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Determination of cutoff of Alzheimer's dementia biomarkers in cerebrospinal fluid
No full textU ovom diplomskom radu analizirane su koncentracije Tau proteina, fosforiliranog Tau
proteina i Aβ42 u cerebrospinalnoj tekućini pacijenata s različitim oblicima demencija, kao i u
kontrolnoj skupini. Određivanje ovih biomarkera ključno je za rano otkrivanje, praćenje i
potvrdu Alzheimerove demencije. Cilj rada bio je odrediti granične vrijednosti za sva tri
biljega za testove proizvođača Euroimmun iz Njemačke te ispitati dijagnostičku vrijednost
svakog pojedinog biljega.
Istraživanje je provedeno na skupini od 220 ispitanika s ranije utvrđenim dijagnozama, a
skupina je obuhvaćala 59 pacijenata s potvrđenom Alzheimerovom demencijom, 14 s
miješanom demencijom, 93 s ostalim demencijama ili neurodegenerativnim bolestima te 54
pacijenata bez dokazane neurodegenerativne bolesti koji su predstavljali kontrolnu skupinu.
Statističkom analizom rezultata dobivene su granične vrijednosti za Tau protein <414 ng/L, za
pTau protein 734 ng/L. Analizom ROC krivulje pokazano je i da biljezi
imaju dobar odnos osjetljivosti i specifičnosti.
Također, dobiveni rezultati potvrdili su dijagnostičku vrijednost navedena tri biljega za
razlikovanje Alzheimerove demencije od ostalih oblika demencije pri čemu se kao
najpouzdaniji biljeg pokazao pTau protein.In this thesis, the concentrations of Tau protein, phosphorylated Tau protein, and Aβ42 were
analyzed from the cerebrospinal fluid of patients with various forms of dementia as well as in
a control group. Determining these biomarkers is crucial for the early detection, monitoring,
and confirmation of Alzheimer's dementia. The aim of the study was to establish cutoff values
for all three biomarkers using tests from the Euroimmun manufacturer in Germany and to
assess the diagnostic value of each individual biomarker.
The research was conducted on a group of 220 subjects with previously established diagnoses.
The group included 59 patients with confirmed Alzheimer's dementia, 14 with mixed
dementia, 93 with other dementias or neurodegenerative diseases, and 54 patients without
proven neurodegenerative disease, who represented the control group.
Statistical analysis of the results yielded cutoff values for Tau protein at <414 ng/L, for pTau
protein at 734 ng/L. ROC curve analysis also showed that the
biomarkers have a good sensitivity and specificity ratio.
Additionally, the results confirmed the diagnostic value of the three biomarkers in
distinguishing Alzheimer's dementia from other forms of dementia, with pTau protein proving
to be the most reliable biomarker
The association of two polymorphisms in the endothelial nitric oxide synthase gene and paediatric haemorrhagic stroke
No full textHemoragijski moždani udar (HMU) je akutni neurološki poremećaj koji nastaje uslijed rupture krvne žile, a stanje
je uzrokovano brojnim nasljednim i stečenim poremećajima. Endotelna sintaza dušikovog oksida ima ključnu
ulogu sintezi dušikovog oksida koji sudjeluje u cerebrovaskularnoj regulaciji, modulaciji razvoja neurona,
postnatalnom razvoju, ali je i medijator hipoksično-ishemijskoga oštećenja i oksidativnoga stresa. Polimorfizmi
NOS3 utječu na izražaj endotelne sintaze dušikovog oksida i dostupnost dušikovog oksida. Cilj ovoga istraživanja
bio je ispitati povezanost pojedinačnih polimorfizama NOS3 -786 T>C i 894 G>T i potencijalnoga haplotipa s
pojavom HMU-a u djece u Hrvatskoj. U istraživanje je bilo uključeno 32 djece s dijagnozom HMU-a starosne
dobi do 18 godina i 32 zdrave djece podudarne po dobi i spolu. U sve djece su genotipizirani polimorfizmi NOS3
-786 T>C i 894 G>T molekularno-genetičkom metodom, korištenjem kompleta za analizu CVD StripAssay® A
(ViennaLab Diagnostics GmbH, Beč, Austrija). U ispitivanju su se koristili uzorci genomske deoksiribonukleinske
kiseline svih ispitanika prethodno izdvojeni iz preostalih uzorka periferne krvi koji su korišteni za rutinske
laboratorijske analize. Rezultati statističke obrade podataka pokazali su podjednaku distribuciju genotipova i alela
za pojedinačne polimorfizme NOS3 -786 T>C i 894 G>T u djece s HMU-om i u kontrolnoj skupini, zbog čega
nije utvrđena statistički značajna povezanost ispitivanih polimorfizama s pojavom HMU-a u djece. Analiza
haplotipova NOS3 nije provedena budući da dobivena vrijednost parametra D' = 0,454 nije zadovoljila kriterije za
uključivanje dva polimorfizma u daljnju analizu. Daljnjim ispitivanjem na većem broju ispitanika i uključivanjem
analize više polimorfizama ili cijelih gena, primjerice, primjenom metoda sekvenciranja sljedeće generacije, moći
će se detaljnije istražiti povezanost endotelne sintaze dušikovoga oksida s pojavom HMU-a u djece.Haemorrhagic stroke (HS) is an acute neurological disorder that occurs due to the rupture of a blood vessel. It is a
condition, caused by numerous hereditary and acquired disorders. Endothelial nitric oxide synthase plays a key role in
the synthesis of nitric oxide, which is involved in cerebrovascular regulation, modulation of neuronal development,
postnatal development, and also acts as a mediator of hypoxic-ischemic injury and oxidative stress. NOS3
polymorphisms affect the expression of endothelial nitric oxide synthase and the availability of nitric oxide. The aim of
this study was to examine the association between the NOS3 -786 T>C and 894 G>T polymorphisms, including its
haplotype, and the occurrence of haemorrhagic stroke in children in Croatia. The study included 32 children diagnosed
with HS up to the age of 18 years and 32 healthy children matched by age and sex that represented the control group. In
all children included in the study, NOS3 -786 T>C and 894 G>T polymorphisms were genotyped using a moleculargenetic method, the CVD StripAssay® A analysis kit (ViennaLab Diagnostics GmbH, Vienna, Austria). In this study,
samples of genomic deoxyribonucleic acid from all participants were used that were previously extracted from residual
peripheral blood samples used for routine laboratory analyses. The results of the statistical analysis showed an equal
distribution of genotypes and alleles for the individual NOS3 -786 T>C and 894 G>T polymorphisms in children with
HS and in the control group. Therefore, no statistically significant association was found between the examined
polymorphisms and the occurrence of HS in children. Analysis of NOS3 haplotypes was not performed since the obtained
value of the parameter D' = 0.454 have not meet the criteria for including two polymorphisms in further analysis. Further
studies with a larger number of participants and the inclusion of analyses of more polymorphisms or entire genes, for
example, using next-generation sequencing methods, could offer detailed investigation of the association between
endothelial nitric oxide synthase and the occurrence of haemorrhagic stroke in children
Importance of detecting IDH1 and IDH2 mutations in patients with acute myeloid leukemia diagnosis
No full textAkutna mijeloična leukemija visoko je heterogena hematološka zloćudna bolest, koju karakterizira abnormalna
proliferacija nezrelih stanica mijeloidne loze. AML je bolest starije životne dobi s medijanom od 67 godina. U pozadini
AML-a stoje broje genetske promjene pa najnovije smjernice za stratifikaciju rizika i postavljanje dijagnoze ističu
važnost određivanja citogenetskih i genskih promjena. Iako je došlo do znatnog napretka u dijagnostici i razumijevanju
patofiologije AML-a, osnovna terapija (intenzivna kemoterapija) ostaje ista dok je petogodišnje ukupno preživljavanje
i dalje nisko posebno kod recidiva, starijih osoba, sekundarnog i rezistentnog AML. Odobrene spasonosne terapije
postoje samo za one s FLT3 te IDH1/2 mutacijama. Terapija IDH inhibitorima nije se pokazala učinkovitijom od
standardnih terapija kod novodijagnosticiranog AML-a, ali kod bolesnika s R/R AML-om omogućuje postizanje CR u
oko 20 % slučajeva dok stopa ukupnog odgovora uključujući i hematološko poboljšanje iznosi oko 40 %. Zbog toga je
rana detekcija ovih mutacija postala bitna. ELN preporuča da se genska analiza na prisutnost IDH1/2 mutacija provede
unutar 3 do 5 dana od zaprimanja uzoraka. Prema tome, cilj ovog rada bio je uspostaviti dijagnostički postupak za
dokazivanje najčešćih mutacija u genima IDH1 i IDH2 u rutinskoj laboratorijskoj dijagnostici. Odabran je brzi i osjetljivi
komercijalni test, kojim je analizirano 40 uzoraka izdvojene DNA bolesnika s dijagnozom AML-a kojima je prethodno
dokazana NPM1 mutacija. Dokazane su ukupno 22 mutacije – 12 u IDH1 genu (7 na kodonu R132 i 5 varijanti G105G)
i 10 u IDH2 genu (7 na kodonu R140 i 3 na kodonu R172). Dio rezultata potvrđen je metodom Sangerovog sekvenciranja
uz potpuno slaganje. Utvrđena je očekivana učestalost IDH mutacija u skupini bolesnika s NPM1 mutiranim AML-om
te test zadovoljava temeljne kriterije za uporabu u rutinskoj laboratorijskoj dijagnostici.Acute myeloid leukemia (AML) is a highly heterogeneous hematologic malignancy marked by abnormal proliferation
of myeloid progenitors, primarily affecting elderly patients, with a median age of 67 years. Given the numerous genetic
abnormalities in AML, current guidelines for risk stratification and diagnosis underscore the importance of identifying
cytogenetic and genetic alterations. Although significant advances have been made in AML diagnostics and
understanding AML's pathophysiology, intensive chemotherapy remains the standard treatment. Still, 5-year overall
survival remains low, particularly for the elderly, relapsed, secondary, and resistant AML. Currently, approved targeted
therapies exist only for patients with FLT3 and IDH1/2 mutations. While IDH inhibitors have not demonstrated superior
efficacy to standard therapy in newly diagnosed AML, in relapsed/refractory AML, they enable complete remission in
about 20% of cases, with an overall response rate of approximately 40% when including hematologic improvements.
Thus, early detection of these mutations is critical. The European LeukemiaNet (ELN) recommends that genetic analysis
for IDH1/2 mutations be conducted within 3 to 5 days of sample collection. This thesis aimed to establish a diagnostic
protocol for the routine laboratory detection of IDH1 and IDH2 mutations. A fast, sensitive commercial test was
employed to analyze DNA from 40 AML patients with a previously confirmed NPM1 mutation. 22 mutations were
detected—12 in the IDH1 gene (7 at codon R132 and 5 G105G variants) and 10 in the IDH2 gene (7 at codon R140 and
3 at codon R172). Sanger sequencing confirmed a subset of results with full concordance. The study confirmed the
expected frequency of IDH mutations among NPM1-mutated AML patients and demonstrated that the chosen test meets
essential criteria for use in routine laboratory diagnostics
Influence of inflammation and hypoxia on hepcidin concentration in patients with COVID-19
No full textKod bolesnika s COVID-19 istovremeno su prisutni upala i hipoksija kao signali sa suprotnim učinkom na
ekspresiju hepcidina. Istraživanje se temeljilo na hipotezi da je u skupini hipoksičnih bolesnika s COVID-19
koncentracija hepcidina u krvi snižena i da je razina parametara statusa željeza promijenjena u odnosu na skupinu
normoksičnih bolesnika. Glavni cilj istraživanja bio je ispitati postoji li razlika u koncentraciji hepcidina i
čimbenika koji reguliraju koncentraciju hepcidina kod normoksičnih i hipoksičnih bolesnika s COVID-19 te u
skupini zdravih ispitanika. Također, željelo se ispitati postoji li povezanost koncentracije hepcidina i parametara
statusa željeza, eritropoeze, hipoksije i sustavne upale u ispitivanim skupinama. Primijenjeni su omjeri koji su
uključivali hepcidin i molekule koje su povezane s hepcidinom (hepcidin/željezo, feritin/hepcidin,
hepcidin/interleukin-6 (IL-6), hepcidin/C-reaktivni protein (CRP), hepcidin/eritropoetin (EPO)) kako bi se
istodobno pratio njihov suodnos i međusoban utjecaj, ali i omjeri kojima se povezuju i stavljaju u suodnos različiti
upalni parametri (CRP/IL-6, omjeri CRP-a i IL-6 s leukocitnim podskupinama, omjeri neutrofila i limfocita
(NLR), neutrofila i monocita (NMR) te monocita i limfocita (MLR)) kako bi se pokušalo što bolje razlikovati
skupine bolesnika s COVID-19 koje imaju različit obrazac kliničke prezentacije i ishode bolesti.
U istraživanje je uključeno 96 bolesnika s COVID-19 i 47 zdravih ispitanika podudarnih po dobi i spolu.
Uzorkovanje je provedeno po primitku na Hitni infektološki prijem te su bolesnici s COVID-19 podijeljeni u
skupinu normoksičnih i hipoksičnih bolesnika na temelju zasićenje hemoglobina kisikom (SpO2), a daljnjim
praćenjem tijeka bolesti podijeljeni su u skupine prema težini bolesti.
Najvažniji rezultati ovog istraživanja ukazuju da su plazmatske koncentracije hepcidina, feritina, EPO-a, CRP-a i
IL-6 te omjeri hepcidin/željezo i feritin/hepcidin bili značajno viši, dok su omjeri hepcidin/IL-6 i hepcidin/CRP
bili značajno niži u hipoksičnih u odnosu na normoksične bolesnike te u bolesnika s teškim ili kritičnim oblikom
COVID-19 u odnosu na one s blagim i umjerenim oblikom bolesti. Nađena je dobra povezanost hepcidina s CRPom i IL-6 kod normoksičnih bolesnika, dok je u skupini hipoksičnih bolesnika povezanost s CRP-om bila slaba, a
s IL-6 odsutna. Vrijednosti omjera CRP/neutrofilni granulociti, CRP/limfociti, CRP/monociti, NLR i NMR na
prijemu bile su najviše kod bolesnika koji su imali teški i kritični tijek bolesti. Omjeri IL-6/neutrofilni granulociti,
IL-6/limfociti, IL-6/monociti bili su statistički značajno viši u skupini ispitanika koja je imala kritični tijek bolesti
u odnosu na ostale skupne bolesnika. Multiparametarski model za predviđanje razvoja kritičnog oblika COVID19 koji je uključivao EPO i feritin/hepcidin ispravno je klasificirao 88 % slučajeva.
Prisutnost izraženije sustavne upale u skupini hipoksičnih bolesnika vjerojatno je uzrok više koncentracije
hepcidina u odnosu na normoksične bolesnike s COVID-19. Uz to, razlike u vrijednostima pojedinih omjera
upalnih biljega među skupinama bolesnika s COVID-19 ukazuju da bi ovi omjeri mogli biti korisni u
predviđanju težine bolesti.In patients with COVID-19, inflammation and hypoxia are simultaneously present, having opposite effects on
hepcidin expression. This study is based on the hypothesis that hepcidin concentration is lower and concentration
of iron status parameters is changed in hypoxic COVID-19 patients compared to normoxic patients. The main aim
of the study was to determine the difference in hepcidin concentration and the level of parameters regulating its
concentration between normoxic and hypoxic COVID-19 patients and healthy subjects. Additionally, the study
investigated the association of hepcidin concentration with iron status parameters, erythropoiesis, hypoxia and
systemic inflammation. Ratios involving hepcidin and molecules related to hepcidin (hepcidin/iron,
ferritin/hepcidin, hepcidin/interleukin 6 (IL-6), hepcidin/C-reactive protein (CRP), hepcidin/erythropoietin (EPO))
were also examined to observe their interrelationship as well as the ratios of inflammatory parameters (CRP/IL-6,
ratios of CRP and IL-6 with leukocyte subpopulations, neutrophil to lymphocyte ratio (NLR), neutrophil to
monocyte ration (NMR), monocyte to lymphocyte (MLR)) to distinguish subgroups of COVID-19 patients with
different clinical presentations and disease outcomes.
The study included 96 COVID-19 patients and 47 healthy subjects matched by age and gender. Sampling was
conducted on admission to the emergency unit of the Department of Infectious Diseases. COVID-19 patients were
subdivided into normoxic and hypoxic groups based on haemoglobin oxygen saturation (SpO2), and, after followup, into disease severity groups.
The key findings of this study indicate that concentrations of hepcidin, ferritin, EPO, CRP, IL-6, and values of
hepcidin/iron and ferritin/hepcidin were significantly higher, while values of hepcidin/IL-6 and hepcidin/CRP were
significantly lower in hypoxic patients compared to normoxic patients as well as in severe and critical COVID-19
patients compared to mild and moderate COVID-19 patients. Hepcidin correlated well with CRP and IL-6 in the
normoxic patient group, whereas the correlation with CRP was weak and that with IL-6 was absent in hypoxic
patients. The ratios of CRP/neutrophil granulocytes, CRP/lymphocytes, CRP/monocytes, NLR, and NMR on
admission were the highest in patients with severe and critical disease course. The IL-6/neutrophil granulocytes,
IL-6/lymphocytes, and IL-6/monocytes ratios were significantly higher in the group with a critical disease course
compared to other patient groups. Multiparameter model for prediction of the risk for critical COVID-19 that
included EPO and ferritin/hepcidin correctly classified 88 % cases.
The presence of more pronounced systemic inflammation in the hypoxic patient group is probably the cause of
higher hepcidin concentrations found in this patient group compared to normoxic COVID-19 patients. In
addition, differences in the values of inflammatory markers’ ratios among COVID-19 patient groups suggest
their potential value in predicting disease severity
Analysis of fecal elastase and fecal calprotectin results in patients with and without inflammatory bowel disease
No full textUpalne bolesti crijeva (IBD), ulcerozni kolitis i Crohnova bolest, složeni su kronični upalni poremećaji koji zahvaćaju različite
dijelove sluznice gastrointestinalnog trakta, uz kontinuirane izmjene stanja remisije i relapsa. Većina kliničkih simptoma nije
specifična za IBD i mogu se javiti kod drugih upalnih bolesti. Egzokrina insuficijencija pankreasa može je javiti kao posljedica
brojnih poremećaja, a često se javlja kao ekstraintestinalna manifestacija upalnih bolesti crijeva. Fekalna elastaza je enzim pankreasa
koji se ne razgrađuje prolaskom kroz crijeva i korelira s egzokrinom funkcijom pankreasa. Fekalni kalprotektin je protein koji
sudjeluje u stimulaciji upalnog odgovora, a njegova koncentracija u fecesu neizravna je mjera infiltrata neutrofila u sluznici crijeva.
Cilj ovog rada je bio analizirati rezultate fekalne elastaze i fekalnog kalprotektina kako bi se ispitao njihov potencijal u razlikovanju
gastrointestinalnih poremećaja ovisno o prisutnosti odnosno odsustvu IBD. Ispitanici su podijeljeni u dvije skupine (prva uključuje
pacijente s dijagnozom IBD-a, a druga gastrointestinalna oboljenja uz isključenu dijagnozu IBD). Iz ostatnih ekstrahiranih uzoraka
stolice 59 ispitanika određene su koncentracije fekalne elastaze i fekalnog kalprotektina imunoturbidimetrijskim metodama na
analitičkom sustavu Beckman Coulter AU 5800 (Beckman Coulter, Brea, SAD). Rezultati istraživanja su pokazali da nema razlika
u koncentraciji fekalne elastaze između pacijenata s dijagnozom IBD-a i pacijenata s ostalim gastrointestinalnim poremećajima.
Bolesnici s dijagnozom IBD-a imali su visoke koncentracije fekalnog kalprotektina u odnosu na skupinu bolesnika koji nemaju
navedeni poremećaj. Snižene koncentracije fekalne elastaze pokazale su slabu korelaciju s povišenim koncentracijama fekalnog
kalprotektina kod pacijenata s dijagnozom aktivnog IBD-a. Fekalni kalprotektin ima značajan dijagnostički potencijal te može s
visokom dijagnostičkom osjetljivošću (87,88 %) i specifičnošću (88,46 %) odvojiti pacijente s IBD-om od pacijenata s drugim
gastrointestinalnim poremećajima. Na temelju dobivenih rezultata zaključuje se da fekalna elastaza nema dodatnu vrijednost u
diferencijalno dijagnostičkom pristupu IBD-a, dok fekalni kaprotektin ima izvrsnu dijagnostičku točnost u otkrivanju IBD-a.Inflammatory bowel disease (IBD), ulcerative colitis and Crohn's disease, are complex chronic inflammatory disorders that affect
different parts of the gastrointestinal tract mucosa, with continuous changes in the state of remission and relapse. Most of the clinical
symptoms are not specific to IBD and can occur in other inflammatory diseases. Pancreatic exocrine insufficiency can appear as a
result of numerous disorders, and often as an extraintestinal manifestation of inflammatory bowel diseases. Fecal elastase is a
pancreatic enzyme that is not degraded when passing through the intestines and correlates with the exocrine function of the pancreas.
Fecal calprotectin is a protein that participates in the stimulation of the inflammatory response, and its concentrations in feces are an
indirect measure of the neutrophil infiltrate in the intestinal mucosa. The aim of this work was to analyze the results of fecal elastase
and fecal calprotectin in order to examine their potential in differentiating gastrointestinal disorders depending on the presence or
absence of IBD. Subjects were divided into two groups (the first included patients with a diagnosis of IBD, and the second included
patients with gastrointestinal diseases without IBD). From the remaining extracted stool samples of 59 subjects, the concentrations
of fecal elastase and fecal calprotectin were determined by immunoturbidimetric methods on the analytical system Beckman Coulter
AU 5800 (Beckman Coulter, Brea, USA). The research results showed that there were no differences in fecal elastase concentrations
between patients with a diagnosis of IBD and patients with other gastrointestinal disorders. Patients with a diagnosis of IBD had high
concentrations of fecal calprotectin compared to the group of patients without the mentioned disorder. Decreased fecal elastase
concentrations showed a weak correlation with elevated fecal calprotectin concentrations in patients diagnosed with active IBD.
Fecal calprotectin has significant diagnostic potential and can distinguish patients with IBD from patients with other gastrointestinal
disorders with high sensitivity (87.88%) and specificity (88.46%). Based on the obtained results, it is concluded that fecal elastase
has no additional value in the differential diagnostic approach of IBD, while fecal calprotectin has excellent diagnostic accuracy in
detecting IBD
Citrinin effect on Chk2 and FANCD2 proteins in human cell lines HepG2 and A549
No full textMikotoksini su produkti sekundarnog metabolizma filamentoznih gljivica. Citrinin, čest onečišćivač ljudske i stočne
hrane, poliketidni je mikotoksin koji pokazuje antibakterijska, antitumorska i neuroprotektivna svojstva, ali je
istovremeno jako nefrotoksičan i genotoksičan. Mehanizmi kojima ostvaruje svoju toksičnost su nedovoljno poznati, a
pretpostavlja se da je jedan od njih, kao i kod drugih mikotoksina, poticanje apoptoze te moduliranje različitih
signalnih puteva. U ovom radu istražen je učinak citrinina na ukupnu količinu i fosforilaciju dva proteina, Chk2 i
FANCD2, koji su ključni u mehanizmima dvolančanog popravka DNA i to na dvije stanične linije humanih tumorskih
stanica, HepG2 (hepatocelularni karcinom) i A549 (plućni adenokarcinom). Za ispitivanje je korišten s enzimom
povezani imunosorpcijski test (ELISA), CytoGlow™ koji se izvodi izravno na stanicama uzgajanima mikrotitarskim
pločicama. Dobiveni podaci analizirani su u statističkom programu GraphPad Prism. Utvrđeno je da citrinin može,
ukoliko je dovoljne koncentracije, uzrokovati povećanje ukupne količine proteina Chk2 i FANCD2 i u stanicama
HepG2 kao i u stanicama A549 te potaknuti njihovu fosforilaciju odnosno aktivaciju. Intenziteti uočenih promjena su
relativno blagi do osrednji, ovisni o primijenjenim koncentracijama i vrsti stanica. S obzirom da aktivaciju proteina
Chk2 i FANCD2 potiču dvolančani lomovi DNA, možemo pretpostaviti da je jedan od mehanizama kojima citrinin
ostvaruje svoju toksičnost upravo izazivanje takvih dvolančanih lomova DNA i moduliranje signalnih puteva u kojima
sudjeluju proteini Chk2 i FANCD2.Mycotoxins are products of the secondary metabolism of filamentous fungi. Citrinin, a common food and animal feed
pollutant, is a polyketide mycotoxin, demonstrating antibacterial, antitumour and neuroprotective properties. It is also
highly nephrotoxic and genotoxic. Mechanisms of its toxicity are still insufficiently explored, but it is assumed that
one of those mechanisms, like in other mycotoxins, is apoptosis induction and modulation of various signaling
pathways. In this study, it was explored how citrinin impacts the total amount and phosphorylation of two proteins,
Chk2 and FANCD2, which are essential in double-strand DNA break repairs. The experiments were performed on two
human tumour cell culture lines, HepG2 (hepatocellular carcinoma) and A549 (lung adenocarcinoma), using
CytoGlow™ enzyme-linked immunosorbent assay (ELISA), directly on cells seeded in 96-well plates. The data was
analysed using the GraphPad Prism statistical software. It has been shown that citrinin can, if present in a high enough
dosage, cause the increase in total amounts of Chk2 and FANCD2 proteins, as well as induce their phosphorylation
and activation in both tested cell lines. The intensity of these changes is relatively mild to moderate, depending on the
concentration of citrinin which was applied and the type of cells. Because the activation of Chk2 and FANCD2 is
induced by double-stranded DNA breaks, we can assume that one of citrinin’s mechanisms of toxicity is precisely the
induction of double-stranded DNA breaks and the modulation of signaling pathways in which Chk2 and FANCD2
take part in
Verification of methods for the determination of antibodies to acetylcholine receptors (anti-AChR) and antibodies to muscle-specific tyrosine kinase (anti-MuSK)
No full textMiastenija gravis autoimuna je bolest koja najčešće nastaje zbog pojavnosti autoantitijela na acetilkolinski
receptor (anti-AchR) i autoantitijela na mišićno-specifičnu tirozin-kinazu (anti-MuSK) što uzrokuje
poremećeni neuromuskularni prijenos zbog čega nastaje mišićna slabost i umor. Cilj ovog rada je bio provesti
postupak verifikacije za dvije nove metode prije uvođenja u rutinski rad u Kliničkom zavodu za kemiju, KBC
Sestre milosrdnice. Ispitivane metode su kvantitativna ELISA metoda za određivanje anti-AChR i
kvalitativna metoda indirektne imunofluorescencije (IIF) na transfeciranim stanicama za određivanje antiMuSK. Nepreciznost ELISA metode za anti-AChR je ispitana mjerenjem komercijalnih kontrolnih uzoraka.
Rezultati su pokazali da ne zadovoljava kriterije deklarirane od proizvođača, ali zadovoljava kriterij kliničke
interpretacije kao i općeprihvaćeni kriterij nepreciznosti ELISA metoda. Istinitost ELISA metode za antiAChR je ispitana usporedbom s istovjetnom metodom implementiranom u Kliničkom zavodu za
laboratorijsku dijagnostiku, KBC Zagreb, na temelju 41 uzorka seruma. Ustanovljena je izvrsna podudarnost
u kategorizaciji rezultata između metoda. Regresijskom analizom ustanovljeno da u području klinički
značajnih pozitivnih rezultata ne postoji statistički značajno odstupanje između metoda. Ponovljivost IIF
metode za detekciju anti-MuSK antitijela je ispitana na 20 rezultata ponavljanja negativnih i pozitivnih
kontrolnih uzoraka te je zadovoljila postavljene kriterije prihvatljivosti prema CLSI EP12-A2. Istinitost IIF
metode je ispitana usporedbom s ELISA metodom implementiranom u Kliničkom zavodu za laboratorijsku
dijagnostiku, KBC Zagreb, na temelju 33 uzorka seruma. Ustanovljena je zadovoljavajuća usporedivost, ali
zbog malog broja pozitivnih uzoraka (mala pojavnost anti-MuSK) nije zadovoljen preduvjet za pravilnu
interpretaciju statističkog testa. Rezultati provedene verifikacije potvrđuju da su ispitivane metode
prihvatljive za uvođenje u rutinski rad.Myasthenia gravis is an autoimmune disease that most often occurs due to the appearance of autoantibodies
to acetylcholine receptor (anti-AChR) and muscle-specific tyosine kinase (anti-MuSK), which causes
impaired neuromuscular transmission resulting in muscle weakness and fatigue. The aim of this study was to
conduct a verification procedure for two new methods before their implementation into routine work in the
Department of Clinical Chemistry, Sestre milosrdnice University Hospital. Tested methods were the
quantitative ELISA method for the determination of anti-AChR antibodies and the qualitative method of
indirect immunofluorescence (IIF) on transfected cells for the determination of anti-MuSK antibodies. The
imprecision of the ELISA method for anti-AChR was tested by measuring control samples. The imprecision
did not meet the criteria declared by the manufacturer, but meets the one of clinical interpretation as well as
the generally accepted criterion of imprecision of ELISA methods. The trueness of the ELISA method for
anti-AChR was tested by comparison with the identical method implemented in the Department of Laboratory
Diagnostics, University Hospital Centre Zagreb, based on 41 serum samples. A perfect agreement in the
categorization of the results between the methods was established and regression analysis revealed that there
is no statistically significant difference between the methods in the range of clinically significant results. The
reproducibility of the IIF method for the detection of anti-MuSK antibodies was tested on 20 repetition results
of negative and positive control samples and met the acceptance criteria according to CLSI EP12-A2. The
trueness of the IIF was tested by comparison with the ELISA method implemented in the Department of
Laboratory Diagnostics, University Hospital Centre Zagreb, based on 33 serum samples. A satisfactory
comparability was established, but due to the small number of positive samples (low incidence of anti-MuSK
antibodies), the precondition for the correct interpretation of the statistical test was not met. The results of the
verification confirm that the tested methods are acceptable for implementation into routine work
Validation of Methods for Genotyping Polymorphisms in Factor V, Prothrombin, Plasminogen Activator Inhibitor-1, and Methylenetetrahydrofolate Reductase Genes
No full textVenski tromboembolizam je potencijalno po život opasno stanje čija etiologija uključuje brojne stečene i
nasljedne rizične čimbenike. Nasljedni rizični čimbenici uključuju genske polimorfizme faktor V Leiden (F5
G1619A) i protrombin G20210A (F2 G20210A). Katkad se u rizične faktore za venski tromboembolizam
ubrajaju i genski polimorfizmi PAI-1 5G/4G i MTHFR C677T iako se oni ne mogu jednoznačno povezati s
povećanim rizikom venskog tromboembolizma. U Kliničkom zavodu za kemiju Kliničkog bolničkog centra
Sestre milosrdnice došlo je do promjene analitičkog sustava za genotipizaciju navedenih polimorfizama te je
uveden LightCycler® 480 (Roche Diagnostics GmbH, Švicarska). Cilj ovog rada bio je validirati analitičke
karakteristike mjernih postupaka genotipizacije F5 G1619A, F2 G20210A, PAI-1 5G/4G i MTHFR C677T
metodom lančane reakcije polimeraze uz analizu krivulje taljenja na sustavu LightCycler® 480 prije
korištenja u rutinskom radu. Ispitani su analitička nepreciznost, analitička osjetljivost, analitička specifičnost,
analitička točnost, usporedivost drugom metodom i granica detekcije. Svi ispitani parametri u potpunosti su
zadovoljili unaprijed definirane analitičke kriterije kvalitete i stoga se ispitani mjerni postupci mogu prihvatiti
za rutinski rad.Venous thromboembolism is a potentially life-threatening condition whose etiology includes numerous
acquired and hereditary risk factors. Hereditary risk factors include gene polymorphisms factor V Leiden
(F5 G1619A) and prothrombin G20210A (F2 G20210A). Gene polymorphisms PAI-1 5G/4G and MTHFR
C677T are also sometimes considered a risk factor for venous thromboembolism although those can not
be consistently associated with increased risk of venous thromboembolism. In the Clinical Chemistry
Department of the Sestre milosrdnice University Hospital Center, the analytical system being used for
genotyping mentioned polymorphisms was changed by introducing LightCycler® 480 (Roche Diagnostics
GmbH, Switzerland). This study aimed to evaluate analytical characteristics of genotyping
polymorphisms F5 G1619A, F2 G20210A, PAI-1 5G/4G, and MTHFR C677T by real-time polymerase
chain reaction with melting curve analysis on LightCycler® 480 before putting it into routine operation.
Analytical imprecision, analytical sensitivity, analytical specificity, analytical accuracy, repeatability
using another method, and limit of detection were examined. All examined parameters fully met the predefined analytical quality criteria and therefore the evaluated methods can be accepted for routine work
Competitive binding fluorescence polarization assay for determination of association constants of dipyridamole and warfarin to human serum albumin
No full textCilj ovog rada bio je odrediti konstante ravnoteže reakcija vezanja dipiridamola i varfarina na humani serumski albumin
(HSA). Dobro poznavanje vezanja lijekova za proteine krvne plazme, posebice za HSA koji je među njima
najzastupljeniji, od velike je važnosti za predviđanje farmakokinetičkih svojstava lijekova koji se za njega vežu. S
obzirom na to da je samo nevezani udio lijeka farmakološki aktivan, učinak lijeka uvelike ovisi o svojstvima vezanja pa
stoga kompeticija za vezna mjesta na proteinu može biti uzrok interakcija među lijekovima. Metoda korištena u ovom
radu za proučavanje afiniteta vezanja lijekova za HSA naziva se polarizacija fluorescencije. Zasniva se na svojstvu malih
fluorescentnih molekula, među koje spada dipiridamol, da u slobodnom obliku rotiraju velikom brzinom i zahvaljujući
tome smanjuju polarizaciju fluorescencije. Prilikom vezanja u kompleks s velikom molekulom, u ovom slučaju HSA,
brzina rotacije se značajno smanjuje što dovodi do veće polarizacije fluorescencije. Dodatkom varfarina koji s
dipiridamolom ulazi u kompeticiju za vezno mjesto dipiridamol se iz kompleksa oslobađa u otopinu gdje ponovno rotira
velikom brzinom i smanjuje polarizaciju fluorescencije. Mjereći polarizaciju fluorescencije bilo je moguće kvantificirati
udio vezanog dipiridamola i izračunati konstante ravnoteže reakcija dipiridamola i varfarina za HSA na temelju mjerenja
dobivenim prvo titracijom otopine dipiridamola otopinom HSA, a zatim otopine HSA i dipiridamola otopinom varfarina.
Izračun je izveden u programu SPECFIT i razmotrena su dva modela vezanja. Pokazalo se da model s dva vezna mjesta
za dipiridamol na HSA bolje opisuje eksperimentalne podatke te da je polarizacija fluorescencije pogodna metoda za
proučavanje afiniteta vezanja lijekova za HSA.The main goal of this work was to determine the binding equilibrium constants of dipyridamole and warfarin to human
serum albumin (HSA). A good understanding of blood plasma protein's drug binding properties, especially of HSA's
which is the most abundant among them, is of great importance for the prediction of the pharmacokinetic properties of
drugs bound by it. Since only the free fraction of the drug can be pharmacologically active, the drug's effect is greatly
dependent on its binding properties and because of that the drug's competition for the protein's binding sites can be a
cause of drug interactions. The method used in this work to study HSA's drug binding is called fluorescence polarization.
It is based upon the property of small fluorescent molecules, such as dipyridamole, to rotate at great speed when unbound
and because of that to reduce fluorescence polarization. By binding to a larger molecule, in this case HSA, dipyridamole's
rotation speed is greatly reduced, and fluorescence polarization is increased. With the addition of warfarin,
dipyridamole's binding site competitor, dipyridamole is released from HSA allowing it to rotate at great speed once
again and reduce fluorescence polarization. By measuring fluorescence polarization, it was possible to quantify the
bound fraction of dipyridamole and to calculate the binding equilibrium constants based on the measurements obtained
first by a titration of a dipyridamole solution with a HSA solution and then by a titration of both dipyridamole and HSA
solution with a warfarin solution. The calculations were performed in SPECFIT and two binding models were
considered. It was shown that the model in which HSA has two binding sites for dipyridamole better describes the
experimental data and that fluorescence polarization is a convenient method for studying HSA's drug binding properties
Analysis of abemaciclib, ribociclib and palbociclib using capillary zone electrophoresis
No full textZbog sve veće pojavnosti raka dojke u populaciji, postoji potreba za razvijanjem analitičkih metoda koje će moći koristiti u terapijskom praćenju lijekova kako bi se pospješio uspjeh terapije.
Cilj ovog istraživanja bio je razvoj nove kapilarnoelektroforetske metode kako bi se na jednostavan, brz i točan način mogao detektirati ili kvantificirati lijekove iz skupine cikliba – abemaciklib, ribociklib ili palbociklib. Korištena tehnika je zonska kapilarna elektroforeza.
Korištena je kapilara od izvučenog kvarca duljine 32,5 cm unutrašnjeg promjera 50 μm. Uzorci su u kapilaru injektirani pod tlakom od 50 mbar, pri temperaturi 25 °C tijekom 6 s. Valna duljina na kojoj su provođene analize iznosi 320 nm.
Optimizirani su sljedeći parametri - vrsta i pH pufera, koncentracija pufera, napon, temperatura i uvjeti ispiranja. Pratio se njihov utjecaj na vrijeme analize, površinu pika, broj teorijskih tavana i simetriju pika. Kao optimalne vrijednosti uzete su 50 mM fosfatni pufer pH 2,5, napon 27,5 kV i temperatura 30 °C. Za optimalne uvjete ispiranja odabrano je da se na početku dana ispire 10 min s 10% fosfornom kiselinom, 10 min s ultračistom vodom i 20 min s radnim puferom, a na kraju dana 20 min s ultračistom vodom te da se između mjerenja radi prekondicioniranje 5 min s radnim puferom. Pokazalo se da su za uspješnu i reproducibilnu analizu dostatni i uvjeti rada s citratnim puferom pri pH 3,17, koncentracije pufera 113 mM, temperature 25 °C, napona 20 kV i pH 4,11, koncentracije pufera 35 mM, temperature 25 °C i napona 25 kV.Considering the growing incidence of breast cancer in our population, there is a need to develop an analytical method that can be used in therapeutic drug monitoring to enhance the success of therapy.
The goal of this research was to develop a new capillary electrophoresis method so that any of the observed drugs from the ciclib group – abemaciclib, ribociclib or palbociclib could be detected or quantified in a simple, fast and accurate way. The technique used is zone capillary electrophoresis.
This analysis was performed on a 32,5 cm long capillary of extracted quartz with an inner diameter of 50 μm. The samples were injected into the capillary under a pressure of 50 mbar, at a temperature of 25 °C for 6 seconds. A wavelength of 320 nm was used.
The following parameters were optimized through the study - type and pH of the buffer, buffer concentration, voltage, temperature and flushing conditions. Their influence on the analysis time, peak area, number of theoretical plates and peak symmetry was monitored. The optimal values found included 50 mM phosphate buffer, voltage 27.5 kV and temperature 30 °C. For optimal flushing conditions, it was chosen to flush at the beginning of the day for 10 minutes with 10% phosphoric acid, 10 minutes with ultrapure water and 20 minutes with the working buffer, and at the end of the day 20 minutes with ultrapure water and to precondition for 5 minutes between measurements with the working buffer. It turned out that for the successful and reproducible analysis, the working conditions with the citat buffer at pH 3.17, buffer concentration 113 mM, temperature 25 °C, voltage 20 kV and pH 4.11, buffer concentration 35 mM, temperature 25 °C and voltage 25 kV were also sufficient