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Multipla skleroza (engl. multiple sclerosis, MS) je kronična upalna demijelinizacijska bolest središnjeg živčanog sustava (SŽS). Uzrok MS je nepoznat, no smatra se da je MS multikomponentna bolest do koje dolazi prisutnošću genske predispozicije i autoimunosnim odgovorom koji je potaknut infekcijom. Razvoj novih metoda i biljega omogućuje uvid u dosad nepoznatu patogenezu bolesti. Standardi za dijagnostiku MS su oligoklonalne vrpce, IgG indeks i MRZ reakcija u kliničkoj praksi. Prognostički biomarkeri za MS mogu pružiti informacije o tijeku aktivnosti bolesti i ukazati na konverziju u drugi oblik MS (detekcija oligoklonalnih vrpca IgG u likvoru, detekcija oligoklonalnih vrpca IgM u likvoru, CHI3L1, NF, N-CAM, MMP). Biomarkeri terapije (CXCL13, NFL, vitamin D, BDNF) u postupku su istraživanja i implementacije. U istraživanjima je pokazana mogućnost primjene novih biomarkera, cfDNA i miRNA, prilikom dijagnoze, praćenja tijeka bolesti i terapije MS koji bi kao takvi bili neinvazivni biomarkeri. Razvoj metode sekvenciranja nove generacije, omogućio je otkrivanje novih čimbenika uključenih u patogenezi MS, potencijalnih biomarkera gena i novih meta za razvoj lijekova. Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system (CNS). The cause of MS is unknown, but MS is thought to be a multicomponent disease caused by the presence of a genetic predisposition and an autoimmune response triggered by infection. The development of new methods and markers provides insight into the unknown pathogenesis of the disease. Standards for the diagnosis of MS are oligoclonal bands, IgG index and MRZ reactions in clinical practice. Prognostic biomarkers for MS can provide information on the course of disease activity and indicate conversion to another form of MS (detection of oligoclonal IgG bands in cerebrospinal fluid, detection of oligoclonal bands IgM in cerebrospinal fluid, CHI3L1, NF, N-CAM, MMP). Biomarkers of therapy (CXCL13, NFL, vitamin D, BDNF) are in the process of research and implementation. Research has shown the possibility of using new biomarkers, cfDNA and miRNA, in the diagnosis, monitoring of the course of the disease and therapy of MS, which as such would be non-invasive biomarkers. The development of a new generation sequencing method has made it possible to discover new factors involved in the pathogenesis of MS, potential gene biomarkers and new targets for drug development.

Karcinom dojke najučestalije je maligno oboljenje u Hrvatskoj, a dijagnoza se vrlo često postavlja kasno što utječe na daljnji tijek bolesti. Važno je poboljšati laboratorijsku dijagnostiku da bi se otkrile i standardizirale metode koje bi omogućile specifično i rano postavljanje dijagnoze. Biomarkeri, koji se danas koriste, naišli su na svoja ograničenja, stoga se želi unaprijediti laboratorijski proces u ranom otkrivanju karcinoma dojke kao i svih ostalih malignih bolesti. Slobodna cirkulirajuća DNA, cfDNA, potencijalni je biomarker karcinoma budući da se velikim dijelom u cirkulaciju otpušta iz tumorskog tkiva. Prednosti, u odnosu na trenutno poznate biomarkere, su u tome što je metoda neivazivna jer je potreban samo uzorak krvi, daje uvid u trenutni molekularni status što omogućuje personalizirani pristup, bolje praćenje statusa bolesti i terapije. Ovim radom željelo se ispitati kako predanalitička faza utječe na kvalitetu i kvantitetu cf DNA. Koristili su se uzorci plazme i seruma pacijentica s karcinomom dojke sa i bez metastaza te kontrolni zdravi uzorci. Uzastopnim odmrzavanjem i zamrzavanjem uzoraka vidjelo se da se time narušava integritet DNA pa tako i kvaliteta te kvantiteta izoliranog materijala. Primjenom dviju vrsta kolona za izolaciju, ustanovljeno je da je za izolaciju cf DNA potrebno koristiti specifične kolone namijenjene za cfDNA da bi doprinos bio veći jer genomska DNA nepovoljno utječe na izolaciju cfDNA. Nadalje, različiti stadiji bolesti utječu na integritet cfDNA što otežava izolaciju. Dodatno se javlja problem povećane koncentracije cfDNA kod zdravih pojedinaca što znači da postoje fiziološka stanja popraćena povećanom koncentracijom cfDNA. Svi navedeni rezultati ukazuju na potrebu za specifičnim genetičkim analizama. Breast cancer is the most common malignant disease in Croatia and it is often diagnosed too late which affects the course of disease. It is important to improve laboratory diagnostics to detect and standardize methods that would allow a specific and early diagnosis. The biomarkers, that are used nowadays, have their limitations, so there is an urge in advancing the laboratory process of early detection of breast cancer as well as other malignant diseases. Free circulating DNA, cfDNA, is a potential cancer biomarker since it is released into the circulation from tumor tissue. The advantages over the currently-known biomarkers are that the method is non invasive because only blood sample is needed, it provides an insight into the molecular status at theparticular moment and enables personalized medicine, better monitong of disease progression and therapy. This research investigated how the pre-analytical phase affects the quality and quantity of cfDNA. Samples of plasma and serum from breast cancer patients with and without metastases were used as the samples from healthy controls. Several freezing and defrosting affected in a way that the quality and quantity of DNA were poor. Two different colons for isolation were used. It was found that isolation of cfDNA requires specific columns intented for specific isolation of cfDNA so that DNA yield would be bigger and more stable despite the presence of genomic DNA. Moreover, different stages of the disease affect DNA integrity which makes isolation more difficult. In addition, there is a problem of increased cfDNA concentration in healthy individuals which means there are some physiological states with elevated cfDNA concentration. All these results suggest specific genetic analysis.

Od otkrića cfDNA (od eng. cell-free DNA) do danas, razmatrane su i istraživane mnoge njene primjene u molekularnoj medicini: od dijagnoze i praćenja tumora te predviđanja ozdravljenja pomoću specifičnih biomarkera, do otkrivanja i predviđanja poteškoća u razvoju ploda putem neinvazivne prenatalne dijagnostike. Danas su poznati mnogi biomarkeri specifični za određene maligne tumore i pomoću nekih od njih moguće je doći do zaključaka o pojavnosti/otpornosti na liječenje/stadiju tumora, no još uvijek se ne zna dovoljno da bi takvo što u medicini postalo široko primjenjivano. U prenatalnoj dijagnostici, cfDNA se pokazao kao iznimno vrijedan alat pri otkrivanju genomskih abnormalnosti. Kako bi se u budućnosti sprječila zloupotreba ove tehnologije, potrebno je uspostaviti detaljni etički kodeks. Općenito, cfDNA porijeklom iz tumora/fetusa, kraća je od normalne/majčine cfDNA, što daleko olakšava pročišćavanje i analizu. Since the discovery of cfDNA (cell-free DNA), many of its applications in molecular medicine have been considered and researched: from diagnosing and monitoring tumors, and predicting disease outcome using specific biomarkers, to the detection and prediction of aberrant fetus development using noninvasive prenatal diagnostics. Today, many biomarkers specific for certain tumors types have been discovered and some of them can be used to help the decision making process about the presence/treatment resistance/stage of a tumor. Still, there is an insufficient amount of knowledge about the subject, which will have to be thoroughly analysed before wide use in medical practice. cfDNA has become an immensely valuable tool in prenatal diagnostics. To prevent future misuse, establishment of a detailed ethical codex is vital. Generally, tumor/fetal cfDNA is shorter than normal/maternal cfDNA, which makes purification and analysis far easier.