Dr. sc.Marija Grdi Rajkovi, docent Sveu iliata u Zagrebu Farmaceutsko-biokemijskog fakulteta.Dr. sc.Sandra `upraha Goreta, docent Sveu iliata u Zagrebu Farmaceutsko-biokemijskog fakulteta. Rad prihvaen: lipanj 2017. Basic documentation card University of Zagreb Faculty of Pharmacy and Biochemistry Study: Medical biochemistry Department of Medical Biochemistry and Hematology Domagojeva 2, 10000 Zagreb, Croatia Diploma thesisRELATIONSHIP BETWEEN STRESS AND ACTIVATION OF THE COAGULATION SYSTEMMarta KmetSUMMARY24-h work shifts are known to be one of the major stressors for physicians because of the circadian rhythm disruption and tiredness. Besides that, they include responsibility in making decisions and working with patients who are not always cooperative. Studies have shown that work stress leads to the activation of the coagulation system, and that the resulting hypercoagulable state favors the development of cardiovascular damage. The aim of this study was to examine the effects of a 24-h work shift on global coagulation parameters: prothrombin time (PT), activated partial thromboplastin time (APTT) and thrombin time (TT), but also the fibrinogen concentration and fibrinolysis time. The study group included 29 physicians on education. The blood was sampled three times: before, during and after the work shift. Our results have shown that there was no coagulation system activation that would affect global tests and fibrinogen concentration. However, fibrinolysis time obtained with the euglobulin test was extended in two morning measurements compared to the evening measurement. It is not clear if this is caused by work stress or the physiologic oscillations in fibrinolysis.The thesis is deposited in the Central Library of the University of Zagreb Faculty of Pharmacy and Biochemistry.Thesis includes:37 pages, 9 figures, 3 tables and 37 references. Original is in Croatian language.Keywords:Work stress, shift work, hypercoagulabilityt, fibrinolysis, coagulation testsMentor:Nada Vrki, Ph.D. Associate Professor, University of Zagreb Faculty of Pharmacy and Biochemistry Reviewers:Nada Vrki, Ph.D. Associate Professor, University of Zagreb Faculty of Pharmacy and Biochemistry Marija Grdi Rajkovi, Ph.D. Assistant Professor, University of Zagreb Faculty of Pharmacy and BiochemistrySandra `upraha Goreta, Ph.D. Assistant Professor, University of Zagreb Faculty of Pharmacy and BiochemistryThe thesis was accepted: June 2017.      PAGE \* MERGEFORMAT 0  PAGE \* MERGEFORMAT 37 Slika  SEQ Slika \* ARABIC 1. Pojednostavljeni prikaz koagulacijske kaskade (preuzeto i prilagoeno s http://www.frca.co.uk/images/clotting_cascade.gif, pristupljeno 3.4.2017.) Slika  SEQ Slika \* ARABIC 2. Fibrinoliza (preuzeto i prilagoeno s https://www.ebi.ac.uk/interpro/potm/2006_11/Page1.htm) P = 0,773 Slika  SEQ Slika \* ARABIC 4. Usporedba rezultata PV-a s obzirom na vrijeme uzorkovanja. To ke uzorkovanja: 15 min prije po etka de~urstva te 12 i 24 sata nakon po etka de~urstva P = 0,749 Slika  SEQ Slika \* ARABIC 5. Usporedba koncentracija fibrinogena s obzirom na vrijeme uzorkovanja. To ke uzorkovanja: 15 min prije po etka de~urstva te 12 i 24 sata nakon po etka de~urstva P = 0,761 Slika  SEQ Slika \* ARABIC 6. Usporedba rezultata TV-a s obzirom na vrijeme uzorkovanja. To ke uzorkovanja: 15 min prije po etka de~urstva te 12 i 24 sata nakon po etka de~urstva P = 0,714 Slika  SEQ Slika \* ARABIC 7. Usporedba rezultata APTV-a izra~enog u sekundama s obzirom na vrijeme uzorkovanja. To ke uzorkovanja: 15 min prije po etka de~urstva te 12 i 24 sata nakon po etka de~urstva Slika  SEQ Slika \* ARABIC 8. Usporedba rezultata fibrinolize s obzirom na vrijeme uzorkovanja. To ke uzorkovanja: 15 min prije po etka de~urstva te 12 i 24 sata nakon po etka de~urstva Slika  SEQ Slika \* ARABIC 9. 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